Effects of preharvest applications of natural antimicrobial products on tomato fruit decay and quality during long-term storage

Scientia Horticulturae 222 (2017) 193–202

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Effects of preharvest applications of natural antimicrobial products on tomato fruit decay and quality during long-term storage

MARK

Carmela Anna Miglioria, Luca Salvatib, Luigi Francesco Di Cesarec, Roberto Lo Scalzoc, ⁎ Mario Parisid, a Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca Ingegneria e Trasformazioni agroalimentari (CREA-IT), Sede di Torino, Strada delle cacce 73, 10135, Torino, Italy b Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca Agricoltura e Ambiente (CREA-AA), Sede di Roma, Via della Navicella 2-4, 00184, Roma, Italy c Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca Ingegneria e Trasformazioni agroalimentari (CREA-IT), Sede di Milano, Via Venezian 26, 20133, Milano, Italy d Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca Orticoltura e Florovivaismo (CREA-OF), Sede di Pontecagnano, Via Cavalleggeri 25, 84098, Pontecagnano, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Solanum lycopersicum L GRAS substances Fruit senescence Phytochemicals Total solids content Aromatic compounds

The effects of preharvest sprays of Thyme essential oil, Propolis and Chitosan on postharvest quality and decay of a “long-storage” tomato, called “Vesuviano”, stored at room temperature for 120 days, were investigated. Postharvest fruit quality [number of swollen-healthy fruits (SF), withered-healthy fruits (WF) and rotten fruits (RF)], organoleptic-related indexes (dry matter, soluble sugars, organic acids, volatiles) and health-related compounds (total carotenoids and phenols) were investigated at 40, 80 and 120 days post-harvesting (T40, T80, T120). Propolis and Chitosan were able to reduce rotten fruits starting from T80, while the effect of Thyme was evident as early as T40. Furthermore Chitosan delayed fruit senescence (as expressed by SF/WF ratio) during the long-storage period. All treatments did not affect the overall postharvest quality, nevertheless some compounds (such as total soluble sugar for Chitosan and Thyme; total carotenoids and flavonols for Chitosan; total organic acids, 2-(E)-hexenal, 2-isobutylthiazole and terpenes for Propolis), were better retained than the control during postharvesting period. Among the three natural fungicides, Chitosan was most effective in reducing fruit senescence, maintaining a good quality of the fruits over a long-term.

1. Introduction Tomato (Solanum lycopersicum L.) is one of the most produced and extensively consumed vegetable crops in the world (Sacco et al., 2015). Fresh fruits and tomato-based foods provide basic organoleptic features, as well as a wide variety of nutrients and health-related phytochemicals. Sugars and organic acids include the majority of the total dry matter content of tomato fruit. The most abundant sugars are fructose and glucose, while major organic acids are citric and malic, with predominance of the first acid (Davies and Hobson, 1981). Earlier studies have shown that the level of sugars and acids affect not only tomato taste attributes, but also the overall flavour (Hobson and Bedford, 1989). A relevant contribution to tomato flavour is also given by volatiles: hexanal, 2(E)-hexenal and 2-isobutylthiazole are considered as characteristic tomato compounds (Dirinck et al., 1976). Moreover, terpenes have been demonstrated to contribute to product’s

⁎

Corresponding author. E-mail address: [email protected] (M. Parisi).

http://dx.doi.org/10.1016/j.scienta.2017.04.030 Received 30 January 2017; Received in revised form 21 April 2017; Accepted 25 April 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

quality (Baldwin et al., 2000). The most important health-related compounds in tomato fruit are carotenoids with lycopene being the most representative (Shi and Maguer, 1999). Epidemiological studies have highlighted a strong association between consumption of carotenoids-rich foods and prevention of different cancers such as cervical, breast, colon, prostate, rectal and stomach (Dorais et al., 2008). Phenolic compounds, such as naringenin chalcone, quercetin-type flavonols and hydroxycinnamic acids, are another important class of phytochemical compounds of tomato fruit (Giovannelli et al., 1999). Their potential benefit for human health, likely due to the antioxidant activity, is widely acknowledged (Siracusa et al., 2012). Despite benefits that can be derived from the crop, postharvest losses make its production in most parts of the world unprofitable. Postharvest losses in tomatoes can be as high as 25–42% globally and are due to physiological disorders, physical injury and fungal infections (Arah et al., 2015). Fruits are susceptible to attacks of several

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fermentation-senescence markers (ethanol).

pathogenic fungi [such as Alternaria alternata (Fr.:Fr.) Keissl., Botrytis cinerea (Pers), etc.] making them unsuitable for consumption by producing mycotoxin, in addition to causing rots (Ibrahim and AlEbady, 2014). Although public opinion strongly demands for a reduction of synthetic agents, fungicides are still the primary means of controlling postharvest diseases. Negative effects of chemical fungicides call for alternative products that reduce losses due to postharvest decay. Among naturally derived substances, i.e. GRAS (Generally Recognized as Safe), essential oils, propolis and chitosan were recently considered with the aim to controlling biological spoilage and extending the storage life of different fresh commodities (Tripathi and Dubey, 2004). Plant-derived essential oils are considered non-phytotoxic compounds and are potentially effective as natural pesticides for crop protection (Pane et al., 2013). They contain complex mixtures of secondary metabolites, which are biologically active, endowed with antimicrobial, allelopatic, antioxidant and bioregulatory properties (Antunes and Cavaco, 2010). Propolis is a natural resinous substance obtained from leaf buds and bark of poplar and conifer trees. It has antibiotic, antibacterial and antifungal activity (Tripathi and Dubey, 2004). Application of propolis has shown positive effects on reducing decay and extending the shelf life of different vegetables and fruits (Yang et al., 2016). Chitosan is a cationic polysaccharide produced by alkaline Ndeacetylation of chitin and it has been reported to have plant protective and antifungal properties on different crops (Pichyangkura and Chadchawan, 2015). The efficacy of postharvest applications of different natural antimicrobial substances on reducing postharvest losses have been documented in earlier studies (Aminifard and Mohammadi, 2012; Reddy et al., 2000; Ordóñez et al., 2011). Conversely, studies regarding the preharvest use are reported for few fresh commodities excluding tomato crop (Romanazzi et al., 2002, 2012, 2013; Saavedra et al., 2016; Tezotto-Uliana et al., 2014; Meng et al., 2008). An improved investigation on preharvest uses depend on varieties or landraces belonging to each fruit or vegetable species, since they differ in term of shelf lives extension and storage condition (temperature, relative humidity) can be very diversified. Therefore the effectiveness of GRAS substances adopted to control postharvest decay and quality can be severely affected by varying background conditions. Several cherry-like tomato landraces showing high drought tolerance are traditionally cultivated in Mediterranean countries, and especially in southern Italy, under non-irrigated conditions. Peculiar textural properties, such as high firmness and thickness of skin, allow an extended shelf life of the fruits, which after the harvest are stored in the typical hung-shaped appearance for 3–4 months under no-conditioned atmosphere (“long-storage tomatoes”) (Siracusa et al., 2012; Mercati et al., 2015). The most important and profitable “long-storage” landrace is cultivated on the slopes of the Vesuvio volcano (Campania region) and is called “Vesuviano” or “Pomodorino del piennolo del Vesuvio” (labeled as PDO − Protected Designation of Origin; Reg. 1238, 2009; Ercolano et al., 2008, 2014). This high-value product is commercialized throughout the Italian peninsula and in other European countries starting from winter to early spring, when high-quality fresh tomatoes are not available on the markets. However, despite the prolonged shelf life, unprofitable fruit rots and quality decay are currently observed during the typical storage of “Vesuviano” landrace. Therefore the adoption of means to limit these losses are highly desirable. Based on these premises, the objectives of this study were (i) to examine the effects of preharvest spraying of propolis, chitosan and thyme essential oil for controlling natural decay and (ii) to evaluate postharvest quality during the typical prolonged storage at room temperature by measurement of dry matter, soluble sugars, organic acids, by the main health-related compounds (phenols and total carotenoids), and evaluating volatile substances both for tasting (hexanal, 2(E)-hexenal, 2-isobutylthiazole and total terpenes) and for

