Origanum dubium Boiss. (Cypriot oregano) Use for the Preservation of Fresh Spearmint Quality and Safety

Abstract

Very little is known about the preservation and storage of fresh medicinal and aromatic plants (MAPs) and/or herbs. As with many leafy vegetables, i.e., lettuce, fresh spearmint is a very perishable product throughout the production line and requires special handling/processing. The current study aimed to examine the antibacterial activity of selected MAPs (Origanum dubium, Salvia fruticosa and Sideritis cypria) grown in Cyprus towards foodborne pathogens. Then, the most effective MAPs’ essential oil (EO) and hydrosol (at different doses; combination of concentration and time of dipping submerge) were tested to preserve fresh spearmint quality and safety. The results showed that O. dubium EO and hydrosol presented great in vitro antibacterial activity against four foodborne pathogens and those products were further selected for application on fresh spearmint. During sensory evaluation, it was observed that higher O. dubium EO concentrations and longer time of application resulted into a less marketable product (less acceptable with less spearmint-like aroma), while hydrosol resulted in a more marketable product even at high doses. In general, EO and hydrosol applications increased spearmint’s antioxidants (including polyphenols, flavonoids, and ascorbic acid), resulting in a product of increased nutritional value. The bacterial populations of Salmonella enterica and Listeria monocytogenes inoculated on fresh spearmint were decreased with the EO and hydrosol application doses applied, and their effects were evident even after six days of storage at 4 °C. From the findings of this study, it can be concluded that O. dubium EO and hydrosol could be a potential sanitation method for fresh spearmint preservation.

1. Introduction

There is increased interest by the food industry and the scientific community on the utilization of natural components such as essential oils (EOs), plant extracts and natural compounds to preserve fresh produce [1,2]. Essential oils are secondary metabolites derived from various medicinal and aromatic plants (MAPs) and present a plethora of bioactive properties including antimicrobial (antibacterial, antifungal, virucidal), antioxidant, analgesic and anti-inflammatory activity, among others [1]. Many EOs and natural compounds are generally characterized as safe (GRAS) for use in food according to the European Commission Regulation on flavorings and certain food ingredients with flavoring properties (EC No.1334/2008) [3,4].

Previous reports indicated that the use of EOs and other natural extracts from MAPs such as sage (Salvia trilova), and oregano (Origanum vulgare, Origanum dubium) resulted in the preservation of fresh produce, minimizing quality losses and ensuring safe-for-consumption products [5,6,7,8]. The search for natural products that can be applied on fresh commodities has also explored the properties and use of hydrosol from MAPs on fresh produce. Hydrosols (also called aromatic water, floral water and distillate water) are one of the by-products of the EO hydrodistilation procedure presenting similar activities as their EOs [3]. These products have been proven to possess antioxidant and antimicrobial activities, which are attributed to their primary and secondary components (mainly water-soluble compounds) [3,9]. The composition of hydrosols might be similar or completely different from the EO of the respective plant since they can contain traces of the EO [9]. Moreover, the investigation of endemic, unexploited MAPs and their properties is rapidly gaining more interest worldwide, including in Cyprus. Origanum dubium, Salvia fruticosa and Sideritis cypria are examples of MAP species cultivated in Cyprus that present a variety of beneficial properties (i.e., antibacterial, antifungal, antioxidant) [10,11,12]. However, limited data are available for their use on fresh produce preservation (especially leafy greens and fresh MAPs).

Spearmint (Mentha spicata) is a common culinary MAP used in food and drinks, cosmetics, perfume, and medicines [13]. It can be used on its own fresh or dry, and its products and preparations such as EOs, extracts, infusions and decoctions can be used as well, due to its antimicrobial (antibacterial, antifungal), antioxidant, and insecticidal activity, among others [13,14,15]. As with many other MAPs, spearmint can be sold as fresh-cut herbs in bundles, trays and/or bagged in modified atmosphere packaging [15]. Fresh MAPs (including spearmint) are highly perishable products with a high nutritional value that deteriorates rapidly during storage (within five days up to a couple of weeks of storage). Little to no information is available regarding the postharvest preservation and safety of these products during storage. In the fresh product sector, an estimated 30–40% of fruits and vegetables are lost between harvest and consumption due to various factors, including inappropriate infrastructure, transportation, limited knowledge of postharvest handling, market inefficiencies, and technological gaps [16].

The aims of the present study were (i) to assess the in vitro antibacterial activity of EOs and hydrosols of three MAP species grown in Cyprus against four main foodborne pathogens, (ii) select the most effective MAP species and examine the effects of the selected MAP species’ EO and hydrosol on fresh spearmint’s quality attributes, as well as (iii) assess the efficacy of selected MAPs’ EO and hydrosol against Salmonella enterica and Listeria monocytogenes on fresh spearmint stored at 4 °C.

2. Materials and Methods

2.1. Plant Material and Bacterial Cultures’ Preparation

Fresh spearmint (Mentha spicata) was obtained from a local producer (Limassol, Cyprus, 34°39′ N, 32°57′ E, 25 m). Plants were grown under an organic cultivation system for five months during spring, without any synthetic pesticides and fertilizers/chemicals applied. The organic fertilizer used was OASIS BIO (Italpollina Spa, Biandrate, Italy), with four applications (1.33 L/m³ H₂O each time) during the cultivation period. For crop protection, TRACER (Dow Agrosciences, Drusenheim, France) was applied three times and KONFLIC (Atlantica Agricola, Alicante, Spain) one time. Spearmint was harvested (early morning, with a sharp knife manually) at the 2nd harvesting cycle, and the plants had a height of approximately 18 cm. Irrigation took place every four days or according to the plants’ needs. The soil characteristics included organic matter of 3.12%; available CaCO₃ of 22.14%; pH of 8.37; and EC (electrical conductivity) of 0.81 mS/cm. The plants were transferred to the laboratory and homogenously sorted for no physical defects or leaf wilding, washed with chlorinated water (sodium hypochlorite—NaOCl 0.05%), and rinsed with sterile water three times (to minimize native microflora).

Fresh Origanum dubium (Cypriot oregano = Origanum majorana L.), Salvia fruticosa (sage) and Sideritis cypria (Cypriot mountain tea) plant tissue, at the early flowering stage, was obtained from the Department of Aromatic Plants, Ministry of Agriculture, Rural Development and Environment (Nicosia, Cyprus) and the experimental farm/greenhouse of Cyprus University of Technology (Limassol, Cyprus). Plant material was dried at 42 °C in an air-ventilated oven to achieve biochemical stability of the plant tissue after moisture removal in a short time. A Clevenger apparatus was used for hydro-steam distillation (3 h) of the dried plant material of the tested MAP to obtain the essential oil. The EOs were stored at −20 °C until use. The water that remained after distillation (i.e., hydrosol) was also collected after filtration with a cheesecloth. The hydrosols were stored at 4 °C until use. The EO composition was determined with Gas Chromatography–Mass Spectrometry (GC/MS; Shimadzu GC2010 gas chromatograph interfaced Shimadzu GC/MS QP2010 plus mass spectrometer, Kyoto, Japan) following the procedure previously described in [14].

Bacterial strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 11632), Salmonella enterica subsp. enterica (ATCC 51741) and Listeria monocytogenes were obtained from the Department of Nursing (Cyprus University of Technology, Limassol, Cyprus). Fresh bacterial cultures (8 log cfu/mL) were prepared in a brain–heart infusion broth (BHI, HiMedia, Mumbai, India) after overnight incubation at 37 °C according to the procedure described by Chrysargyris et al. [14].

2.2. In Vitro Antibacterial Activity

The antimicrobial activity of the examined EOs and hydrosols against four foodborne pathogens (i.e., E. coli, S. aureus, S. enterica and L. monocytogenes) was assessed with (i) the disc diffusion assay and (ii) the microdilution method as previously described [14]. For the disc diffusion assay, a sterile filter paper (6 mm in diameter) was impregnated with 10 μL of pure EO or hydrosol and was placed on solid BHI agar inoculated with 100 μL bacterial culture, and the plates were left to dry. Following 24 h of incubation at 37 °C, the diameter of the inhibition zone (DIZ) was recorded (in mm).

