**3. Discussion**

Tomato is an important food crop, characterized by high consumption numbers worldwide, and numerous uses and health benefits [30]. As a fresh produce commodity, tomato has a relatively short shelf life and its quality is affected by many pre and postharvest factors. During postharvest handling many parameters can influence tomato's quality attributes resulting in rapid deterioration. In this study, dipping tomato fruits in 0.4% EP increased product's weight loss after the 12th day of storage; however, the weight loss was less than 1.2%, which is not considered of great issue in postharvest storability of fresh produce. Similarly, Tzortzakis et al. [23] reported increased weight loss with dipping tomatoes in 0.5% sage EO compared to 0.1% EO and control fruits after seven and 14 days of storage at 11 ◦C. In another study, the vapor application of oregano EO did not result in any significant differences in tomato's weight loss [31]. These effects can be attributed to the similar EO composition of the main components of rosemary (isoborneol, α-pinene, α terpineol, 1.8-cineole), eucalyptus (1.8-cineole, α-pinene and δ-3 carene) and sage (α-thujone, camphor, 1.8-cineole, camphene and α-pinene) in comparison to oregano EO (carvacrol, *p*-cymene, γ-terpinene) [4,23,31,32].

Tomato, as a typical climacteric fruit, is characterized by increased respiration rate followed by increased ethylene production while ripening [33]. In that sense, vapor application increased respiration rates mainly at high EP levels and chlorine after seven days and at chlorine application at 14 days. Such changes in respiration were not evidenced in dipping application, indicating a non-lasting impact of the dipping, compared to the vapor method. We speculate that any boost in respiration rates of dipped tomatoes could possibly happen before the seven days, as indicated by the increased ethylene levels at days 7 and 14. However, considering the effects of EP product, vapor application of EP (0.4%, 0.8%) increased ethylene emission on the seventh day, while respiration increase was observed only at the 0.8% EP vapor application, on the same day. Both vapor and dipping application methods retarded ethylene emission on the last day of storage compared to day zero (fruits were at room temperature), and this is evidenced by the low temperatures used in the present study in comparison to room temperature, by retarding the ripening process and slowing down metabolic changes of the fruit. In a previous study, it has been shown that respiration rate and ethylene emission of tomato fruit were increased after dipping the fruits in 0.5% sage EO, compared to control and 0.1% EO, after the seventh day and up to the last day (14th) of storage at 11 ◦C [23]. The differences in respiration rate and ethylene emission of these products can be attributed to disturbance of gas exchange and cell wall degradation that can be caused by the duration of the EO application and/or even the method of application [34,35]. The increased respiration and/or ethylene production are processes related to increased fruit metabolism and ripening.

Tomato fruit firmness decreases through storage and maturation as fruit gets softer, while it has been previously suggested that EO treated fruits maintained higher firmness values compared to non-treated fruits [35]. Oregano EO when applied to tomato fruits was found not to affect fruit's firmness in comparison with control fruits while at the same time it decreased fruit decay after 14 days of storage [31], highlighting a prolonged postharvest storage period. Vapor application of EP (0.4%) resulted in firmness maintenance compared to vapor chlorine on the seventh day of storage. Moreover, in the present study, no symptoms of decay were observed on the examined tomatoes in all treatments and methods of applications (vapor vs. dipping). Similarly, in another study, eucalyptus EO vaportreated tomatoes maintained their firmness [36]. No significant differences regarding firmness were reported with the application (via dipping) of sage EO (0.5% and 0.1%) on tomato fruits even up to 14 days of storage at 11 ◦C [23]. These results are in accordance with the observations of dipping application of the EP (0.4% and 0.8%) in the present study. Indeed, EO effectiveness from the previous mentioned EOs on fruit firmness is attributed to the common main components of the tested EOs, such as 1,8-cineole and α-pinene; however, that statement needs further substantiation by testing individual chemical components and/or mixtures of them in ratios similar to the examined EOs. The Eos' effectiveness is not related only to the main component of the oil, but to the synergistic action of the major components, usually numbering 3–5 components in each EO. Nevertheless, EOs effectiveness can vary, and that can be attributed to the different species, even varieties, to the cultivation practices, and components' composition.

Acidity (organic acids) is one of the main taste characteristics of tomatoes that influence fruit quality and decreases throughout storage, as fruit ripens [37,38]. Tomato's TA was decreased on the seventh day by the vapor application of 0.4% EP. The use of sage EO decreased tomato's TA, while increasing its sweetness (ripening index) after 14 days of storage at 11 ◦C [23]. Similarly, Adams et al. [35] mentioned a decrease in TA in EO-treated (ginger EO) tomatoes compared to control. Aminifarda and Mohammadi [39] reported that tomato fruits treated (dipping method) with ammi and anise EOs (concentration range: 200–800 μL L<sup>−</sup>1) presented higher TSS compared to the control ones. During the ripening of tomato fruits, sugars accumulate above the required levels for respiration purposes and this is reflected in an increased TSS value [37]. In the present study, both application methods (dipping and vapor) did not affect tomato's TSS throughout storage. However, it

has been previously mentioned that EO-treated tomatoes seem to have a slightly decreased TSS due to respiratory metabolism [35].