2. Material and methods 2.1. Field trial, treatments and storage conditions The study was performed adopting a previously described accession of “Vesuviano” landrace (PV-ISCI 10) (Ruggieri et al., 2014). Tomato plants were grown in open field, according to the traditional practices, on the slopes of Vesuvio volcano (Massa di Somma, 40° 50′ 0″ N, 14° 22′ 0″ E, 175 m a.s.l.). Transplant was performed in single rows with a density of 5.0 plants m2. A single drip irrigation was applied in support of rainfall along the growing season (115 mm). This experiment included three preharvest treatments: Chitosan (Chitoplant®, Agritalia − Villa Saviola, Italy), Propolis (Propoli®, Serbios − Badia Polesine, Italy) and Thyme (Thymus vulgaris essential oil, A.C.E.F. − Fiorenzuola d’Arda, Italy). These substances were diluted by water to give final concentrations of 0.3%, 0.2% and 1% (water emulsion) respectively, and were compared to untreated control (water). The commercial product Chitoplant® was an aqueous solution containing 95% of chitosan (47–65 kDa), 2.5% of boron and 2.5% of zinc; Propoli® was a propolis glycolic extract with a flavonoid content (expressed as galangin) of 20 mg ml−1, while Thyme was Thymus vulgaris essential oil containing 57.4% of thymol and 2.8% of carvacrol. The abovementioned natural antimicrobial substances were spread on the plants (at 5, 15, 25 and 35 days before the harvest) until all fruits were wet to runoff. Field trials were carried out in a completely randomized experimental design with three replicates and 30 plants for each replicate. At harvest (July 31, 2013), successively mentioned as T0, ripe bunched fruits were harvested taking care not to detach them from peduncles. For each experimental plot, 8 kg of fruits (320 in number, approximately) were selected based on uniformity of size and colour and absence of physical injury or lesions caused by pathogen. These quantities were subdivided in two wooden boxes, each containing 4 kg approximately of fresh product, used for quali-quantitative market assessment and chemical-physical analysis. The overall samples (without any packaging) were then transferred in an unconditioned and wellventilated shed up to 120 days after harvest, where temperature and relative humidity were recorded, every 60 min, by a wireless data logger (EL-USB-2 model, Lascar Electronics Ltd. − United Kingdom) (Supplementary Fig. 1). 2.2. Postharvest decay assessment In order to evaluate the decay rate, both treated and untreated fruits of each experimental plot were visually observed at 40 (September 10, 2013), 80 (October 20, 2013) and 120 (November 30, 2013) days post harvest (thereafter mentioned as T40, T80 and T120). At these time points fruits were classified in three categories: swollen-healthy fruits (SF) (ripe fruits without abiotic/biotic damages and showing high firmness), withered-healthy fruits (WF) (wrinkled/senescent fruits without abiotic/biotic damages and showing low firmness) and rotten fruits (RF) caused by physiological or biological alterations (Supplementary Fig. 2). Number of fruits falling in these classes were recorded at each time point and the relative incidence (%) was expressed as cumulative number of each category on number of fruits at harvest (T0). RF were discarded each time as waste product, while WF and SF represented the commercial yield. A preliminary report (Carrieri et al., 2016) provided technical details and specific analysis on post harvest decay microorganisms. 2.3. Analytical methods 2.3.1. Tomato samples For each treatment, healthy tomato fruits were sampled in duplicate 194

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significant peaks. The clean coloured organic layer was filtered (0.45 μm regenerated cellulose filter) and analyzed by HPLC (JascoItaly, Lecco, Italy models PU980, diode array detector mod MD-2010, the used column was a C30 YMC Carotenoid 0,8 ml/min, 35 °C, YMC Europe, Dinslaken, Germany) using the methodology described by Ishida et al. (2001). The concentration of total carotenoids, obtained by the sum of β-carotene and all-trans lycopene, the major carotenoids in fresh tomato fruits, was calculated from the experimental peaks area by analytical interpolation, using standard calibration curves and were expressed as mg/100 g dw.

(around 600 g/sample) and within 12 h from harvesting, they were quickly frozen in a forced-air blast tunnel (Thermo-Lab, Lodi, Italy) at −50 °C. Subsequently, sampled fruits were divided in two aliquots, one stored at −80 °C and the other subjected to lyophilisation. Dry matter content was calculated by the ratio lyophilized product weight vs fresh one. The frozen material, before analysis, was homogenized in a blade mill blender, sieve of 1 mm and kept at −20 °C. 2.3.2. Soluble sugars and organic acids The extraction was carried out on 500 mg of lyophilized sample, homogenized with 10 ml deionised water and separated by centrifugation. The supernatant was filtered (0.45 μm nylon filter), diluted and analyzed via high-performance liquid chromatography (Jasco-Italy, Lecco, Italy models PU980, RI 930 refractive index detector, the used column was a Biorad Aminex HPX-87C, 0,7 ml/min, 85 °C, Bio-Rad Laboratories, Milano, Italy) under the conditions described by Forni et al. (1992). As for organic acid analysis, the extraction was performed as for soluble sugars and the HPLC analysis (Jasco-Italy, models PU980, UV1570 detector setted at 214 nm, the used column was a GL Sciences Inertsil ODS-3 C18, 0,6 ml/min, 30 °C, Microcolumn, Monza, Italy) was performed as described by Lo Scalzo et al. (2012).

2.3.5. Extraction-concentration and analysis of volatile substances The volatile fraction from tomatoes samples was extracted and concentrated by a combined microwave-resin-solvent and the extracts obtained were analyzed by GC/MS (Agilent Mod. 6890N and MS 5973N, Agilent Technologies Italia, Milano, Italy, the used column was a capillary DB-1, 60 m, 0.25 mm 0.25 μm film thickness, J & W Scientific, Folsom, California, USA), following an already validated protocol (Migliori et al., 2012). The volatile components were identified using the retention times of chromatographic peaks, by comparing their mass spectra with those in a commercial library (Wiley 7 n. 1 Library, Mass Spectral Data Base, Hewlett-Packard, Vienna, Austria) and by using commercial standards when available. For quantitative analysis, standard solutions of the components at known concentrations were used to calculate the response factors. For the components whose standards were not commercially available, the internal standard procedure was followed, by using solutions of methyl palmitate. The hexanal, 2(E)-hexenal and 2-isobutylthiazole were considered as characteristic tomato volatile compounds, according to Dirinck et al. (1976). The level of total terpenes, given by the sum of 2,3-epoxigeranial, neral, geranial, β-damascenone, nerylacetone, β(Z)-ionone, (E, E)pseudoionone have been reported and discussed.

