Reversible Electroporation and Post-Electroporation Resting of Thai Basil Leaves Prior to Convective and Vacuum Drying

Abstract

Pretreatment by reversible electroporation followed by resting (storage under saturated moisture at 21 ± 2 °C) was evaluated for modification of the properties of dried and rehydrated Thai basil leaves. The treated leaves were dried by convection at 40 °C or in a vacuum at room temperature. The results showed that vacuum drying provoked more cell damage and tissue collapse than convective air drying at a moisture ratio (MR) of 0.2 and 0.1. Under this level of MR, the pulsed electric field (PEF) and resting pretreatment exerts a protective effect of the tissue for both drying methods. However, under complete dehydration (water activity, aw = 0.05) damage seems to be similar for both drying methods despite the PEF pretreatment. Remarkably, reversible electroporation followed by resting resulted in higher trichome preservation. At MR of 0.05, the area of trichomes on the surface of convective-dried, PEF-rested and fresh samples were not statistically different at 2267 ± 89 µm² and 2218 ± 65 µm², respectively, showing that this pretreatment still exerts a protective effect on trichomes when complete dehydration is achieved.

1. Introduction

A pulsed electric field (PEF) has been used as pretreatment to increase the rate of mass transfer during drying of foodstuffs such as vegetables, meat, fruit, and herbs [1,2,3,4,5,6,7,8,9,10,11]. However, the majority of these PEF applications were designed to cause irreversible electroporation of cells, which would greatly increase mass transfer but result in numerous changes in food quality, including aroma, color, and texture [4]. Irreversibly electroporated sweet basil leaves appear to lose their aroma and color significantly when compared to untreated and reversibly electroporated samples upon drying in air at 50 °C [4].

By inducing electroporation on guard cells of the stomata and, at the same time, keep the rest of the cells viable, reversible electroporation could be used to improve the drying of plant leaves [4]. The electroporated guard cells cause long-term stomatal opening, which aids in the drying process [12]. Telfser and Gómez Galindo [12] evaluated the effects of reversible electroporation on the structure, rehydration capacity, color, and sensory quality of basil leaves dried using convective drying at 40 °C, vacuum drying, and freeze-drying. The authors found that reversible electroporation causing stomatal opening of sweet basil leaves reduced the drying time in all studied drying methods, and PEF resulted in better preservation of the leaf structure when used prior to convective and vacuum drying, as compared to the untreated control. In a previous paper, Thamkaew et al. [13] reported that reversible electroporation followed by resting (storage under saturated moisture at 21 ± 2 °C) allows survival of cells in Thai basil leaves at certain levels of dehydration (aw > 0.6). Resting after PEF and prior to drying allowed the cells to establish protective mechanisms in response to the temporary loss of metabolic homeostasis caused by the electroporation process. Maintenance of cell integrity after rehydration would allow keeping the fresh characteristics of the product.

Structural integrity of the epidermal and cuticle layers of herbs may be an important aspect for the preservation of aromatic volatile compounds during drying, mainly regarding the integrity of trichomes that produce and accumulate terpenoid oils [14]. The integrity of the trichomes is strongly influenced by the drying conditions and the drying method in lemon verbena [15]. In basil, vacuum drying resulted in a better preservation of the integrity of trichomes than hot air drying at 40 °C [12]. Vacuum drying is suitable for heat-sensitive food materials [16], having the advantage of low drying temperatures and time, improving the preservation of color [16].

In the present investigation, the advantages of reversible permeabilization and post-electroporation resting documented by Thamkaew et al. [17] at MR of 0.2 and 0.1 on cell preservation were tested for complete dehydration (aw = 0.05) using both air drying at 40 °C and vacuum drying at room temperature. High-resolution optical microscopy with digital 3D surface reconstruction was used as a non-invasive and non-destructive method for evaluating the integrity of trichomes in the leaves after dehydration and rehydration. Non-destructive methods are especially relevant when trichome damage is the investigation’s primary goal. Other strategies for trichome evaluation such as chemical fixation, scanning electron microscopy, and transmission electron microscopy require extensive sample preparation processes, which may significantly affect the trichomes [18]. We aim at comparing the properties of the dried and rehydrated products using reversible PEF and resting prior to dehydration.