Color is one of the most important quality attributes of tomato fruit. The development of pigments changes during tomato fruit maturation, with the production of carotenoids and the breakdown of chlorophylls. In the present study, both application methods (dipping and vapor) did not affect tomato's AA, *β*-carotene and lycopene content throughout storage, which is important for maintaining the fruit's high nutritive value. These results are of agreement with another study in which no significant differences were reported on the carotenoid content of peaches and nectarines with the application of thyme and savory EO (1% and 10% in vapor phase) [26]. Carotenoid content decreases during storage and exposure of fruits to light, however the use of EOs has been proven to prevent oxidation processes (scavenging free radicals) and even preserve carotenoid levels due to their antioxidant activities [8]. On the other hand, the application of 0.1% sage EO resulted in higher AA levels of tomato fruits compared to control and 0.5% EO treated fruits [23]. Interestingly, AA and carotenoid content (*β*-carotene and lycopene) were found to be increased with the dipping of tomato fruits in ammi and anise EOs (concentrations range: 200–800 μL L<sup>−</sup>1) compared to control fruits, initiating the antioxidative metabolism of the fruit [39]. Moreover, application of oregano EO (0.40 mL L<sup>−</sup>1) resulted in tomato's increased lycopene and AA content (compared to control fruits) after 14 days of storage [31]; however, oregano EO levels were 10-fold lower than the ones used in the present study (i.e., 0.4%). Santoro et al. [26] reported that the reduced loss of the nutritional value macromolecules such as AA and carotenoids might be attributed to the EOs and their components (i.e., 1,8-cineole, α-pinene and camphor) that possess antioxidant activities, protecting sensitive nutrients from oxidation.

The results of this study indicate that both application methods (dipping and vapor) did not cause significant changes in total phenolic content. Considering that AA, carotenoids (*β*-carotene and lycopene), and total phenolics remained unchanged in general, this indicates that non-enzymatic antioxidants compounds were not influenced by the examined treatments. Therefore, any possible stress, as indicated by MDA and H2O2 increases, could alter the enzymatic antioxidant capacity of the fruit (for example, the increased CAT and POD values). Tzortzakis et al. [23] also reported no significant differences in phenolic content of tomato fruits treated with sage EO (0.1% and 0.5% via dipping). Additionally, in the same study no significant differences in antioxidant levels (DPPH, FRAP) were observed on the seventh day of storage, while increased antioxidants were reported on the last day of storage when tomato fruits were dipped in 0.1% sage EO (compared to 0.5% EO and control) [23], being in agreement with our results of 0.4% EP (vapor method) for DPPH activity at seven days of storage. The application of EOs on fresh produce can enhance the content as well as the production of antioxidant compounds when applied to fresh produce due to their own antioxidant capacity [23]. However, the increase in antioxidants during fresh produce storage is also attributed to other extrinsic postharvest factors such as chilling or adverse storage temperatures and low RH, among others.

Considering damage indices, dipping in EP product decreased tomato's H2O2 levels on the seventh day of storage, whereas dipping in 0.8% EP decreased MDA levels on the same day. The decreased MDA levels observed in EO-treated fruits can be attributed to the induced activation of defense-related enzymes, towards the oxidative stress challenge [40]. When citrus fruits were treated with 0.5% clove EO, increased H2O2 levels were revealed up to four days of storage at 25 ◦C [40]. At the same study, clove EO-treated fruits presented lower MDA levels compared to non-treated fruits from 12 h after the application and up to four days [40]. Another study mentioned that kiwifruits treated with 0.6 μL mL−<sup>1</sup> citral presented decreased H2O2 and MDA levels when stored at 1 ◦C for up to 90 days [41]. On the other hand, our findings indicate that vapor application of the EP increased MDA levels (increased lipid peroxidation) during the seventh day of storage. Shao et al. [42] reported that vapor application of tea tree EO (0.9 g L<sup>−</sup>1) increased H2O2 levels of strawberry fruits stored at 20 ◦C for three days. Increased oxidative stress—production of reactive oxygen

species (ROS) such as H2O2—can be reported throughout storage of fresh produce and this might be attributed to the ripening process, the time and conditions of storage as well as the applied treatments (including the application of EOs). EOs when applied in fruits seem to directly and indirectly initiate the activation of fruit defense mechanisms, including the activity of antioxidant enzymes [42].