2.3.3. Phenols Analysis of phenol content was performed according to Van der Rest et al. (2006), with some modifications. For the extraction, 300 mg of lyophilized powder was dissolved in 10 ml of a 1:1 v/v mixture of EtOH and 0.12 N HCl. The mixture was vortexed for 1 min and subjected to shaking for 2 h. The mixture was centrifuged at 25000g for 5 min at 4 °C, and an aliquot (400 μl) was diluted with an equal volume of 80% EtOH plus 50 μl of 50% CH3COOH prior to HPLC injection. The HPLC analysis was performed using a JASCO system equipped with a diode array detector (MD-910 JASCO). The pump (PU-980 JASCO) was coupled to a ternary gradient unit (LG-1580-02 JASCO). The data were evaluated using a software-management system for chromatographic data (ChromNAV, Jasco). The separation was performed by reversedphase chromatography using an ODS-3 Lichrosorb 250 × 4 mm column. The flow rate was 0.7 ml/min, the injection volume was 30 μl, and the oven temperature was 45 °C. The mobile phase consisted of 5% CH3COOH in water (solvent A) and methanol/water/CH3COOH (90:5:5) (solvent B). The gradients were as follows: (A/B): 95/5 for 0–3 min, from 95/5 to 70/30 in 5 min, 70/30 for 20 min, from 70/30 to 35/65 in 7 min, 35/65 for 5 min, from 35/65 to 95/5 in 10 min, and 95/5 for 12 min. The total analysis time was 59 min. The peaks were identified by direct comparison with commercial standards and by inspection of spectral and chromatographic properties with respect to relevant data reported in the literature. The main compound observed were chlorogenic acid, rutin, two rutin derivatives, tentatively identified as rutin-O-hexoside and rutin-O-pentoside, from chromatographic data of previous works (Moco et al., 2006; Van der Rest et al., 2006; Vallverdú-Queralt et al. 2010), and chalconaringenin, whose presence in tomato is well known. Quantification was based on the calibration curves of external standards. The levels of total flavonols (mg/100 g dw), resulting from the sum of contents of rutin and the two rutin derivatives, were reported.

2.3.6. Ethanol The ethanol measurement was performed by a static headspace GCFID (DANI Instruments, Milano, Italy, GC Mod. 6400 equipped with an HSS 86.50 headspace apparatus, the used column was a wide-bore DBWAX, 30 m, 0.32 mm 0.25 μm film thickness, J & W Scientific, Folsom, California, USA. The carrier gas was helium pure for gas-chromatography 5.0) method, already validated on other plant materials (Lo Scalzo et al., 2007). In this case, the used material was represented by homogenized frozen tomatoes, added with 10% NaCl w/w and conditioned at 80 °C for 20 min in gas-tight 20 ml vials. The dosage was made applying an external standard methodology. 2.3.7. Statistical analysis A quantitative analysis based on Chi-square contingency tests was used to evaluate the effect of four treatments (Control, Thyme, Propolis and Chitosan) on the number of collected fruits falling into three (RF, SF and WF) or two [RF, (SF + WF)] commercial categories over time (T40-T80-T120). The effectiveness of natural substances on fruit senescence delaying was investigated considering changes in the SF/WF ratio over different time points. This variable was analyzed using a Kruskal-Wallis nonparametric analysis of variance with significance tested at p ≤ 0.05, adopting two designs: (a) four comparison groups (1 = control, 2 = Thyme, 3 = Propolis, 4 = Chitosan) or (b) three comparison groups [1 = control, 2 = (Thyme + Propolis), 3 = Chitosan]. In both cases, the post-hoc Median Test was applied testing for significance at p ≤ 0.05 level. All chemical determinations were performed in triplicate for each replicate thesis. Data were subjected to the analysis of variance (ANOVA) and the comparison of the average values was carried out using the Tukey test. Statistical differences at P ≤ 0.05 were considered significant; standard deviation were also reported. Statistical analysis was performed using Statistica version 6.0 (Tulsa, Oklahoma, US).

2.3.4. Total carotenoids These lipophilic compounds were extracted under controlled conditions (in darkness at 0–1 °C to avoid sample decomposition) from 10 g frozen homogenized sample, using 100 ml n-hexane/acetone/ethanol (2:1:1 v/v/v) solution, added with 1 mg/ml butylated hidroxytoluene (BHT), as described by Shi et al. (1999). The mixture was separated by centrifugation at 5000 rpm for 10 min at low temperature. As the method followed made only one extraction, it was validated by laboratory trials with subsequent extractions, checking the colour of the pellet, which remained uncoloured after the first solvent treatment. Chromatograms of the second and subsequent extracts gave no 195

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Table 1 Chi-square contingency table analysis showing comparison between three treatments and untreated control for three and two fruit commercial categories under three time-points. Comparision

T

(1)

RF vs WF vs SF

(2)

RF vs (WF + SF)

χ2

p

d.f.

χ2

p

d.f.

Thyme vs control

40 80 120

20.25 35.04 25.53

0.0270 0.0001 0.0044

10 10 10

18.74 24.61 23.49

0.0021 0.00016 0.00027

5 5 5

Chitosan vs control

40 80 120

8.90 20.63 30.80

0.5354 0.02383 0.0006

10 10 10

6.89 10.05 18.17

0.2286 0.0738 0.0027

5 5 5

Propolis vs control

40 80 120

11.14 19.25 31.78

0.3465 0.0371 0.0004

10 10 10

9.92 12.01 16.10

0.0774 0.0347 0.0066

5 5 5

Legend: (1) T = days post-harvest; SF = swollen-healthy fruits.