2. Materials and Methods

Figure 1 summarizes the complete methodology used in this investigation

2.1. Thai Basil Leaves Handling

Before being transferred to our lab, potted Thai basil (O. basilicum cv. thyrsiflora) was cultivated for 28 days at a local grower’s greenhouse. To avoid sugar starvation, the plants were placed under LED growth lamps with a light intensity of 200 µmol m⁻² s⁻¹ for 16 h/day at 21 ± 2 °C for 2–5 days before experimentation. Leaves measuring 2.5 ± 0.2 cm × 3.5 ± 0.3 cm in length and weighing 0.18 ± 0.05 g were harvested and examined within 15 min. To prevent moisture loss, the leaves were kept in a closed plastic container with wet tissue on the bottom prior to experimentation.

2.2. Electrical Treatment

The PEF treatment was performed on three leaves, which were placed together in an electroporation chamber. The stainless-steel chamber, with a 0.5 cm space between electrodes, was filled with 50 mL of NaCl solution with a conductivity of 130 µS/cm to cover the electrodes and leaves. The solution to sample mass ratio was 100 to 1. For PEF treatments, the chamber was connected to a pulse generator (ADITUS AB, Lund, Sweden). The PEF protocol, established in [13], was used: 200 monopolar, rectangular pulses with a 50 µs pulse duration, 760 µs space between pulses, and a nominal field strength of 650 V/cm (specific energy input of 4.81 kJ/kg). This PEF protocol (optimized with microscopy observations of propidium iodide fluorescence and fluorescein diacetate vital staining of cells) was found to cause reversible electroporation of epidermal cells in Thai basil leaves and cause the opening of stomatal guard cells [13]. The temperature did not significantly increase during the PEF treatments. The treated samples were washed with distilled water and absorbent paper was used to remove excess salt solution. The PEF procedure was performed 11 times to obtain sufficient treated leaves for the drying experiment.

2.3. Resting

After the PEF treatment, the leaves were stored at room temperature (21 ± 2 °C) for 24 h in the dark in an air-sealed container with wet tissue on the bottom (“PEF-rested” leaves). Untreated leaves (“control-rested”) were stored under the same conditions in another container. Fresh leaves (“control”) were harvested and dried without resting. The containers were placed under the growth lamps for two hours before drying.

2.4. Drying

2.4.1. Convective Drying

Thai basil leaf samples were dried at 40 °C with an air flow of 3 m/s in a convective dryer built at Lund University. Each batch of leaves was evenly distributed on a metal drying tray (23 × 33 cm, wire mesh with 5 × 5 mm square holes) without overlapping. The sample load was 0.076 kg/m². The tray was placed on a scale connected to a software (RS232 Monitor, EVM Software) that continuously recorded the weight loss. The drying time was determined by the treatment and the final moisture ratio (MR). For each treatment: “PEF-rested”, “control-rested” and “control”, three drying procedures were carried out for each experimental condition and for each MR level (MR of 0.05, 0.1, and 0.2), for a total of 18 drying procedures.

2.4.2. Vacuum Drying

Thai basil leaf samples were dried in a Gallenkamp vacuum oven (Fistreem international Ltd., Leicestershire, UK) at ambient temperature (21 ± 2 °C). The chamber was set to a vacuum pressure of 13 Pa. The leaf samples were arranged on the metal tray in a manner similar to that for convective drying. In preliminary experiments, the drying times were established with the goal of producing samples with similar water activity and MR to air-dried samples. Three drying procedures were carried out for each experimental condition and for each MR level (MR of 0.05, 0.1 and 0.2), for a total of 18 drying procedures.