For the determination of the minimum inhibitory concentration (MIC), the microdilution method was performed. Briefly, sterile 96-well plates were prepared by dispersing 45 μL of each dilution (from EOs and hydrosols) in each respective well and adding 50 μL of BHI broth. Then, 5 μL of bacterial culture was added in each well (6 log cfu/mL) (final volume of each well: 100 μL). Two negative controls were used, which consisted of (i) 100 μL BHI broth, and (ii) 45 μL diluted EO/hydrosol and 55 μL BHI broth. The positive control consisted of 5 μL bacterial culture and 95 μL BHI broth. The plates were sealed with a sterile plastic membrane and placed for incubation at 37 °C for 22 h, measuring the optical density (OD) at 630 nm every 30 min with an absorbance microplate reader (ELx808 BioTek Instrument Inc., Highland Park, VT, USA). Growth curves were produced, and the MIC was defined as the lowest concentration that inhibited the bacterial growth with respect to the positive control. After incubation, the bacterial growth was confirmed by plating drops (10 μL each) from each well on BHI agar, and further incubated for another 24 h at 37 °C [17]. Examining the bacterial growth after incubation, the lethal dose 90 (LD₉₀) was determined as the concentration that was able to reduce the bacterial population by 90%. The examined EOs were diluted in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) to achieve a stock concentration of 20% and serial two-fold dilutions were prepared in sterile water. Due to the use of DMSO, another control was prepared consisting of 45 μL diluted DMSO, 50 μL BHI broth and 5 μL of bacterial culture in order to ensure that the DMSO level used did not interfere with the bacterial growth. Hydrosols were diluted only in sterile water.

2.3. Preliminary Screening

Following the in vitro screening, O. dubium EO and hydrosol were selected as the most effective means to further investigate the effects on fresh spearmint preservation. Fresh spearmint bundles (approx. 35 g) were dipped in the tested solutions in various concentrations (0.001%, 0.01% and 0.1% v/v) and for various periods (1, 5 and 10 min) at room temperature. For control, distilled water was employed. An emulsifier (Tween 20) was used at 0.1% v/v to dilute the EO into the water. Then, the bundles were left to dry at room temperature (approximately 30 min) and enclosed in 5 L polypropylene plastic containers. The containers were stored for six days at 4 °C and 90% relative humidity (RH) (accomplished via putting a wet piece of paper into each container).

2.3.1. Weight Loss and Respiration Rate

During storage, the bundles’ weight loss and respiration rate were recorded at 0, 3, and 6 days. Each bundle was weighed on each of the above-mentioned days and the weight loss percentage was computed.

Respiration rates were determined as previously described [18] by measuring the produced carbon dioxide (CO₂) after hermetically enclosing each bundle at room temperature for 1 h in a 5 L plastic container using a dual gas analyzer (GCS 250 Analyzer, International Control Analyser Ltd., Kent, UK). The weight and volume of each bundle were recorded, and the results of respiration rates were expressed as mL of CO₂ produced per kg per h (mL CO₂/kg/h).

2.3.2. Sensory Evaluation

At least four panelists (aged 21–32 years old, considering gender balance, not professionals, but an introduction/training for spearmint storage conditions was conducted) assessed the aroma, appearance and marketability of spearmint on different days (0, 3 and 6 day) throughout storage [19]. Aroma evaluation was implemented using a 10-point scale (1-point interval): 1: not spearmint-like and very unpleasant aroma, 3: not spearmint-like and slightly unpleasant aroma, 5: not spearmint-like but pleasant aroma, 8: less spearmint-like aroma, and 10: intense spearmint-like aroma. Spearmint’s appearance (visual quality and color) was assessed using a 10-point scale (1-point interval): 1: yellow color of 50%, 3: yellow-green, 5: light green, 8: green, and 10: deep green. Overall quality (i.e., marketability) was evaluated with the use of a scale of 1–10 (1-point interval): 1: not marketable quality (i.e., malformation, wounds, infection); 3: low marketability with malformation; 5: marketable with few defects, i.e., small size, decolorization (medium quality); 8: marketable (good quality); and 10: marketable with no defects (extra quality).

2.3.3. Quality Parameters

On the initial (day 0) and last day of storage (days 6), the quality parameters were also assessed. The leaf’s surface color was evaluated by recording the L* (brightness/lightness; 0: black/100: white), a* (−a*: greenness and +a*: redness) and b* (−b*: blueness and +b*: yellowness) values (CIELAB uniform color space) with the use of a colorimeter (Chroma meter CR400 Konica Minolta, Tokyo, Japan). Hue (h) represents the common distinction between colors around a color wheel (h = 0°: red-purple; h = 90°: yellow, h = 180°: bluish-green, h = 270°: blue) and it was determined using the following equation: h(°) = 180 + tan⁻¹(b*/a*) [20,21]. Chroma value (C) represents the degree of the departure from gray to pure chromatic color and it was calculated as C = (a*² + b*²)^1/2 [20]. The color index (CI) was determined as follows: CI = (a* × 1000)/(L* × b*), where color index (CI): ≥ −40 and <−20: blue-violet to dark green; ≥−20 and <−2: dark green and yellowish green; ≥+2 <+20: pale yellow and deep orange; and ≥+20 and <+40: deep orange and deep red color [21,22].

The procedure for leaf pigment extraction and determination was performed using methanol (Merck, Darmstadt, Germany) as reported by Wellburn [23]. To estimate the chlorophyll a (chl a), chlorophyll b (chl b), total chlorophylls (tot chl) and total carotenoids (tot car) content, the solution’s optical density was measured at 480, 649 and 665 nm, and the results were expressed as mg of chlorophyll (or carotenoids) per g of fresh weight (mg/g).

Polyphenols and antioxidants were extracted using methanol 50% (v/v) with the Folin–Ciocalteu method as previously described [24]. The absorbance of the reaction was measured at 755 nm using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific Oy, Vantaa, Finland) and the results were expressed as mg of gallic acid (Scharlab, Sentmenat, Spain) equivalents (GAE) per gram of fresh weight (mg GAE/g).

The antioxidant capacity of spearmint’s methanolic extracts was determined with two different assays, (i) 2,2-diphenyl-1-picrylhydrazyl (DPPH) and (ii) ferric-reducing antioxidant power (FRAP), that were performed as described by Chrysargyris et al. [14]. The reduction of the DPPH free radical by the methanolic extracts was assessed at 517 nm. The formation of the blue-colored [Fe(TPTZ)2]²⁺ complex with the FRAP assay was determined at 593 nm. For both antioxidant assays, the results were expressed as mg of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) (Sigma-Aldrich, Taufkirchen, Germany) per gram of fresh weight (mg trolox/g).

Flavonoids were estimated using the aluminum chloride colorimetric method as described by Chrysargyris et al. [14]. The formation of pink-colored complexes was measured at 510 nm and the results were expressed as mg of rutin per gram of fresh weight (mg rutin/g).

The ascorbic acid (AA) content of spearmint was determined using the 2,6-dichloroindophenol (DCPIP) titrimetric method, as previously mentioned [25]. The results were expressed as mg of ascorbic acid per 100 g of fresh weight (mg AA/100 g).

In order to determine the damage index induced by abiotic and biotic stress of the applied treatments, hydrogen peroxide (H₂O₂) levels and lipid peroxidation were estimated. The levels of H₂O₂ were determined by the procedure described by Loreto and Velikova [26], measuring the absorbance at 390 nm and results were expressed as μmol H₂O₂ per g of fresh weight (μmol H₂O₂/g). Lipid peroxidation was determined by measuring the production of malondialdehyde (MDA) [27]. The absorbance was measured at 532 nm (subtracting non-specified absorbance at 600 nm) and the results were expressed as nmol MDA per g of fresh weight (nmol MDA/g).

2.4. In Vivo Antibacterial Activity

2.4.1. Procedure

From the preliminary screening with 10 EO and 10 hydrosols combinations, the two most promising treatments for each product (EO and hydrosol) were selected based on their effects on spearmint’s visual quality and overall marketability, as well as other positive effects on the investigated parameters (i.e., respiration rate, antioxidant status). The selected doses (concentration and duration of application) from the preliminary screening that presented the similar positive effects on produce quality attributes were further investigated for their ability to lower and/or eliminate the populations of S. enterica and L. monocytogenes inoculated on fresh spearmint. For this, the following treatments were assessed: (i) sterile distilled water (control), (ii) chlorine (0.02%), (iii) EO Dose A (0.01% for 5 min), (iv) EO Dose B (0.001% for 10 min), (v) hydrosol Dose A (0.1% for 5 min), and (vi) hydrosol Dose B (0.01% for 10 min). Chlorine was employed as a common postharvest sanitizer in postharvest sector.

The procedures of inoculation and treatment application were performed as previously described [28], with slight modifications. Spearmint bundles were washed with 0.05% chlorinated water (NaOCl), rinsed with sterile water, and then air-dried in a laminar flow cabinet at room temperature for approximately 30 min. Each bundle (25 g) was then placed in a labeled sterile stomacher bag. Afterwards, 2 mL of inoculum (8 log cfu/mL) was sprinkled evenly on the surface and the bags were left open for 1 h, allowing for bacterial attachment on the leaves’ surface. An appropriate volume of the treatment solution (in a ratio of 1:10 w/v) was added into the bags, and the bags were sealed and left for the appropriate time at room temperature. Then, the treatment solution was discarded, and the bags were closed and stored at 4 °C for six days. Sampling and microbiological analysis were performed on the first and sixth days in order to record the direct and a late effect of the examined treatments.