(2)

RF = rotten fruits; WF = withered-healthy fruits;

percentages of SF were detected likewise for Thyme (39.6%), Propolis (40.9%) and Chitosan (44.9%) in respect to the control (34.7%) (Fig. 1C). Results of the statistical analysis highlighted a great influence at T120 (χ2 = 74.1; d.f. = 22; p < 0.0001) and at T80 (χ2 = 51.1; d.f. = 22; p = 0.0004). Grouping all commercial fruits (WF + SF) and comparing them against rotten fruits, a valuable effect of the treatments was observed over the three time points [T40 (χ2 = 21.5; d.f. = 11; p = 0.028), T80 (χ2 = 29.8; d.f. = 11; p = 0.002), T120 (χ2 = 43.6; d.f. = 11; p < 0.0001)]. In order to detect the most effective treatment capable to reduce rotting fruits, a Chi-square contingency test was finally run comparing each thesis against the untreated control, for each of the time points, using separately three (RF vs WF vs SF) and two variables [RF vs (SF + WF)]. By considering three homogeneous variables (RF vs WF vs SF) by treatments (Thyme, Propolis and Chitosan), the overall impact on the proportion of the RF, WF and SF fractions was significant against the control at T80 and T120 and significant at 40 days post-harvesting for Thyme only (Table 1). Pooling together WF and SF fractions against RF, all treatments impacted significantly the proportions of commercial categories at T120. Propolis was effective also at T80 and Thyme was effective at both 40 and 80 days post-harvesting. Fig. 2 shows the time-course of the SF/WF ratio by treatment. For all treatments, fruit senescence negatively impacted the level of the SF/WF ratio over time. However, high values of the SF/WF ratio were observed for Chitosan in respect to the control at each point in time (2.60 vs 1.99 at T40; 2.17 vs 1.36 at T80; 1.59 vs 0.99 at T120, respectively). The Kruskal-Wallis test run on four single treatments (untreated Fig. 1. Postharvest time-course of three fruit commercial categories (RF, WF and SF).RF = rotten, WF = withered-healthy and SF = swollen-healthy at three time-points (A = 40; B = 80; C = 120 days post-harvesting) as affected by preharvest treatments with Thyme, Propolis, Chitosan and water (control). Data correspond to the mean ± SE of three independent replicates.

3. Results 3.1. Postharvest decay The effect of three preharvest treatments on fruits number falling into three commercial categories (RF, WF and SF) is shown in Fig. 1(A–C). Despite a relevant difference in the incidence of both rotten and healthy fruits (Fig. 1A), a Chi-square contingency test showed no significant difference among treatments at T40 (χ2 = 24.75; d.f. = 22; p = 0.309). At T80 the tomato samples treated with Thyme, Propolis and Chitosan showed higher incidences of SF than the untreated control (53.0%, 55.1% and 52.6% vs 43.6%, respectively) (Fig. 1B), as well as a lower percentages of rotten fruits (16.3%, 18.8% and 17.2% vs 24.5%, respectively). At T120, high

Fig. 2. Postharvest time-course of SF/WF ratio as affected by preharvest treatments with Thyme, Propolis, Chitosan and water (control).WF = withered-healthy fruits; SF = swollen-healthy fruits. Data correspond to the mean ± SE of three independent replicates.

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Table 2 Postharvest changes in dry matter, soluble sugars and organic acids content of “Vesuviano” tomatoes treated with Thyme, Propolis, Chitosan and water (control) during preharvest. Treatment

Timepoint (1)

Dry matter (%)

SOLUBLE SUGARS (g/100 g d.w.) Glucose

Fructose

ORGANIC ACIDS (g/100 g d.w.)

Total amount

change % (2)

Fructose/ glucose

Malic

Acetic

Citric

Total amount

± ± ± ±

6.7 bA 6.4 aB 7.2 aA 6.4 bB

Control

T0 T40 T80 T120

10.8 ± 0.1 aA 9.4 ± 0.4 aA 8.9 ± 0.1 bA 9.8 ± 0.3 aA

17.8 18.7 14.2 12.6

± ± ± ±

1.7 bA 0.8 bA 1.8 bB 3.8 aC

19.7 ± 0.2 aA 18.5 ± 0.5 abA 21.6 ± 2.1 aA 21.2 ± 4.9 aA

37.5 bA 37.2 bA 35.8 bA 33.8 aB

−0.8 −4.5 −9.9

1.1 aB 1.0 aB 1.5 aA 1.7 bA

1.3 1.6 2.2 2.5

± ± ± ±

0.4 cD 0.2 aC 0.3 aB 0.2 bA

0.1 0.4 0.7 1.1

± ± ± ±

0.0 cD 0.1 bC 0.2 cB 0.2 bA

5.3 4.3 4.1 2.9

Thyme

T0 T40 T80 T120

10.6 ± 0.5 aA 9.7 ± 0.5 aA 10.2 ± 0.3 aA 8.7 ± 0.4 bB

21.6 22.1 17.1 13.1

± ± ± ±

0.7 aA 0.5 aA 1.9 aB 1.3 aC

22.4 ± 0.1 aA 20.4 ± 0.5 aA 21.7 ± 1.1 aA 21.4 ± 4.7aA

44 aA 42.5 aA 38.9 aB 34.6 aC

−3.4 −11.6 −21.4

1.0 aB 0.9 aB 1.3 bA 1.6 bA

1.9 1.3 2.1 2.4

± ± ± ±

0.4 aC 0.5 bD 0.5 aB 0.5 bA

0.1 0.5 0.9 1.1

± ± ± ±

0.0 bD 0.0 aC 0.1 bB 0.2 bA

5.9 ± 1.7 aA 4.7 ± 0.05 aB 3.2 ± 0.7 bC 3.2 ± 0.3 bC

7.9 6.5 6.2 6.7

Propolis

T0 T40 T80 T120

9.8 ± 0.3 aA 10.0 ± 1.2 aA 9.4 ± 0.8 aA 8.2 ± 0.2 bB

22.6 18.2 17.6 11.1

± ± ± ±

5.1 aA 0.8 bB 2.0 aB 1.7 aC

24.5 17.8 21.3 21.4

± ± ± ±

3.3 0.2 1.5 1.5

aA bC aB aB

47.1 36.0 38.9 32.5

aA bB aB bC

−23.6 −17.4 −31.0

1.1 1.0 1.2 1.9

1.2 ± 0.08 cD 1.7 ± 0.4 aC 2.3 ± 0.09 aB 2.9 ± 0.2 aA

0.1 0.4 0.9 1.2

± ± ± ±

0.0 bD 0.1 bC 0.2 bB 0.1 aA

6.4 4.7 4.0 3.7

± ± ± ±

0.6 aA 0.6 aB 0.2 aC 0.2 aD

7.8 aA 6.8 aB 7.3 aA 7.9aA

Chitosan

T0 T40 T80 T120

10.3 ± 0.0 aA 10.1 ± 0.6 aA 10.2 ± 0.3 aA 9.4 ± 0.0 aA

18.6 21.0 17.6 11.8

± ± ± ±

1.5 bB 0.2 aA 1.7 aB 1.6 aC

20.6 21.1 21.8 19.4

± ± ± ±

1.9 1.2 0.7 0.5

aA aA aA aB

39.3 42.1 39.4 31.1

bB aA aB bC

+7.1 +0.3 −20.9

1.1 aC 1.0 aC 1.2 bB 1.6 bA

1.5 1.7 2.3 2.3

0.2 0.4 1.0 1.1

± ± ± ±

0.0 aC 0.0 bB 0.1 aA 0.0 bA

5.9 4.0 3.3 2.9

± ± ± ±

0.0 0.4 0.4 0.8

7.6 6.2 6.7 6.3

aC aC bB aA

± ± ± ±

0.5 bB 0.1 aB 0.2 aA 0.2 bA

0.0 bA 0.3 aB 0.4 aB 0.5 cC

aA bB bC cD

aA aB bB bB

aA bB bB bB

For each treatment, different capital letters indicate significant differences between the storage time points (p ≤ 0.05). For each storage time point, different lower-case letters indicate differences between the treatments (p ≤ 0.05). Data correspond to the mean ± SD of three independent replicates. Legend: (1) T0 = harvest; T40-T80-T120 = 40-80–120 days postharvest; (2) percentage change respect to T0.