It has been previously mentioned that the application of clove EO (0.4%) on citrus fruits enhanced the activity of plant defense-related enzymes including POD, phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and lipoxygenase (LOX) [40]. In the present study, the application of EP (based on rosemary and eucalyptus essential oil components) revealed an increase in the CAT and POD activity of tomato's fruits during the seventh day of storage, highlighting the induction of enzymatic antioxidant mechanisms of the fruit. Similarly, tea tree EO vapor treated (0.9 g L<sup>−</sup>1) strawberries showed higher SOD, PAL and POD activities during a three-day storage at 20 ◦C [42]. In our study, a decrease in POD activity was reported on the seventh day with dipping application of 0.4% EP, followed by an increase in POD activity on the 14th day. These results differ from a previous study where increased POD activity was recorded throughout storage of citral-treated (0.6 μL mL<sup>−</sup>1) kiwifruits [41], and such EO variation in its effectiveness is related to the EO composition, application practices and/or tested commodities. However, both tomatoes and kiwifruits are climacteric fruits and a speed-up of the ripening metabolism is taking place during storage, as indicated by the increased respiration and ethylene emission rates, mainly after seven days of storage. EO-treated fruits tend to present increased activity of antioxidant enzymes in order to suppress the accumulation of ROS during ripening and senescence of fruits during storage conditions and processing. Moreover, the activity of POD has also been correlated with fresh produce quality attributes (including color) [8]. This might also partially explain the lower appearance scoring values of tomato fruits vapor treated with the EP.

Sensory evaluation (aroma, optical/visual appearance) of fresh produce is one of the main factors affecting consumers' buying decisions. In the present study, dipping tomato fruits in 0.4% EP decreased marketability on the seventh day compared to control fruits. In contrast, the application of ginger EO (application time 20 and 30 min) increased acceptance of tomato fruit compared to control fruits [35]. Furthermore, the application of 0.8% EP (vapor method) in our study resulted in a less tomato-like aroma on the seventh day, while all applied vapor treatments also decreased aroma (less tomato-like aroma) on the last day of storage. When fruits were treated with the vapor method (all treatments), the appearance of the fruits decreased on the last day of storage indicating a less red product (less acceptable by consumers). Tzortzakis et al. [23] mentioned that tomato fruits treated with 0.1% sage EO presented greater appearance, aroma, texture and marketability compared to control fruits. On the other hand, the same study reports that increased sage EO concentration (0.5%) resulted in a less acceptable product [23]. The time of application can also affect the quality of the end product. In a previous study, the dipping application of ginger EO for 20 min resulted in a less acceptable ("sour") product compared to 30 min application which resulted in a more acceptable product [35]. Further investigation of EO and EP applied to postharvest fresh produce is needed, as it is essential that fresh produce and applied EOs are being combined in a harmonious manner enhancing fresh produce quality attributes.

#### **4. Materials and Methods**

#### *4.1. Plant Material and Experimental Set Up*

The present study took place at the laboratory facilities of Food Science and Technology of the Cyprus University of Technology, Limassol, Cyprus. Tomato (*Solanum lycopersicum* cv. F179) fresh produce were purchased from a commercial greenhouse. Crops were grown under common cultivation practices in a clay sandy-loam soil and drip irrigation and fertigation were applied according to crop needs. The cultivation took place during winter–spring months and the temperature ranged between 19 ◦C and 31 ◦C.

An eco-product (EP; named as ''Agriculture Green-tech E", Meydan Solution Ltd., Larnaca, Cyprus) based on rosemary (*Rosmarinus officinalis* L.) and eucalyptus (*Eucalyptus crabra* L.) essential oil was used. Individual EOs were analyzed by gas chromatography-mass spectrometry (GC/MS-Shimadzu GC2010 gas chromatograph interfaced Shimadzu GC/MS QP2010 plus mass spectrometer) and constituents were determined [43] and presented in Table S1. *Rosemarinus officinalis* essential oils used in the tested formula were found rich in isoborneol (30.29%), α pinene (25.71%), α terpineol (14.89%) and 1.8-cineole (10.81%), while the dominant compounds of the essential oils from *Eucalyptus crabra* were 1.8-cineole (26.51%), α pinene (24.12%) and δ-3 carene (20.10%), being in accordance with previous studies [44,45]. This product was a mixture of these two essential oils (eucalyptus: rosemary in approximately 2:1 *v*/*v* ratio) and it also contained vinegar < 5% *w*/*w* as well as emulsifier treated water (<80%). Chlorine was used as a commercial sanitizer at 0.02% (*v*/*v*).

Fresh produce was selected based on uniform size, appearance, and absence of physical defects and used immediately in the different experiments. The fruits were disinfected with a chlorine (0.05% *v*/*v*) solution for 4 min and washed four times with distilled water before use (to avoid any microbial load). A preliminary screening and main experiments were implemented.