2.4.2. Microbiological Analysis

The remaining (surviving) inoculum population was determined as described previously [28]. Briefly, the plant tissue was homogenized (1:10 w/v) with maximum recovery diluent (MRD; Merck, Darmstadt, Germany) and serial decimal dilutions were performed (in MRD). An appropriate volume of dilutions (100 μL) was spread on appropriate growing media for S. enterica (Xylose-lysine-deoxycholate agar-XLD agar; Merck, Darmstadt, Germany) and L. monocytogenes (PALCAM agar; Merck, Darmstadt, Germany), and incubation took place at 37 °C (24 h) and 37 °C (48 h), respectively, and the results were expressed as log cfu/g.

2.5. Statistical Analysis

The experimental set up of this study was a Completely Randomized Design (CRD) consisting of four biological replications (for each treatment). The IBM SPSS version 25.0 was used for performing one-way analysis of variance (one-way ANOVA) and comparing the treatment means on each day (Duncan’ multiple range test for p = 0.05). In addition, independent samples t-test was performed for comparing with the control on the initial and final days of the experiment.

3. Results and Discussion

3.1. Essential Oils’ Main Components

The EO analysis and the main EO components were reported previously for O. dubium EO [10], for S. fruticosa EO [29] and for S. cypria EO [30], and were in line with the composition of the used EOs. In the present study, the main components of O. dubium EO were carvacrol (54.09%), γ-terpinene (8.21%), and p-cymene (7.13%). S. fruticosa EO’s main components included camphor (35.77%), 1,8-cineol (15.81%), and α-thujone (8.29%). The main compounds of S. cypria EO were α-pinene (29.82%), β-phyllandrene (27.37%), and β-pinene (23.27%).

3.2. In vitro Antibacterial Activity

Previous studies have shown that EOs and hydrosols from MAPs present great antibacterial activity towards foodborne and spoilage bacteria during in vitro investigation [2,3,10,31]. From the present study, it was found that oregano (O. dubium) EO was the most effective against the tested bacteria (

Table 1. Screening of examined EOs and hydrosols’ antibacterial activity against foodborne pathogens in vitro.

	Plant	E. coli			S. enterica			L. monocytogenes			S. aureus
		MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)
EOs	O. dubium	0.62 ± 0.00 b	1.25 ± 0.00 b	41.67 ± 3.33 a	0.16 ± 0.00 c	1.25 ± 0.00 b	30.00 ± 0.88 a	1.25 ± 0.00 a	5.00 ± 0.00 b	40.00 ± 0.58 a	0.31 ± 0.00 b	0.62 ± 0.00 b	55.00 ± 6.81 a
	S. fruticosa	1.25 ± 0.00 a	2.50 ± 0.00 a	12.33 ± 0.88 c	2.50 ± 0.00 a	5.00 ± 0.00 a	7.50 ± 0.29 c	1.25 ± 0.00 a	2.50 ± 0.00 c	35.00 ± 0.58 b	0.62 ± 0.00 a	1.25 ± 0.00 a	17.67 ± 1.45 b
	S. cypria	1.25 ± 0.00 a	2.50 ± 0.00 a	21.33 ± 0.88 b	1.25 ± 0.00 b	5.00 ± 0.00 a	25.00 ± 0.58 b	0.16 ± 0.00 b	10.00 ± 0.00 a	12.00 ± 0.58 c	0.62 ± 0.00 a	1.25 ± 0.00 a	21.00 ± 2.31 b
		MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)	MIC (%)	LD90 (%)	DIZ (mm)
Hydrosol	O. dubium	50.00 ± 0.00 b	100.00 ± 0.00	10.33 ± 0.33 a	1.56 ± 0.00 b	25.00 ± 0.00	6.00 ± 0.00	12.50 ± 0.00 b	50.00 ± 0.00	6.50 ± 0.29 b	6.25 ± 0.00 b	12.50 ± 0.00	7.00 ± 0.00
	S. fruticosa	100.00 ± 0.00 a	-	6.00 ± 0.00 b	25.00 ± 0.00 a	-	6.00 ± 0.00	50.00 ± 0.00 a	-	8.00 ± 0.00 a	100.00 ± 0.00 a	-	6.00 ± 0.00
	S. cypria	6.25 ± 0.00 c	25.00 ± 0.00	6.00 ± 0.00 b	3.12 ± 0.00 b	-	6.00 ± 0.00	50.00 ± 0.00 a	-	6.00 ± 0.29 b	6.25 ± 0.00 b	12.50 ± 0.00	6.00 ± 0.00

Values presented as mean ± standard error (two biological replicates per treatment). On each column and each treatment (EOs and hydrosols), significant differences (p < 0.05) are indicated with different Latin letters. MIC: minimal inhibitory concentrations; DIZ: diameter of inhibition zone; LD90: The 90% lethal dose.

). Oregano EO and carvacrol (a GRAS monoterpene and the main compounds of oregano EO) have been known to reveal great antimicrobial activity against a plethora of bacteria and fungi [8,10,32]. From our study, it was found that S. aureus had high sensitivity in the presence of EOs (followed by L. monocytogenes). The antibacterial activity of S. fruticosa EO is attributed to its main compounds (i.e., camphor and 1,8-cineol) that have been previously shown to present antimicrobial activity [33]. In addition, two of the main compounds of S. cypria EO α-pinene and β-pinene presented great antibacterial activity [34]. It is worth mentioning, that the antimicrobial activity of EOs is attributed to the synergistic effects of their main components and/or to the presence of less abundant components [2]. Under the investigated conditions, Gram-positive bacteria (i.e., L. monocytogenes and S. aureus) were more susceptible to the EOs than Gram-negative bacteria (i.e., E. coli and S. enterica), whilst hydrosols presented a variance in their antibacterial activity. Among the tested hydrosols, O. dubium hydrosol was the most effective one, presenting lower MIC values, i.e., 1.56%, followed by S. cypria with MIC of 3.12% (v/v) for S. enterica (Table 1). O. dubium hydrosol presented great antibacterial activity against E. coli and S. aureus (DIZ: 10.33 and 7.00 mm, respectively), while S. cypria hydrosol was found to affect the growth of L. monocytogenes more (DIZ: 8.00 mm). The difference between the activity and effectiveness of EOs and hydrosols is attributed to the chemical composition of those products and the structural composition of the bacterial cell wall outer membrane [1,3]. Generally, Gram-positive bacteria (i.e., L. monocytogenes and S. aureus) are more sensitive to the presence of EOs compared to Gram-negative bacteria (i.e., E. coli and S. enterica) due to the lack of a specific cellular structure (i.e., outer lipid bilayer) [1,31]. Moreover, it has been mentioned that the shape of bacterial cells also influences the sensitivity to EOs action, with coccoid cells (i.e., S. aureus) being more susceptible than rod-shaped cells (L. monocytogenes, E. coli, and S. enterica) [31]. These observations confirm our findings in the current study. Both EOs and hydrosols contain hydrophobic molecules of low molecular weight (i.e., phenylpropanoids, terpenoids, amino acid and fatty acid derivatives) that can penetrate the cell wall and retard bacterial cell processes and growth [1]. Interestingly, the differences observed between the disc diffusion assay and the microdilution assay in the present work may be ascribed to the different diffusion of the EO and hydrosol on the growth medium, as well as their volatile nature [35].