values for Thyme (44.0 g/100 g dw) and Propolis (47.1 g/100 g dw) at harvest (T0), instead no significant variation was observed in Chitosantreated sample compared with the Control. Considering the time-course of total sugar contents, pre-harvest treatments with Chitosan resulted in better retention than the Control and the other natural products both at T40 and T80 (+7.1 and +0.3%, respectively). The fructose/glucose ratio increased along the storage period, without strong differences among treatments. As concern organic acids, both malic and acetic acids reached the highest values at T120 for all theses. By contrast, citric acid constantly decreased for all treatments during the postharvest period (from 5.9 g/ 100 g dw to 3.2 g/100 g dw). As shown in Table 1, preharvest treatments with Thyme, Propolis and Chitosan positively affected total content of organic acids at harvest in respect to the control (7.9, 7.8, 7.6 and 6.7 g/100 g dw, respectively) due to the higher amount of citric acid (5.9, 6.4 and 5.9 g/100 for the three treatments against 5.3 g dw of the control). Moreover the malic acid content in Thyme-treated fruits was increased at T0 respect to the untreated fruits (1.9 and 1.3 g/100 g dw, respectively). A good retention of total organic acids were noticed up to T120, as a result of pre-harvest treatments with Propolis, in relation to a slower decrease in citric acid contents and stronger increases of malic acid in respect to the control (Table 2).

Fig. 3. Postharvest changes in total carotenoids concentration of “Vesuviano” tomatoes treated with Thyme, Propolis, Chitosan and water (control) during preharvest.T0 = harvest; T40-T80-T120 = 40-80-120 days postharvest. Data correspond to the mean ± SD of three independent replicates.

control, Thyme, Chitosan and Propolis) showed no significant effects on the SF/WF ratio for each time point (T40-T80-T120). By adopting a less stringent assumption that include three homogeneous groups (1 = control, 2 = Thyme + Propolis, 3 = Chitosan), the Kruskal-Wallis test was non-significant (T40, p = 0.11; T80, p = 0.09; T120, p = 0.15). By contrast, the median test applied on the same dataset was significant to the alternative hypothesis that at least one group median was different from the population median of at least one other group at both T80 (χ2 = 6; d.f. = 2; p = 0.049) and T120 (χ2 = 6; d.f. = 2; p = 0.049).

3.3. Total carotenoids The time-course of total carotenoids (from T0 to T120) showed significant increases up to T120 in Control, Thyme- and Propolistreated fruits (Fig. 3). For each treatment, different capital letters indicate significant differences between the storage time points (p ≤ 0.05). For each storage time point, different lower-case letters indicate differences between the treatments (p ≤ 0.05). Data correspond to the mean ± SD of three independent replicates. Legend: T0 = harvest; T40-T80-T120 = 40–80–120 days post-harvest A different pattern was found adopting preharvest treatments with Chitosan on “Vesuviano” tomatoes. This biopolymer induced a weak decrease between T0 and T40, with no significant variations being detected for T80 and T120 (175.5 and 179.9 mg/100 g dw). Considering the differences among the treatments and the control

3.2. Dry matter, soluble sugars and organic acids The dry matter content of tomato fruits was around 10% with negligible changes among the different treatments at T0 (Table 2). Anyway, good retains were found up to T120 in Chitosan-treated fruits (10.3% at T0 and 9.4% at T120) as well as happened in the Control (10.8% and 9.8% at T0 and T120, respectively). The total soluble sugars content, given by the sum of glucose and fructose, reached the highest 197

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Table 3 Postharvest changes in single polyphenols content of “Vesuviano” tomatoes treated with Thyme, Propolis, Chitosan and water (control) during preharvest. All values are expressed in mg/ 100 g dw. Treatment

Time-point

(1)

Chlorogenic acid

Chalconaringenin Rutin-O-hexoside

Rutin-O-pentoside

Rutin

Total Flavonols

Control

T0 T40 T80 T120

10.0 ± 1.5 aA 2.7 ± 0.4 aB 0.8 ± 0.2 bD 2.0 ± 0.5 aC

4.7 3.1 0.2 0.3

2.4 aA 1.1 bB 0.1 bD 0.0 aC

9.1 ± 3.2 aC 11.4 ± 3.1 aB 7.7 ± 1.6 cC 20.7 ± 5.9 aA

16.0 ± 2.5 aB 20.6 ± 5.2 aA 8.1 ± 1.8 cC 16.3 ± 5.3 aB

45.4 ± 4.8 aC 72.1 ± 17.9 aA 23.9 ± 6.7 cD 61.5 ± 15.9 aB

Thyme

T0 T40 T80 T120

9.3 1.8 0.9 1.5

± ± ± ±

2.5 0.1 0.2 0.4

1.4 ± 0.3 cB 1.8 ± 0.28 cA 0.5 ± 0.3 aC 0.2 ± 0.08 aD

7.0 ± 2.4 cC 5.1 ± 0.5 dD 9.0 ± 0.3 bB 16.2 ± 1.5 bA

10.3 ± 2.2 cC 13.8 ± 3.0 bA 12.1 ± 1.1 bB 7.9 ± 1.1 dD

27.4 37.0 43.7 40.7

± ± ± ±

3.9 cC 5.5 dB 7.2 bA 2.2 cA

44.7 56.0 64.8 64.8

Propolis

T0 T40 T80 T120

8.0 2.6 0.8 1.4

± ± ± ±

0.9 bA 0.4 aB 0.2 bD 0.4 bC

4.3 5.8 0.3 0.3

± ± ± ±

1.6 2.8 0.1 0.1

aB aA bC aC

8.5 ± 3.5 bD 9.4 ± 1.6 bC 10.5 ± 3.9 aB 16.2 ± 2.1 bA

13.0 ± 3.5 bB 16.0 ± 2.9 bA 13.4 ± 2.7 aB 8.8 ± 1.5 cC

38.9 57.8 44.0 40.4

± ± ± ±

8.2 bB 9.2 bA 8.8 bB 4.4 cB

60.4 ± 15.1 bB 83.2 ± 13.4 bA 67.9 ± 15.2 bB 65.5 ± 8.0 cB

Chitosan

T0 T40 T80 T120

7.5 ± 0.9 bA 1.9 ± 0.21 bB 1.1 ± 0.0 aD 1.7 ± 0.4 bC

2.0 1.4 0.6 0.3

± ± ± ±

0.7 bA 0.2 cB 0.3 aC 0.1 aC

7.7 ± 2.5 cC 7.0 ± 1.1 cC 10.9 ± 2.7 aB 19.5 ± 4.9 aA

15.4 15.1 14.1 11.6

43.6 ± 6.4 aB 51.4 ± 5.4 cA 51.0 ± 7.9 aA 51.7 ± 15.2 bA

66.7 ± 10.2 aB 73.6 ± 7.7 cA 76.0 ± 11.9 aA 82.8 ± 22.9 bA

aA bB bC bB

± ± ± ±

FLAVONOLS

± ± ± ±

2.8 1.8 2.2 2.8

aA bA aA bB

70.5 ± 9.2 aB 104.0 ± 26.2 aA 39.7 ± 10.0 cC 98.5 ± 26.9 aA ± ± ± ±

8.1 cC 8.9 dB 8.6 bA 4.2 dA

For each treatment, different capital letters indicate significant differences between the storage time points (p ≤ 0.05). For each storage time point, different lower-case letters indicate differences between the treatments (p ≤ 0.05). Data correspond to the mean ± SD of three independent replicates. Legend: (1) T0 = harvest; T40-T80-T120 = 40-80–120 days postharvest.