2.5. Analysis

2.5.1. Moisture Ratio

The Page model [19] was used to calculate the MR of the samples during drying. The equilibrium moisture content was assumed to be negligible. The MR was calculated using the following equation:

$$\mathrm{MR}=\frac{M_t-M_{\mathrm{e}}}{M_0-M_{\mathrm{e}}}=\frac{M_t}{M_0}=\exp \left(-k{t}^n\right)$$

(1)

where MR is the dimensionless moisture ratio, M_t is the moisture content at any time (kg water/kg dry mass), M_e is the equilibrium moisture content (kg water/kg dry mass), M₀ is the initial moisture content (kg water/kg dry mass), k is the drying rate constant (min⁻¹), t is the drying time (min), and n is the empirical constant.

2.5.2. Moisture Content and Water Activity

Moisture content was determined using the AOAC method [20], by placing 2 g of samples at 105 °C for 24 h in an air convection oven (AB Termo-Glas, Gothenburg, Sweden). The water activity of the leaf samples (2 g) was measured at room temperature (20 °C) using an Aqualab water activity analyzer (Model CX-2, Decagon devices Inc., Pullman, WA, USA). Measurements of each experimental condition were repeated three times.

2.5.3. Rehydration

The rehydration capacity was determined according to Thamkaew et al. [17]. Each dried leaf was weighed individually before being placed in a 50 mL plastic tube (2.9 cm in diameter and 11.4 cm in length) filled with 19 mL distilled water and kept at room temperature (approximately 100:1 water-to-leaf ratio). Leaf samples were taken from each tube every hour, and the excess water on the leaf surface was gently removed with tissue. Then, the leaf was weighed and returned to the tube. This procedure was repeated until the leaf weight remained constant. The experiment was done in triplicate.

To ensure that every leaf sample was rehydrated to its maximal rehydration capacity and for the same period of time, the longest rehydration time among all treatments was used as a rehydration time for subsequent experiments.

2.5.4. Conductivity

The electric conductivity of the rehydration water (leaked ions from the samples) was measured with a conductometer (Orion Research Inc., Jacksonville, FL, USA) at 21 °C after rehydration was completed. The leaf samples were returned to their rehydration tubes until the photosynthesis and respiration tests were performed (not longer than 30 min). The conductivity of the samples was compared to the conductivity of leaves that were damaged by frozen at –18 °C for 30 min and thawed at room temperature for 1 h.

2.5.5. Photosynthesis and Respiration

The photosynthesis and respiration rates of leaf samples were determined as described in Panarese et al. [21] with some modifications. The leaf samples were stored in the dark for 20 min prior to the measurement. Measurements were performed at 20 °C with an oxygen electrode (S1 O₂ electrode, Hansatech, Norfolk, UK) with a thermostated electrode chamber (LD2/3, Gas-Phase Oxygen Electrode Chamber) and an integrated light source providing 380 µmol m⁻² s⁻¹ (LS3 Computer Controlled UV Light Source, Hansatech, Norfolk, UK). The samples were cut into 3.5 cm-diameter discs and placed on a fabric plate saturated in a pH 9 bicarbonate buffer. The experiment started with a 10-min dark respiration (light turned off) measurement, followed by a 10-min photosynthesis measurement (light on). Seven rehydrated leaves were randomly selected from each drying procedure and their photosynthesis and respiration rates were determined.