3.3. Preliminary Screening

3.3.1. Effects on Weight Loss and Respiration Rate

Weight loss and leaf wilting are among the main symptoms of fresh produce deterioration and possible decay (especially in leafy vegetables), shortening fresh produce shelf life. Water loss causes weight loss, which is accompanied by increased respiration and transpiration rates. This evidently results in the wilting, senescing, discoloration (yellowing and/or browning) of leafy vegetables [36]. In the current research, EO application did not affect the spearmint weight loss on the third day compared to control treatment (Figure 1A). On the final storage day (day 6), EO application of 0.1%-1 min, 0.001%-5 min, 0.1%-5 min, 0.01%-10 min and 0.1%-10 min increased weight loss (up to 2.68% for 0.1%-10 min EO treatment) compared to all the other treatments and control. Notably, weight losses greater than 3% on leafy vegetables are linked with adverse effects on produce quality such as appearance (i.e., wilting), texture and short shelf life [37,38]. Reduced weight loss rate has been mirrored in improved storability and enhanced visual quality [36]. The use of rosemary EO on fresh rosemary resulted in less weight loss even 12 days after storage; however, as a xerophilic plant when stored under high humidity conditions, its fresh weight increases due to water absorbance, as opposed to spearmint [19]. When O. dubium EO was applied on tomato and cucumber fruits, this resulted in great water loss (up to 5.22%) when treated with 0.5% oregano EO for 20 min [6]. The lower water loss found in the current study could be related to the lower EO concentrations and time of dipping application used. In the present study, on the sixth day of storage, spearmint treated with O. dubium hydrosol 0.1%-1 min presented increased weight loss (up to 4.62%) compared to 0.001%-1 min, 0.01%-1 min, 0.1%-10 min, 0.001%-5 min, and control (Figure 1B). However, no differences were found on control and hydrosol-treated spearmint on the third day of storage. Marjoram hydrosol (O. majorana, which is a synonym of O. dubium in Greece) used on shredded carrots (1:15 v/v for 10 min) resulted in less intense water loss, maintaining the product’s moisture levels and acting as a protection against the appearance of surface whitening [39]. The findings in our study might be attributed to the Eos’ high concentration and longer time of application. On the other hand, the hydrophilic nature of the hydrosols (containing EO components and water-soluble compounds) seemed not to be able to rehydrate leaves’ surfaces and prevent water loss [36].

Previous indications showed elevated CO₂ production (i.e., increased respiration rates) of MAPs and fresh herbs (50–300 mL CO₂/kg/h at 20 °C) and this is mainly attributed to the temperature at which the produces were stored [36]. All applied EO treatments (except 0.001%-5 min) increased the respiration rate of spearmint on the third day, while on the last day, an increased respiration rate was found with EO 0.01%-5 min, 0.1%-5 min and 10 min (all concentrations) (Figure 1C). Rosemary EO applied via dipping on fresh rosemary (1:1500 and 1:500 v/v for 10 min) did not cause a dramatic increase in produce respiration rate (similar levels to the non-treated ones) even after 12 days, as rosemary has a slow metabolic process profile, being slow-grown plants in nature [19]. The use of O. dubium hydrosol 0.01%-5 min, 0.1%-5 min and 10 min (all concentrations) increased spearmint’s respiration rate on the third day compared to the control treatment, with 0.01%-10 min showing the highest value (93.48 mL CO₂/kg/h) (Figure 1D). An increase in fresh spearmint’s respiration rate was observed at the end of storage time with hydrosol 0.01% and 0.1% (5 and 10 min) compared to 0.001% (1 and 5 min) and control. Increases in leafy vegetables’ respiration such as lettuce and spinach are linked with increased metabolism processes, senescing and degradation of chlorophylls (yellowing) [40]. In a prior study, the use of marjoram hydrosol on shredded carrots resulted in similar respiration rates those of non-treated ones, while marjoram EO caused an increase in carrot’s respiration [39]. The differences in respiration rates observed in our study might have been caused by the disruption of the plant cell walls that possibly led to the disturbance of gas exchanges by the EO and/or even the hydrosol applications [19,36].

3.3.2. Effects on Sensory Attributes

As with many culinary herbs, spearmint is used for its unique organoleptic characteristics and fresh aroma. Consumers demand fresh and fragrant herbs; however, these products are very susceptible to a limited shelf life. The distinctive aroma of culinary herbs tends to fade during storage (especially at the end of the storage) [19]. In the present study, all applied EO treatments (except 0.001% for 1 and 5 min, and 0.1%-1 min) scored lower values in the aroma scale on the third day of storage (less spearmint-like aroma) (Figure 2A). Moreover, on the last day, lower aroma scores were found after the application of EO 0.1%-5 min, 0.01%-10 min and 0.1%-10 min (less spearmint-like aroma, not pleasant) compared to the control. Decreased scorings on the aroma evaluation scale were reported with 0.1%-1 min, 0.01%-5 min, 0.001%-10 min and 0.1%-10 min hydrosol treatment, indicating a less pleasant aroma on the third day. At the end of storage, the application of hydrosol 0.001%-1 min presented a higher aroma score (more intense spearmint aroma) compared to 0.1% (1 and 10 min) (Figure 2B). The changes in spearmint’s aroma could be a result of the volatile nature of EOs and hydrosols’ components of the applied products and especially their strong aroma [19,41]. Moreover, after storage, leaves are dehydrated (due to water loss), whilst leaf glandular trichomes (storing the plant’s EOs) might be disrupted and volatile EOs can be released to the environment, making fresh spearmint less aromatic. It has been previously mentioned that when using EOs on fresh produce in high concentrations (to ensure low microbial load), a negative impact on organoleptic properties (i.e., aroma and taste) can be noted [42]. This was also observed in our case, where the panelists perceived less spearmint-like (pleasant) aroma.

The vibrant green color of herbs decreases due to the senescing of leaves during storage and the degradation of chlorophylls [15,43]. As shown in Figure 2 and Figure S1, lower scores on the appearance evaluation scale were observed by all EOs in the applied treatments on the third day (Figure 2B). After three days of storage at 4 °C, the application of 0.01%-5 min O. dubium hydrosol resulted in a greener produce (i.e., higher score in appearance) compared to 0.1%-10 min, but at the same time did not differ from the non-treated samples (control). Higher appearance scores (dark green color) were also found on the last day with hydrosol 0.001%-1 min compared to 0.1%-5 min and 10 min (all concentrations) (Figure 2C and Figure S1). The changes in spearmint’s color (i.e., appearance) might have been caused by a possible phytotoxic effect of O. dubium EO at high concentrations and longer time of application. On the other hand, the antioxidant activity of the EO (especially at lower concentrations and short time of application) and hydrosol could be the reason for the preservation of the product’s optical quality during storage [10,19,32].

A less marketable product was found with the application of EO 0.001%-5 min and 10 min (all concentrations) after three days of storage. In addition, the application of 0.1%-10 min presented a lower marketability scoring compared to all other treatments at the end of storage (Figure 2E). All applied hydrosol treatments (except 0.01%-1 min and 0.001%-1 min) resulted in less marketable products (lower scores) compared to the non-treated ones (control). Interestingly, the same observations were made at the end of storage (Figure 2F). Previously, it was shown that lettuce leaves treated with bay leaf and sideritis hydrosols for 60 min were found to be more acceptable by the testing panelists compared to the ones treated with thyme, rosemary and sage hydrosols [41]. These observations, as well as the results from our study, suggest that EOs and hydrosols with distinct and strong aromas (i.e., thyme, rosemary, sage, oregano) could result in less acceptable products (visual and aroma) compared to the ones with a more subtle aroma (i.e., bay and sideritis). As in our case, O. dubium EO and hydrosol have a strong aroma that may interfere with fresh produce aroma; however, due to the volatile nature of their components, the aroma could fade out after six days of storage.

3.3.3. Effects on Quality Parameters

Effects on Color

The main sensory attribute that attracts consumers when buying fresh produce is the visual quality, i.e., color of the product. Color of fresh spearmint was affected by the application of O. dubium EO (

Table 2. Effects of O. dubium EO and hydrosol application on color parameters of fresh spearmint stored at 4 °C for 6 days: L* (brightness/lightness), a* (greenness/redness), b* (blueness/yellowness), hue (h), chroma value (C), and color index (CI).