dw) and Propolis (779 vs 483 μg/100 g dw). As for the time course of 2(E)-hexenal contents, no significant differences were observed for the control, while higher values were detected at T0 than T120 by adopting three preharvest treatments with GRAS substances. Regarding 2isobuthylthiazole (responsible of the “wine-like” aroma of tomato fruits), the use of Thyme strongly increased the content of this volatile compound at T0 which was more than double compared to the untreated control (937 vs 449 μg/100 g dw). The effects of three preharvest treatments at T40 on the contents of 2-isobuthylthiazole were similar to what happened for hexanal; a better retention between T40 and T80 was observed for Thyme- and Propolis-treated fruits compared with the control. As for the terpenes contents, peaks of content were detected at T120 for Thyme (998 mg/100 g dw), at T80 for Propolis (1135 mg/100 g dw) and at T40 for Chitosan (847 mg/100 g dw). For the latter treatment a lower variability than other treatments were also observed over the time (Table 4). In the same Table, ethanol, that can be considered an index of anaerobic metabolism (Park et al., 1994), was also reported. At T0, a significant higher content of this alcohol was found in the control than the other treatments (29 mg/100 g dw vs 21, 19 and 15 mg/100 g dw of Thyme, Propolis and Chitosan, respectively). Moreover, except for the control, in all treated samples strong increases were detected moving from T0 to T40, with the highest variation in Chitosan-treated fruits (174 mg/100 g dw). Starting to 40 days post-harvesting, no clear behaviour was observed for the control, while relevant decreases in ethanol contents were observed in all tomatoes treated with GRAS substances. As a general rule, significant highest values of alcohol contents were always found in Chitosan-treated fruits, until to the end of the storage.

for each time-point, a significant higher level of total carotenoids at harvest (126,5 mg/100 g dw) was detected in Chitosan-treated fruits than other thesis and the control (80.9 mg/100 g dw for Thyme, 88.7 mg/100 g dw for Propolis and 91.6 mg/100 g dw for the control). However, for the following time-points, no notable differences were found between the control and Chitosan treatment. Finally, preharvest treatments with Propolis or Thyme resulted in lower carotenoids content than the control at T40-T80-T120 time-points. 3.4. Phenolic compounds As shown in Table 3, chlorogenic acid significantly decreased during the storage at room temperature and the most relevant decreases were found in all samples from T0 to T40. Furthermore, all preharvest treatments did not affect significantly the decreasing rate of chlorogenic acid up to T120. Regarding the chalconaringenin contents no significant differences were found at T0 between untreated control (4.3 mg/ 100 g dw) and Propolis treatment (4.7 mg/100 g dw). Moreover, despite an overall decrease over time in the concentration of this flavonoid, significant increases were observed from T0 and T40 for Thyme and Propolis treatments (from 1.4 to 1.8 mg/100 g dw and from 4.3 to and 5.8 mg/100 g dw, respectively). The level of total flavonols at T0, as sum of rutin and its two derivatives, resulted about the same, around 70 mg/100 g dw, with a significant lower content in Thymetreated fruits (44.7 mg/100 g dw). Starting to T40, rutin was well retained in Chitosan-treated fruits; a notable increase in rutin-O-hexoside content was also detected up to T120. Moreover, moving from T0 to T120 a better retention of rutin-O-pentoside was found for the same treatment than the control and the other treatments. According to these results, a clear increasing in total flavonols content was observed in Chitosan-treated tomatoes up to the end of storage (from 66.7 to 82.8 mg/100 g dw).

4. Discussion

3.5. Volatile substances

Alternative strategies aiming to control postharvest decay and to reduce dependency on synthetic fungicides are currently investigated in tomato and other perishables. Several in vitro studies have highlighted the inhibitory activities of Propolis on postharvest decay-causing fungi and bacteria of different fruits and vegetables (Basim et al., 2006; Tosi et al., 1996). The effectiveness of this natural substance has been also demonstrated in

Taking into account the evolution of characteristic volatile compounds during the postharvest period (Table 4), highest values of hexanal occurred at T80 in untreated control (1006 μg/100 g dw) or at T40 in the other treatments. Moreover, the content of this aldehyde was higher at T120 than T0 for the untreated control (895 vs 435 μg/100 g 198

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Table 4 Postharvest changes in characteristic volatile compounds, total terpenes and ethanol contents of “Vesuviano” tomatoes treated with Thyme, Propolis, Chitosan and water (control) during preharvest. All values are expressed in μg/100 g dw, with the exception of ethanol, that is expressed as mg/100 g dw. Treatment

Time-point

(1)

Hexanal ± ± ± ±

35 dC 99 bB 141 aA 38 aB

2-(E) hexenal

2-isobutylthiazole

Terpenes

Ethanol

371 ± 6 bA 325 ± 35 cA 356 ± 63 bA 324 ± 21 aA

449 ± 73 bD 761 ± 49 cC 891 ± 114 bB 982 ± 84 aA

713 ± 2 aB 781 ± 2 cB 629 ± 10 cC 877 ± 15 bA

29 ± 1 aA 16 ± 3 cC 24 ± 11 bB 3 ± 1 cD

Control

T0 T40 T80 T120

435 843 1006 895

Thyme

T0 T40 T80 T120

932 ± 128 aB 1281 ± 49 aA 864 ± 56 bB 864 ± 21 aB

309 437 290 256

± ± ± ±

18 cB 38 aA 23 cB 13 bC

937 ± 164 aB 1122 ± 116 aA 879 ± 62 bB 874 ± 38 bB

634 ± 31 bB 928 ± 5 bA 587 ± 8 cB 998 ± 28 aA

21 ± 5 bB 98 ± 6 bA 23 ± 7 bB 5 ± 3 bC

Propolis

T0 T40 T80 T120

482 ± 14 cD 1227 ± 172 aA 983 ± 98 aB 779 ± 132 bC

458 385 399 242

± ± ± ±

16 aA 48 bB 33 aB 20 bB

345 ± 17 cD 953 ± 101 bB 1106 ± 141 aA 655 ± 63 dC

594 ± 33 bC 1108 ± 17 aA 1135 ± 36 aA 839 ± 54 bB

19 99 26 2

Chitosan

T0 T40 T80 T120

597 ± 26 bB 1194 ± 45 aA 617 ± 69 cB 592 ± 40 cB

426 417 192 175

± ± ± ±

22 aA 51 aA 32 dB 11 cB

732 ± 63 aA 847 ± 104 bA 785 ± 50 bA 657 ± 5 cB

15 ± 3 dC 174 ± 52 aA 39 ± 10 aB 6 ± 1 aD

371 981 754 750

± ± ± ±

17 cC 41 bA 40 cB 74 cB

± ± ± ±

14 cC 49 bA 19 bB 1 dD

For each treatment, different capital letters indicate significant differences between the storage time points (p ≤ 0.05). For each storage time point, different lower-case letters indicate differences between the treatments (p ≤ 0.05). Data correspond to the mean ± SD of three independent replicates. Legend: (1) T0 = harvest; T40-T80-T120 = 40-80–120 days postharvest.

in the leakage of intracellular electrolytes and proteinaceous constituents (Palma-Guerrero et al., 2008). Bhaskara Reddy et al. (2000) demonstrated that stem scar application of chitosan inhibits development of blackmold rot [Alternaria alternata (Fr.) Keissl.] of tomatoes and reduces production of pathogenic factors by the fungus, such as cell wall-degrading enzymes (polygalacturonase, pectate lyase and cellulase), organic acids, and host specific toxins responsible for fungal penetration and host tissue damage. Besides antifungal activity, Chitosan has the potential for inducing defense-related enzymes (Bautista-Baños et al., 2006) and phenolics in plants (Romanazzi et al., 2017). Recently, Zhang et al. (2014) suggest that one of the molecular mechanisms involved in the enhancing of resistance to gray mold (Botrytis cinerea Pers.) in cherry tomato fruit was likely associated with activation of the mitogen-activated protein kinase (MAPK) signaling pathway by Chitosan. The high values of the SF/WF ratio in Chitosan-treated fruits may indicate a delaying of fruit senescence over time. Several studies reported that Chitosan coating provides a semi-permeable film around the fruit surface, which modifies the internal atmosphere by reducing oxygen and/or elevating carbon dioxide levels, which decreases the fruit respiration level and metabolic activity, and hence delays the fruit ripening and senescence processes (El-Ghaouth et al., 1992; TezottoUliana et al., 2014). A slower fruit softening, due to low polygalacturonase activity induced by Chitosan, can explain a greater resistance to fungal infection, as reported by different authors (Stevens et al., 2004; Ruoyi et al., 2005). The quality of the tomato fruits is also an important index evaluating the storage effect by different treatments adopted. Quality composition of the “Vesuviano” fruits at harvest was in agreement with the results reported by other authors for the same landrace (Ercolano et al., 2008; Carli et al., 2011; Ruggieri et al., 2014). Moreover, the time-course of total soluble sugars, organic acids and main volatile compounds observed in stored fruits, resembled the behaviour of “Penjar” tomato, a long-storage landrace spreads in Spain and showing similar fruit morphology to “Vesuviano” tomato (Casals et al., 2011; Missio et al., 2015). The long shelf life of the “Penjar” varietal type is controlled by the ripening mutant “alcobaça” (alc), that is an allele of the non-ripening gene (nor) (Casals et al., 2012). As far as the effect of natural fungicides, the decreasing trend in total soluble sugars detected in Propolis-treated tomatoes suggested the occurrence of fermentation and senescence processes and this behaviour could be ascribed to utilization of these carbohydrate as

vivo studies performed on some perishables (such as grapefruit, pear, apple and strawberry), however few studies have investigated the use of Propolis in controlling postharvest diseases in tomato crop (Bakeer et al., 2016; Yang et al., 2016; Ordónez et al., 2011). Our results highlight a positive effect on reducing rotten fruits starting to T80; anyhow preliminary results by Carrieri et al. (2016) reported no efficacy on fungal pathogen at 200 days post-harvesting, hypothesizing a moderate effect over time. Bankova et al. (2002) suggested that composition and biological activities of propolis depend on many factors such as the geographical origin as well as the collection time and plant source. Regarding the effect of Thyme reducing the incidence of fruit rots, this antimicrobial substance showed a relevant efficacy through the entire storage period of “Vesuviano” tomato fruits. Moreover, a satisfactory control on postharvest fungal decay [caused by Alternaria alternata (Fr.) Keissl., Pennicillium spp, Aspergillus spp and Fusarium spp] was also found up to 200 days post-harvesting, by Carrieri et al. (2016). Unlike to in vitro studies, few studies have been performed in vivo conditions to evaluate the fungicidal properties of Thyme in controlling postharvest spoilage of tomatoes (Soylu et al., 2010). Furthermore, the investigations did not assess the antimicrobial effects on very extended periods of storage (a few months). Vitoratos et al. (2013) reported the inhibitory effects of Thyme on spore germination of Penicillium italicum (Wehmer) and Penicillium digitatum [Pers. (Sacc.)] on tomatoes stored at 22 °C for 6 days. Other postharvest fungal pathogens [Aspergillus flavus (Link) and Aspergillus niger (Tiegh.) Sacc.] were satisfactorily controlled by this essential oil, as reported by Ibrahim and Al-Ebady (2014). Most chemical components of essential oils are terpenoids, including monoterpenes, sesquiterpenes, and their oxygenated derivatives. Among them, the most active antimicrobial compounds are terpenes. Thymol and other phenolic compounds (eugenol and carvacrol) may inactivate essential enzymes, reacting with the cell membrane or disturbing genetic material functionality (Davidson, 2001). Finally, Zambonelli et al. (2004) correlated the fungicidal activity of different commercial thyme essential oils with their thymol contents. Regarding the Chitosan, its use resulted in significant reduction in rotten fruit percentages (especially from T80) of “Vesuviano” tomatoes and these findings appear to agree with extended antifungal activity over the time, observed by Carrieri et al. (2016). The mechanism by which chitosan affects the growth of pathogens may be related to the ability of this GRAS substance to interfere with the negatively charged residues of macromolecules exposed on the fungal cell surface, resulting 199

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position are available in literature. Anyway, Baldwin et al. (1995) found that the use of edible coatings on citrus fruit resulted in an increase in desirable flavour compounds after storage, as compared with uncoated fruits. This finding seems in accordance with data here discussed, especially if considering the terpenes amounts which were well retained over the time in Thyme- and Propolis-treated fruits. Since the terpenes are positively related to good organoleptic features, our results highlighted the maintaining of a better quality in “Vesuviano” tomato fruits during the storage by preharvest treatments with Propolis and Thyme essential oil.