	Time (min)	Concentration	L*	a*	b*	h	C	CI
Day 0	0	0.00%	46.21 ± 0.87	−22.89 ± 0.38	33.79 ± 1.06 *	124.18 ± 0.42 *	40.82 ± 1.08	−14.74 ± 0.49 *
EO	0	0.00%	45.01 ± 0.47	−21.32 ± 0.49 *	29.81 ± 0.93	125.61 ± 0.42 ab	36.65 ± 1.01	−15.94 ± 0.40 ab
	1	0.001%	43.67 ± 1.03	−19.56 ± 0.68	26.72 ± 1.93	126.47 ± 0.93 a	33.13 ± 1.96	−17.07 ± 0.90 ab
		0.01%	44.34 ± 1.22	−21.20 ± 0.73	30.51 ± 1.98	125.04 ± 0.98 ab	37.17 ± 2.01	−15.96 ± 0.91 ab
		0.10%	44.36 ± 1.23	−20.82 ± 0.71	28.90 ± 1.55	125.89 ± 0.65 ab	35.63 ± 1.66	−16.42 ± 0.74 ab
	5	0.001%	44.61 ± 0.76	−20.38 ± 0.34	28.69 ± 1.06	125.48 ± 0.55 ab	35.20 ± 1.06	−16.04 ± 0.58 ab
		0.01%	43.05 ± 0.73	−19.57 ± 0.39	26.12 ± 1.27	126.98 ± 0.73 a	32.65 ± 1.26	−17.57 ± 0.68 b
		0.10%	44.68 ± 0.73	−21.32 ± 0.54	30.29 ± 1.12	125.20 ± 0.35 ab	37.05 ± 1.23	−15.83 ± 0.46 ab
	10	0.001%	45.04 ± 0.76	−21.14 ± 0.71	29.84 ± 1.71	125.47 ± 0.63 ab	36.58 ± 1.81	−15.88 ± 0.62 ab
		0.01%	44.92 ± 0.49	−20.31 ± 1.14	30.26 ± 1.02	123.75 ± 1.00 b	36.46 ± 1.41	−14.92 ± 0.59 a
		0.10%	44.99 ± 1.13	−20.12 ± 0.65	28.33 ± 1.46	125.49 ± 0.60 ab	34.76 ± 1.55	−15.95 ± 0.70 ab
Hydrosol	0	0.00%	43.72 ± 1.09 bc	−21.20 ± 0.71 ab	29.51 ± 1.73 abc	125.86 ± 0.73 bc	36.35 ± 1.81 ab	−16.67 ± 0.88 bc
	1	0.001%	44.59 ± 1.51 bc	−20.29 ± 0.66 ab	28.96 ± 2.06 abc	125.30 ± 1.05 bcd	35.38 ± 2.06 ab	−16.09 ± 1.05 abc
		0.01%	44.97 ± 1.23 abc	−20.17 ± 0.77 ab	28.85 ± 1.81 abc	125.15 ± 0.80 bcd	35.21 ± 1.90 ab	−15.79 ± 0.86 abc
		0.10%	41.38 ± 0.60 a	−18.51 ± 0.64 a	24.13 ± 1.33 c	127.64 ± 0.63 ab	30.42 ± 1.44 b	−18.70 ± 0.68 c
	5	0.001%	49.62 ± 1.84 a	−20.61 ± 1.19 ab	30.86 ± 2.79 ab	124.10 ± 1.01 cd	37.14 ± 2.96 ab	−13.80 ± 0.78 ab
		0.01%	49.54 ± 3.15 c	−21.11 ± 0.86 ab	34.38 ± 3.28 a	122.05 ± 1.29 d	40.41 ± 3.25 a	−13.06 ± 1.23 a
		0.10%	43.05 ± 1.27 ab	−19.80 ± 0.79 ab	27.40 ± 1.87 bc	126.08 ± 0.86 bc	33.82 ± 1.96 ab	−17.10 ± 1.01 c
	10	0.001%	42.67 ± 0.79 c	−19.68 ± 0.36 ab	28.09 ± 1.08 abc	125.11 ± 0.67 bcd	34.31 ± 1.07 ab	−16.56 ± 0.70 bc
		0.01%	48.49 ± 0.64 c	−21.90 ± 0.28 ab	33.68 ± 0.91 ab	123.08 ± 0.41 cd	40.17 ± 0.91 a	−13.46 ± 0.40 ab
		0.10%	45.04 ± 1.73 abc	−22.47 ± 2.52 b	27.04 ± 2.15 bc	129.36 ± 2.05 a	35.29 ± 3.03 ab	−18.67 ± 1.81 c

Values are presented as the mean ± standard error (four biological replicates per treatment). The values for day 0 refer to the control (non-treated, 0.00%). Significant differences (p < 0.05) are indicated with different Latin letters for each treatment (EO and hydrosol, separately) on each column. An asterisk (*) indicates significant differences between the initial (day 0) and last day of storage of control (non-treated, 0.00%).

). A higher h value (lighter green color) was recorded on spearmint treated with EO 0.01%-5 min and 0.001%-1 min compared to 0.01%-10 min. Increased CI value was found with EO 0.01%-10 min (darker green color) when compared to 0.01%-5 min. Higher L* values were found with the use of O. dubium hydrosol 0.001%-5 min and 0.01%-5 min indicating a more light-green color. A decrease in a* value was noted with 0.1%-10 min compared to 0.1%-1 min suggesting a greener color leaf (Table 2). Prior report by Chen et al. mentioned that clove EO at 0.05% (v/v) maintained the green color of fresh-cut lettuce for 12 days of storage at 4 °C, whilst presenting lower browning incidence [44]. It seems that lower EOs concentrations can preserve leafy vegetables green color especially due to their antioxidant activities that can prevent the degradation of leaves pigments (i.e., chlorophylls and carotenoids) [10,19]. In the present study, the application of hydrosol 0.1% (1, 5 and 10 min) resulted in lower b* values (more blue color giving a greener-colored product) when compared to 0.01%-5 min. When hydrosol 0.01%-5 min was applied, spearmint’s h value decreased (less green color), whereas 0.1%-10 min increased the product’s h value (more dark green color) (Table 2). Spearmint treated with hydrosol 0.01% (5 and 10 min) presented higher C values than that treated with 0.1%-1 min, indicating a more intense green color. A higher CI value was also found with the application of hydrosol 0.01%-5 min, suggesting a darker color of the leaves. Hydrosols, as by-products of the EO hydrodistilation process, contain molecules of high antioxidant activity (water soluble as well as lipophilic compounds), and this could be one of the reasons for preventing the degradation of leaf chlorophylls and the development of browning and/or yellowing [3,39,41]. In addition, the low storage temperature (i.e., 4 °C) in the present study could have contributed to the slowing of spearmint metabolism, delaying leaf discoloration [43].

Effects on Pigments, Phenols, Antioxidants, Flavonoids and AA Content

Leaf senescing in leafy vegetables and fresh herbs mainly appears as yellowing due to the significant reduction in sugar levels and the degradation of colored pigments (i.e., chlorophylls) [45]. During the present study, spearmint’s pigments were affected by the applied EO treatments (

Table 3. Effects of O. dubium EO and hydrosol application on fresh spearmint’s pigments (chlorophylls—Chls; and total carotenoids—car) stored at 4 °C for 6 days.

	Time (min)	Concentration	Chl a (mg/g)	Chl b (mg/g)	Tot chl (mg/g)	Tot car (mg/g)
Day 0	0	0.00%	0.91 ± 0.01	0.55 ± 0.04	1.46 ± 0.05	0.20 ± 0.01
EO	0	0.00%	0.88 ± 0.02 cd	0.62 ± 0.10	1.51 ± 0.11	0.21 ± 0.01 c
	1	0.001%	0.88 ± 0.01 d	0.62 ± 0.14	1.50 ± 0.13	0.22 ± 0.00 bc
		0.01%	0.92 ± 0.00 abcd	0.58 ± 0.02	1.50 ± 0.03	0.22 ± 0.00 bc
		0.10%	0.93 ± 0.02 ab	0.64 ± 0.03	1.57 ± 0.05	0.23 ± 0.00 ab
	5	0.001%	0.92 ± 0.01 abcd	0.68 ± 0.05	1.60 ± 0.05	0.22 ± 0.01 bc
		0.01%	0.93 ± 0.01 abc	0.63 ± 0.07	1.56 ± 0.08	0.23 ± 0.00 ab
		0.10%	0.88 ± 0.01 cd	0.56 ± 0.06	1.45 ± 0.06	0.22 ± 0.01 bc
	10	0.001%	0.94 ± 0.01 a	0.77 ± 0.04	1.71 ± 0.04	0.24 ± 0.00 a
		0.01%	0.89 ± 0.03 bcd	0.75 ± 0.09	1.64 ± 0.08	0.22 ± 0.00 bc
		0.10%	0.92 ± 0.01 abcd	0.67 ± 0.08	1.59 ± 0.08	0.23 ± 0.00 ab
Hydrosol	0	0.00%	0.59 ± 0.02 abc	0.56 ± 0.03 ab	1.46 ± 0.04 ab	0.21 ± 0.00 b
	1	0.001%	0.93 ± 0.02 ab	0.58 ± 0.03 ab	1.50 ± 0.04 ab	0.21 ± 0.00 b
		0.01%	0.92 ± 0.01 abc	0.61 ± 0.10 ab	1.54 ± 0.10 ab	0.22 ± 0.00 ab
		0.10%	0.89 ± 0.04 bc	0.58 ± 0.12 ab	1.47 ± 0.16 ab	0.22 ± 0.01 ab
	5	0.001%	0.91 ± 0.02 abc	0.65 ± 0.03 ab	1.56 ± 0.01 ab	0.22 ± 0.00 ab
		0.01%	0.88 ± 0.03 bc	0.43 ± 0.05 b	1.31 ± 0.07 b	0.20 ± 0.01 b
		0.10%	0.86 ± 0.02 c	0.65 ± 0.12 ab	1.50 ± 0.14 ab	0.21 ± 0.01 b
	10	0.001%	0.92 ± 0.02 abc	0.64 ± 0.04 ab	1.56 ± 0.05 ab	0.22 ± 0.00 ab
		0.01%	0.92 ± 0.01 abc	0.65 ± 0.02 ab	1.57 ± 0.03 ab	0.23 ± 0.00 ab
		0.10%	0.96 ± 0.01 a	0.70 ± 0.01 a	1.66 ± 0.03 a	0.24 ± 0.00 a

Values are presented as the mean ± standard error (four biological replicates per treatment). The values for day 0 refer to the control (non-treated, 0.00%). Significant differences (p < 0.05) are indicated with different Latin letters for each treatment (EO and hydrosol, separately) on each column.