substrates for fruit respiration. Conversely, reduced rates of respiration, due to coating film around the fruit surface, could explain the good retention of soluble sugars up to T80 in “Vesuviano” tomatoes by Chitosan treatments. Our results are in agreement with those of Zhang et al. (2014) that reported a lower decreasing of soluble sugars in cherry tomatoes coated with chitosan + zinc (5% + 2%) solution and stored for 24 days at 20 °C with 85% R.H. The higher total sugar contents up to T80 in Thyme-treated fruits than the control were in accordance with Tzortzakis et al. (2011), that described the behaviour of cherry tomato fruits treated with eucalyptus (Eucalyptus globulus L.) and cinnamon (Cinnamomum zeylanicum Blume) essential oils and stored at 13 °C for 10 days. Interestingly, a high amount of total organic acid content was detected in Propolis-treated tomatoes at T0 and a good retention was observed until to 120 days post-harvesting. These results were in agreement with those reported by Zahid et al. (2013) on dragon fruits treated with ethanolic extract of propolis at 0.5% and stored for 20 days at 20 ± 2 °C and 80 ± 5% RH. After this treatment the authors observed higher levels of titratable acidity in the fruits than the untreated control. Increased contents of total of organic acids were also detected at harvest in “Vesuviano” tomatoes treated with Thyme: anyhow the inducing effect seemed no so extended in time. Similar results were reported by Perdones et al. (2016), studying the effects of oregano essential oil incorporated into film-forming dispersions based on biopolymers (chitosan and/or methylcellulose) on tomato fruits. The positive effect of Chitosan treatments on carotenoids retention until to the end of storage is in agreement to the results reported by Zhang et al. (2014) on tomato. By treating dragon fruits with 1.0% conventional Chitosan or submicron Chitosan dispersions at 600 nm and 1000 nm, Ali et al. (2013) observed a good retention of total phenols, total flavonoids and lycopene during storage at 10 ± 2 °C and 80 ± 5% RH for 28 days. In our study a steady increase in flavonols contents in “Vesuviano” tomatoes was detected until to the end of storage as resulting of preharvest treatments with Chitosan. Badawy and Rabea (2009), reported decreased activity of polyphenol oxidase (PPO) and a consequent enhancing of phenolic compounds by Chitosan in wounded tomatoes fruit stored at different temperatures until to 21 days. The authors, hypothesized that the effects of this biopolymer on gray mold (Botrytis cinerea Pers.) may be associated with direct fungitoxic properties against the pathogen and the elicitation of biochemical defense responses in fruit. Furthermore, the same authors speculated on the fact that the inhibitory effects of Chitosan on PPO activity were probably a consequence of the adsorption of suspended PPO, its substrates, or its products by the positive charges of Chitosan. Finally, earlier studies investigated the effect of Chitosan applications on the retention of phenolic compounds during the postharvest in different fruits and vegetables (Romanazzi et al., 2017), but no investigation has evaluated the effects of this biopolymer in a longstorage period up to now. As for the postharvest evolution of volatile compounds, the increasing in ethanol contents at T40 for the all treatments, in respect to the control, could be due to the anaerobic metabolism induced by coatings with different preharvest treatments, as previously found by Vanoli et al. (2015) on melon fruit. Similarly, Ali et al. (2011), reported inside fermentation in papaya fruits treated with Chitosan and this phenomena seemed due to block of lenticels due to this GRAS substance. Anyhow, in our research the detected levels of ethanol resulted within the flavour detection threshold (around 80 mg/100 g dw) (Fazzalari, 1978), with the exception of some T40 samples, slightly exceeding this value. Interestingly, a better retention of hexanal, 2-(E)-hexenal and 2isobutylthiazole contents were observed in Propolis-treated fruits than the control, contributing to maintain a good quality during the extended storage of “Vesuviano” tomato. No data about the effect of preharvest treatments with GRAS substances on tomato flavour com-

5. Conclusion Results of the present study indicated that preharvest applications of Thyme, Chitosan or Propolis reduced decay incidence during the longstorage period of “Vesuviano” tomatoes under room temperature. Among the three natural fungicides, Chitosan was the most efficacy to reduce fruit senescence processes and maintain a good overall quality of the fruits over the time. However, further studies are required to investigate different aspects of chitosan application (regarding its solvent, concentrations, molecular weight and deacetylation degree) as well as its antimicrobial activity on the major fungal pathogen causing fruit rotting during the extended storage of “Vesuviano” and other similar tomatoes. Acknowledgements The authors wish to thank “Azienda Agricola Casa Barone” for hosting the experimental field and Dr. Francesco Di Dato, Dr. Riccardo Riccardi and Mr. Raffaele Perreca for assistance in merceological assessment of the fruits. This research was partially supported by the Campania Region (Italy) − Department of Agriculture (grants “Programma delle attività di collaudo e sperimentazione del Centro Orticolo Campano- IV annualità”). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.04.030. References Ali, A., Muhammad, M.T.M., Sijam, K., Siddiqui, Y., 2011. Effect of chitosan coatings on the physicochemical characteristics of Eksotika II papaya (Carica papaya L.) fruit during cold storage. Food Chem. 124, 620–626. http://dx.doi.org/10.1016/j. foodchem.2010.06.085. Ali, A., Zahid, N., Manickam, S., Siddiqui, Y., Alderson, P.G., Maqbool, M., 2013. Effectiveness of submicron chitosan dispersions in controlling anthracnose and maintaining quality of dragon fruit. Postharvest Biol. Tech. 86, 147–153. http://dx. doi.org/10.1016/j.postharvbio.2013.06.027. Aminifard, M.H., Mohammadi, S., 2012. Effect of essential oils on postharvest decay and quality factors of tomato in vitro and in vivo conditions. Arch. Phytopath. Plant Prot. 4511, 1280–1285. http://dx.doi.org/10.1080/03235408.2012.673261. Antunes, M.D.C., Cavaco, A.M., 2010. The use of essential oils for postharvest decay control. A review. Flavour Fragr. J. 25, 351–366. http://dx.doi.org/10.1002/ffj. 1986. Arah, I.K., Amaglo, H., Kumah, E.K., Ofori, H., 2015. Preharvest and postharvest factors affecting the quality and shelf life of harvested tomatoes: a mini review. Intern. J. Agron.. http://dx.doi.org/10.1155/2015/478041. (Article ID 478041, 6 pp). Badawy, M.E.I., Rabea, E.I., 2009. Potential of the biopolymer chitosan with different molecular weights to control postharvest gray mould of tomato fruit. Postharv. Biol. Technol. 51, 110–117. http://dx.doi.org/10.1016/j.postharvbio.2008.05.018. Bakeer, A.T., Elbanna, K., Elnaggar, S.A., 2016. Impact of pre-and post–harvest applications of natural antimicrobial products on apple and pear soft rot disease. Intern. J. Phytopathol. 4, 105–119 ISSN: 2305-106X (Online), 2306-1650 (Print). Baldwin, E.A., Nisperos-Carriedo, M., Shaw, P.E., Burns, J.K., 1995. Effect of coatings and prolonged storage conditions on fresh orange flavor volatiles, degrees brix, and ascorbic acid levels. J. Agric. Food Chem. 43, 1321–1331 (0021-8561/95/14431321$09.00/0). Baldwin, E.A., Scott, J.W., Shewmaker, C.K., Schuch, W., 2000. Flavour trivia and tomato aroma: biochemistry and possible mechanisms for control of important aroma components. HortSci 35, 1013–1022. Bankova, V., Popova, M., Bogdanov, S., Sabatini, A.G., 2002. Chemical composition of

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