). The application of O. dubium EO 0.001%-10 min and 0.1%-1 min presented higher Chl content compared to 0.001%-1 min, 0.1%-5 min treated, and non-treated (control) ones. Spearmint’s leaf carotenoid content at the end of storage (day 6) was found to increase with EO 0.1% (1 and 10 min), 0.01%-5 min, and 0.001%-10 min compared to control. When hydrosol 0.1%-10 min was applied, a higher pigment content (chlorophylls and carotenoids) was found compared to 0.1% (1 and 5 min) and 0.01%-5 min (Table 3). As can be seen from the above observations, both O. dubium products (EO and hydrosol) were found to prevent chlorophyll degradation throughout storage (up to six days at 4 °C). At the same time, some treatments increased carotenoids, which increased the nutritional value of spearmint without affecting its bright green color (as seen from sensory and color evaluation above). Even though increased respiration rates were found in our study, suggesting an increase in spearmint’s metabolism, chlorophylls were not significantly affected. This phenomenon was also observed in peppermint and spearmint stored at 0 °C for up to 21 days [15]. This might be related to the low storage temperature. Moreover, the great antioxidant activity of O. dubium (EO and hydrosol) might have been able to prevent the oxidation and degradation of chlorophylls and the appearance of carotenoids (yellowing) [3,15,19]. These findings further support the observations on spearmints appearance evaluation reported above.

Many MAP species (including spearmint) are rich in antioxidants and contain a plethora of bioactive components such as ascorbic acid, polyphenols, carotenoids, and flavonoids, among others [46,47]. This makes spearmint and other herbs high-nutritional-value products that need to be preserved. In the current study, all applied EO treatments (except 0.01%-1 min and 0.01%-10 min) showed an increase in spearmint’s phenolic content and antioxidants at the end of storage (

Table 4. Effects of O. dubium EO and hydrosol application on fresh spearmint’s phenols, antioxidants (DPPH, FRAP), total flavonoids, and ascorbic acid (AA) content after being stored at 4 °C for 6 days.

	Time (min)	Concentration	Phenols (mg GEA/g)	DPPH (mg trolox/g)	FRAP (mg trolox/g)	Flavonoids (mg rutin/g)	AA (mg AA/100 g)
Day 0	0	0.00%	1.20 ± 0.03	2.99 ± 0.08	1.97 ± 0.15	1.58 ± 0.34	4.39 ± 0.15 *
EO	0	0.00%	2.43 ± 0.37 f	3.45 ± 1.11 e	3.49 ± 0.45 e	4.13 ± 0.07 de *	3.57 ± 0.11 d
	1	0.001%	7.82 ± 0.53 ab	6.99 ± 0.94 d	11.49 ± 1.29 bc	14.30 ± 1.28 b	3.63 ± 0.42 d
		0.01%	2.25 ± 0.48 f	2.78 ± 0.47 e	3.46 ± 0.69 e	2.89 ± 0.60 e	4.65 ± 0.23 c
		0.10%	7.87 ± 0.06 ab	9.87 ± 0.25 bc	12.94 ± 0.33 ab	14.53 ± 0.31 b	5.66 ± 0.20 a
	5	0.001%	8.78 ± 0.83 a	12.42 ± 1.32 a	14.29 ± 1.21 a	17.94 ± 1.69 a	4.53 ± 0.07 c
		0.01%	6.36 ± 0.25 bc	8.38 ± 0.42 bcd	9.03 ± 0.44 c	10.50 ± 0.42 c	5.52 ± 0.14 ab
		0.10%	5.71 ± 0.52 cd	7.65 ± 0.63 cd	9.04 ± 1.02 c	11.23 ± 1.62 c	4.82 ± 0.19 bc
	10	0.001%	8.18 ± 0.71 a	10.65 ± 0.82 ab	13.12 ± 1.08 ab	16.14 ± 1.05 ab	5.96 ± 0.21 a
		0.01%	2.88 ± 0.54 ef	7.23 ± 0.49 d	4.85 ± 0.40 de	3.41 ± 0.36 e	4.58 ± 0.28 bc
		0.10%	4.26 ± 0.62 de	4.41 ± 0.39 e	6.18 ± 0.33 d	6.68 ± 0.50 d	4.78 ± 0.38 bc
Hydrosol	0	0.00%	1.73 ± 0.48 e	3.46 ± 0.32 e	2.23 ± 0.14 f	1.65 ± 0.19 e	4.62 ± 0.26 b
	1	0.001%	4.15 ± 0.30 cd	3.22 ± 0.51 e	6.40 ± 0.61 e	6.32 ± 0.10 d	5.00 ± 0.23 b
		0.01%	5.71 ± 0.60 bc	6.97 ± 0.34 c	10.04 ± 0.99 cd	10.23 ± 1.58 c	5.38 ± 0.24 b
		0.10%	8.95 ± 0.95 a	14.10 ± 0.87 a	16.13 ± 2.19 a	18.85 ± 2.68 a	4.88 ± 0.31 b
	5	0.001%	7.20 ± 0.48 ab	9.94 ± 0.28 b	12.26 ± 0.44 bc	14.88 ± 0.60 b	5.03 ± 0.33 b
		0.01%	3.62 ± 0.43 d	3.52 ± 0.74 e	12.74 ± 0.90 bc	4.87 ± 0.60 de	5.02 ± 0.18 b
		0.10%	5.01 ± 0.32 cd	3.91 ± 0.40 de	7.78 ± 0.62 de	8.35 ± 0.64 cd	5.38 ± 0.21 b
	10	0.001%	3.15 ± 0.36 de	2.83 ± 0.59 e	5.02 ± 0.45 ef	4.59 ± 0.69 de	5.13 ± 0.45 b
		0.01%	7.84 ± 0.50 a	10.53 ± 0.91 b	14.06 ± 1.26 ab	16.73 ± 1.67 ab	7.43 ± 0.30 a
		0.10%	5.92 ± 1.04 bc	5.46 ± 0.74 cd	5.36 ± 0.80 e	5.48 ± 0.82 de	6.68 ± 0.30 a

Values are presented as the mean ± standard error (four biological replicates per treatment). The values for day 0 refer to the control (non-treated, 0.00%). Significant differences (p < 0.05) are indicated with different Latin letters for each treatment (EO and hydrosol, separately) on each column. An asterisk (*) indicates significant differences between the initial (day 0) and last day of storage of control (non-treated, 0.00%).

). The process of fresh produce senescing is linked with increased metabolism and plant defense mechanisms (i.e., antioxidants and antioxidant enzymatic activity) in an attempt to scavenge free radicals caused by oxidative stress [36]. EOs are complex mixtures of a plethora of bioactive compounds that present antioxidant activity, and at the same time, can initiate plant defense mechanisms such as the increased production of antioxidants [48]. A previous study showed an increase in antioxidants and flavonoids in lettuce with the application of thyme EO (dipping in 0.5, 1 and 1.5 g/L) [48]. However, increased concentrations and longer time of application of EOs could cause a decrease in antioxidants. This was observed in tomato and cucumber fruits where O. dubium EO was applied for up to 0.5% for 20 min [6]. As shown in Table 4, all applied hydrosol treatments (except 0.001%-10 min) were found to increase the phenolic content of spearmint after six days of storage at 4 °C. Hydrosols contain, in lower levels, some compounds that are encountered in their respective EOs and present antioxidant and biocidal activities [3]. This could have resulted in an enhancement in spearmint’s antioxidant content. It has been shown that application of EOs and hydrosols could result in an increase in the phenolic content and other antioxidant compounds of fresh produce and at the same time lead to the development of resistance against postharvest diseases [49,50]. Interestingly, the plant tissue could interpret the applied treatments (dipping and/or vapor) on fresh produce as stressors (i.e., abiotic stress), leading to the ignition of mechanisms for the elimination of the stressor factor, i.e., the increase in antioxidant levels among others [51,52].

The content of total flavonoids of herbs may differ during the storage and processing, and this could be also observed between genera and even species of the same genus [15]. Spearmint’s flavonoids content increased with all EO applied treatments apart from 0.01% (1 and 10 min) and 0.1%-10 min, whilst total flavonoids increased with all hydrosol applications (except 0.001%-10 min, 0.01%-5 min and 0.1%-10 min) (Table 4). O. dubium EO (i.e., 5 min applications, 0.001%-1 min, 0.1%-1 min and 0.001%-10 min) and hydrosol (i.e., 1 min applications, 0.001%-5 min, 0.1%-5 min and 0.01%-10 min) caused an increase in total flavonoids; however, higher concentrations and longer time of application did not (i.e., EO: 0.01% (1 and 10 min) and 0.1%-10 min; hydrosol: 0.1%-10 min, 0.01%-5 min, and 0.001%-10 min). Under stress conditions (i.e., oxidative stress), the total flavonoid content of fresh produce tends to increase during fresh produce storage, reaching a peak, and then decreases due to overstress [36,53,54]. This is also known as induced systemic resistance and it takes place as a response to biotic and abiotic stresses, and one of the main responses of this phenomenon is the increase in phenolic compounds levels (including flavonoids) [55]. The increase in flavonoids and other antioxidant compounds could be perceived as desirable as it means that more phytochemicals are present on spearmint; thus, the product´s nutritional value is also considered high.

Ascorbic acid, or vitamin C, is a water-soluble vitamin sensitive to heat and light. A great decrease in AA content is noticed in fresh produce during storage due to degradation. The AA content of spearmint in the present study increased at the end of storage (day 6) under all applied EO treatments (except 0.001%-1 min), highlighting the increased antioxidant capacity of the EO-treated spearmint. In addition, increased AA content was found with the application of hydrosol 0.01%-10 min and 0.1%-10 min (Table 4). It has been previously reported that a variation in mints’ (i.e., Mentha × piperita and Mentha spicata) AA content could be related to the different plant material, maturity levels, and/or even the analytical methods used for its determination [15]. Notably, losses of AA in fresh leafy greens stored at optimum temperature conditions (i.e., 0–2 °C) could be associated with stress induced during harvest, handling, and senescing [48]. A previous study showed a decrease in the AA content of rosemary bundles treated with rosemary EO (dipping at 1:1500 and 1:500 v/v for 10 min) after 12 days of storage at 4 °C. The differences might be attributed to the different plant material, the EOs applied, and/or the application conditions (concentration and time). Furthermore, O. dubium EO presents great antioxidant activity which could potentially result in an increase in AA content as a response to the possible abiotic stress caused by the treatment´s application [10].

The accumulation of free radicals and reactive oxygen species (such as H₂O₂) on leafy vegetables and other plant tissues is an indicator of stress and increased metabolism associated with sensory and quality deterioration (i.e., decrease in green color pigments, loss of aroma, and development of off-odors and off-flavors) [36]. During the present study, H₂O₂ levels were increased by all EO treatments except 0.001%-1 min, 0.1%-5 min, and 0.01%-10 min, whilst at the same time, the lowest H₂O₂ levels were found with the 0.1%-10 min treatment (

Table 5. Effects of O. dubium EO and hydrosol application on fresh spearmint’s damage indexes (H₂O₂ and MDA levels) after being stored at 4 °C for 6 days.

	Time (min)	Concentration	H2O2 (μmol/g)	MDA (nmol/g)
Day 0	0	0.00%	0.67 ± 0.03	6.22 ± 0.25
EO	0	0.00%	0.63 ± 0.04 d	6.72 ± 0.16 bcd
	1	0.001%	0.67 ± 0.07 cd	6.10 ± 0.79 bcd
		0.01%	1.01 ± 0.04 a	7.20 ± 0.50 abc
		0.10%	1.01 ± 0.02 a	8.97 ± 0.20 a
	5	0.001%	0.81 ± 0.01 b	7.77 ± 0.42 ab
		0.01%	0.78 ± 0.02 bc	7.60 ± 1.09 abc
		0.10%	0.68 ± 0.03 cd	6.87 ± 0.56 bcd
	10	0.001%	0.79 ± 0.05 bc	8.87 ± 0.51 a
		0.01%	0.56 ± 0.01 de	5.71 ± 0.57 cd
		0.10%	0.48 ± 0.05 e	5.02 ± 0.47 d
Hydrosol	0	0.00%	0.68 ± 0.02 d	5.22 ± 0.38 bcd
	1	0.001%	0.58 ± 0.00 d	4.67 ± 0.20 cd
		0.01%	1.13 ± 0.04 a	6.76 ± 0.65 ab
		0.10%	0.95 ± 0.04 b	6.45 ± 0.42 abc
	5	0.001%	0.69 ± 0.08 d	7.52 ± 1.34 a
		0.01%	0.62 ± 0.03 d	8.06 ± 0.25 a
		0.10%	0.62 ± 0.01 d	4.33 ± 0.09 d
	10	0.001%	0.65 ± 0.01 d	5.69 ± 0.52 bcd
		0.01%	0.84 ± 0.03 c	6.25 ± 0.33 abc
		0.10%	0.59 ± 0.01 d	5.51 ± 0.32 bcd

Values are presented as the mean ± standard error (four biological replicates per treatment). The values for day 0 refer to the control (non-treated, 0.00%). Significant differences (p < 0.05) are indicated with different Latin letters for each treatment (EO and hydrosol, separately) on each column.

). The decreased levels of H₂O₂ could be attributed to the increased AA content observed under the aforementioned treatments as AA plays an important part in the scavenging of H₂O₂ and the protection of protein and enzymes oxidation [56]. These findings further suggest the reduction in free radicals production and the maintenance of the plant cell wall integrity [57]. Hydrosol application at 0.01%-1 min resulted in increased H₂O₂ production, followed by 0.1%-1 min and 0.01%-10 min (1.13, 0.95, and 0.84 μmol/g, respectively), compared to other treatments. The application of rosemary EO 1:500 (v/v) for 10 min resulted in lower H₂O₂ levels on fresh rosemary stored for 12 days at 4 °C compared to 1:1500 (v/v) EO [19]. Our findings contradict the findings of the prior study, and this might be due to the different products applied (EOs), different produce used (rosemary versus spearmint), as well as the concentration and time of application. Moreover, it has previously been reported that the utilization of EO on fresh produce could result in the induction of produce defense mechanisms (i.e., activation of antioxidant enzymes) to fight oxidative stress [58]. Increased MDA levels were observed with EO application at 0.1%-1 min, 0.001%-10 min, 0.01%-1 min, and 0.01%-5 min (8.97, 8.87, 7.20, and 7.60 nmol/g) compared to 0.1%-10 min (5.02 nmol MDA/g). Moreover, 0.001%-5 min and 0.01%-5 min hydrosol were found to increase MDA levels on the last day of storage compared to other hydrosol treatments (except 0.01%-1 min, 0.1%-1 min, and 0.01%-10 min). In a previous study, rosemary EO (1:500 v/v) was found to decrease MDA levels in fresh rosemary after 12 days [19]. MDA is a secondary product of plant cell wall lipids´ peroxidation and an indication of oxidative stress. These results might be attributed to the antioxidant activities of the Eos; however, due to their volatile nature their effect might fade over time, leading to the increase in MDA values [58]. On the other hand, the increase in MDA levels might be the response of the plant tissue to the applied treatments (i.e., increased concentration and/or longer time of application).

3.4. In Vivo Antibacterial Activity

Fresh herbs, as with other leafy vegetables, can be contaminated and harbor foodborne pathogens (i.e., Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes) throughout the food chain (farm to fork) [59]. During postharvest processing of leafy vegetables, inadequate cleaning by washing could allow bacteria to remain on fresh produce surfaces and form biofilms [60,61]. Eco-friendly, natural means such as EOs are considered potential fresh produce sanitizers due to their strong antimicrobial activities [62,63]. Previous studies on O. dubium EOs and hydrosols suggested increased antioxidant and antimicrobial activities [6,10]. As shown on

Table 6. Effects of O. dubium EO and hydrosol in vivo application against S. enterica and L. monocytogenes (log cfu/g) inoculated on fresh spearmint stored at 4 °C for 6 days. The selected applications were as follows: EO Dose A (0.01% for 5 min), EO Dose B (0.001% for 10 min), hydrosol Dose A (0.1% for 5 min), and hydrosol Dose B (0.01% for 10 min).

	Concentration	S. enterica	L. monocytogenes
Day 1	Control	7.29 ± 0.11 a	6.76 ± 0.17 a
	Chlorine	5.52 ± 0.29 c	6.17 ± 0.05 b
	EO dose A	5.92 ± 0.13 bc	6.07 ± 0.18 bc
	EO dose B	5.95 ± 0.23 bc	5.69 ± 0.10 c
	Hydrosol A	6.07 ± 0.36 bc	6.27 ± 0.09 b
	Hydrosol B	6.49 ± 0.11 b	6.02 ± 0.19 bc
Day 6	Control	7.18 ± 0.08 a	7.40 ± 0.02 a
	Chlorine	5.50 ± 0.11 c	5.32 ± 0.12 d
	EO dose A	4.87 ± 0.10 d	5.90 ± 0.24 c
	EO dose B	5.73 ± 0.27 c	6.43 ± 0.07 b
	Hydrosol A	6.22 ± 0.16 b	6.44 ± 0.04 b
	Hydrosol B	6.45 ± 0.11 b	6.49 ± 0.04 b

Values are presented as the mean ± standard error (four biological replicates per treatment). Significant differences (p < 0.05) between the treatments are indicated with different Latin letters on each day.

, S. enterica numbers on spearmint leaves decreased after one day of storage at 4 °C with all applied treatments, and an especially significant decrease was found with chlorine (up to 1.77 log reduction) compared to hydrosol dose B (0.80 log reduction). At the end of storage, all applied treatments were found to decrease S. enterica population on fresh spearmint, with O. dubium EO dose A presenting greater decrease (2.31 log reduction). The EOs from oregano (O. vulgare) and rosemary (Salvia rosmarinus) alone and/or in combination showed great antibacterial activity against L. monocytogenes, S. Enteritidis, and E. coli inoculated on the surface of fresh leafy vegetables (pool of iceberg lettuce and chard) [7]. Moreover, up to a 3.2 log reduction in Salmonella enterica subsp. enterica serovar Newport on inoculated lettuce and spinach leaves was observed with oregano EO (tested concentrations: 0.1, 0.3, and 0.5% v/v) [64]. The main compound of oregano EO (both O. dubium and O. vulgare) is carvacrol, which is a molecule with significant antibacterial activity, suggesting that the antibacterial activity of oregano EO might be attributed to its presence in high levels [7,8]. Moreover, it has been suggested that EOs could cause injuries to bacterial cells that can eventually become lethal and result in their deaths after some time (i.e., days after exposure) [65]. This could explain the great activity of the EO found in our study.

Notably, the hydrosols´ composition differs from the respective EOs. Some similar compounds (contained in both EOs and hydrosols) might disappear due to the low pH (acidic to neutral pH) of the hydrosol and/or degrade, while others could only be found on hydrosols [9]. This might partially explain the great antibacterial activity of O. dubium hydrosol found in our study (pH: 4.14). Interestingly, S. cypria hydrosol presented a neutral pH value (pH: 7.51), and this could have resulted in the lower antibacterial activity compared to O. dubium (pH: 4.14) and S. fruticosa hydrosols (pH: 4.36). Similar to O. dubium hydrosol, the acidic pH of S. fruticosa (pH: 4.36) could have resulted to its antibacterial activity. Low pH values (below the optimum) in the environment of bacterial cells could lead to decreased enzymatic activity, affecting their survival [66]. A previous study mentioned that after 20 min of thyme and rosemary hydrosol treatment of apple fruits, a significant decrease in E. coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhymurium numbers was observed [67]. The same study also showed that thyme and rosemary hydrosols presented different magnitude of activity against the tested bacteria [67].

A decrease in L. monocytogenes population was observed during the first day of storage with all applied treatments, whereas EO dose B resulted in a greater bacterial decrease than control, chlorine, and hydrosol dose A. At the end of storage, a greater decrease in L. monocytogenes numbers was found with chlorine followed by EO dose A (Table 6). The attachment and growth of L. monocytogenes on fresh produce surfaces depends on surface properties such as topography, surrounding microflora, moisture, and nutrient availability [68]. Even though L. monocytogenes bacterial cells prefer to attach and grow on cut leafy surfaces (higher nutrient availability on cut sides), the possibility of them establishing and growing on intact surfaces is not zero [68]. Foodborne pathogens can survive and proliferate on leaf stomata and other surface niches (crevices and spaces), making sanitizing agents less effective [62,68].

The antimicrobial activity of EOs and hydrosols on bacterial cells could vary according to the bacterial strain, outer cell wall composition (Gram− and Gram+), shape, and produce surface properties, among other factors [1,31,68]. It is well known that EOs are rich in alcohols, terpenes, and ketones, which present great antibacterial activity (especially against Gram-positive bacteria) [1]. Molecules such as carvacrol, 1,8-cineol, carvone, limonene, α-pinene, and β-pinene, alone and in a complex mixture (i.e., EOs), presented antibacterial activities by many different mechanisms and actions, including disruption and reduction in cell wall membrane potential, interruption of proton and ion pumps, changes in cell permeability (damage of the lipid bilayer), intracellular constituents leakage, inhibition of cell metabolism, and interference with enzymatic activities [63,69]. In general, Gram-positive bacteria such as L. monocytogenes are more susceptible to the activities of EOs due to the lack of the outer double lipid layer that Gram-negative bacteria (i.e., S. enterica) possess [1,31], as was evidenced in the present study. These findings are further supported by the observations of another study where O. dubium EO showed greater antibacterial activity against Gram-positive (i.e., Clavibacter michiganensis subsp. michiganensis) seedborne pathogens compared to Gram-negative (i.e., Xanthomonas axonopodis pv. vesicatoria, Xanthomonas axonopodis pv. phaseoli, Xanthomonas axonopodis pv. phaseoli var. fuscans, and Pseudomonas syringae pv. tomato) [32]. In addition to this, Karioti et al. also mentioned significant susceptibility to O. dubium EO of S. aureus, Micrococcus luteus, Sarcina lutea, and Bacillus cereus (Gram-positive bacteria) compared to E. coli, Proteus mirabilis, Agrobacterium tumefaciens, Pseudomonas aeruginosa, Pseudomonas talassi, and S. Enteritidis (Gram-negative bacteria) [10].

It seems that EO and hydrosol from O. dubium could control and reduce the population of S. enterica and L. monocytogenes on fresh spearmint. It is worth mentioning that the EO and hydrosol concentrations selected from the preliminary screening of this study and applied on fresh spearmint (i.e., for EO: 0.01% and 0.001%) were lower than the concentrations that showed antibacterial activity (MIC and LD₉₀) in vitro. For example, the MIC for EO was 0.16% and 1.25% for S. enterica and L. monocytogenes, respectively, compared to that of EO of 0.01%. The MIC value of an antimicrobial agent is the observable minimum concentration that prevents visible bacterial growth and is not a strict minimum value, and thus it cannot be compared with an in vivo-applied concentration [70]. Indeed, a previous study on fruit juice mentioned that concentrations of eucalyptus EO lower than the MIC (2.25 and 1.12 mg/mL) found during the in vitro evaluation (4.5 mg/mL) presented a significant reduction in Saccharomyces cerevisiae [71].

When using EOs, hydrosols, and other natural products, one has to keep in mind that different bacterial strains and/or even serotypes might present different viabilities against EOs and other natural compounds´ action [31]. Increasing the time of application of natural products such as EOs and hydrosols could evidently decrease the bacterial numbers but, at the same time, might negatively affect fresh produce quality attributes and/or even lead to the appearance of phytotoxicity [6,67]. Thus, caution should be taken when using such products, as both quality (indicated by marketability, nutritive value, etc.) and safety (indicated by antimicrobial action) attributes need to be closely observed simultaneously.

4. Conclusions

In the present study, Origanum dubium, Salvia fruticosa, and Sideritis cypria were evaluated for their antibacterial activity against L. monocytogenes, S. aureus, E. coli, and S. enterica. Among the examined EOs and hydrosols from Cypriot MAP, O. dubium EO and hydrosol presented the greatest antibacterial activity in vitro against the tested foodborne pathogens. When applied on fresh spearmint, O. dubium EO was found to result in less-acceptable produce compared to hydrosol treatment, which led to a more marketable and preferable product. The antioxidant capacity of spearmint increased with the applied treatments, and a similar trend was observed for phenols, flavonoids, and ascorbic acid. From this study, it was found that both natural products examined were able to significantly reduce the bacterial populations of S. enterica and L. monocytogenes inoculated on fresh spearmint (up to 2.31 log reduction). Thus, it can be concluded that O. dubium EO and hydrosol could serve as potential sanitation means for fresh spearmint. However, more research is required to investigate the effects on this and other produce, the dose–response effects with produce quality attributes, and the possible phytotoxic effects that might appear.