2.5.6. Color

The colors of fresh and rehydrated Thai basil leaf samples for every treatment were determined using a portable spectrophotometer (CM-700d, Konica Minolta, Konica Minolta Sensing Europe B.V, Nieuwegein, Netherlands.) with 10° standard observer and D65 light source with white plate calibration. An 8 mm width MAV target mask, which is suitable for color measurement of surfaces with uneven color, was used. The measurement was performed perpendicularly to the sample and avoided the main vein on the leaves. Five measurements were performed on each sample. Numerical values of L*, a*, and b* CIELAB color space were used to obtain the total color change (ΔE, Equation (2)), which is a measurement of the color difference between fresh (f) and rehydrated (r) basil leaves.

$$\Delta \mathrm{E}=\sqrt{{\left(L_r^{\ast }-L_f^{\ast }\right)}^2+{\left(a_r^{\ast }-a_f^{\ast }\right)}^2+{\left(b_r^{\ast }-b_f^{\ast }\right)}^2}$$

(2)

2.5.7. High-Resolution Optical Microscopy

Trichomes on the surface of Thai basil leaf samples dried with both methods to a MR of 0.05 and rehydrated to its maximum rehydration capacity were examined using a high-resolution optical digital microscope Keyence VHX-6000 (Keyence international (Belgium) NV/SA, Mechelen, Belgium) equipped with a diffuse light illumination. Peripheral full-ring illumination was used along with digital glare-removal image processing. Micrographs were taken at 100× magnification (for trichome counts), 300× magnification (for surface quality investigation), and 700× magnification (for the measurement of area of the trichome). In a 2 mm² area (see Figure 2) of 10 leaves, the area covered by three individual trichomes was measured using tools integration in the microscope control software. In this way, a total of 30 trichomes were investigated for each treatment. The area covered by a trichome was calculated by drawing a baseline between each edge at the base of the trichomes in the 3D depth profile obtained from the software’s “3D depth composition” function.

2.5.8. Statistical Analysis

SPSS (v.26.0, IBM Corp., Armonk, NY, USA) was used to determine statistical significance (One-way ANOVA) at a significance level of 0.05. Tukey-HSD tests were used to conduct post-hoc analyses.

3. Results

3.1. Drying Time, Moisture Ratio and Water Activity

Table 1. Experimental drying time, moisture ratio (MR), and water activity (aw) of Thai basil leaves samples treated with different treatments (control, control-rested, and PEF-rested). Data are shown as average ± standard deviation of the mean for n = 3 and different letters denote significant differences at p < 0.05.

Treatments	Drying Methods	Experimental Drying Time (min)	Relative Drying Time Compare to Control	Target MR	Experimental MR	aw *
Control	CD	62	Control (1.00)	0.2	0.191 ± 0.027 a	0.841 ± 0.034 a
Control-rested	CD	92	1.48	0.2	0.194 ± 0.018 a	0.864 ± 0.017 a
PEF-rested	CD	49	0.79	0.2	0.212 ± 0.019 a	0.823 ± 0.034 a
Control	CD	123	Control (1.00)	0.1	0.113 ± 0.008 b	0.614 ± 0.013 b
Control-rested	CD	205	1.66	0.1	0.091 ± 0.013 b	0.605 ± 0.013 b
PEF-rested	CD	95	0.77	0.1	0.103 ± 0.018 b	0.612 ± 0.016 b
Control	CD	204	Control (1.00)	0.05	0.049 ± 0.008 c	0.465 ± 0.040 c
Control-rested	CD	368	1.81	0.05	0.046 ± 0.004 c	0.512 ± 0.054 c
PEF-rested	CD	154	0.76	0.05	0.052 ± 0.005 c	0.478 ± 0.035 c
Control	VD	110	Control (1.00)	0.2	0.185 ± 0.006 a	0.807 ± 0.004 a
Control-rested	VD	60	0.55	0.2	0.204 ± 0.024 a	0.849 ± 0.040 a
PEF-rested	VD	40	0.36	0.2	0.202 ± 0.030 a	0.811 ± 0.008 a
Control	VD	390	Control (1.00)	0.1	0.094 ± 0.007 b	0.630 ± 0.031 b
Control-rested	VD	110	0.28	0.1	0.109 ± 0.018 b	0.617 ± 0.030 b
PEF-rested	VD	90	0.23	0.1	0.109 ± 0.014 b	0.604 ± 0.010 b
Control	VD	672	Control (1.00)	0.05	0.045 ± 0.004 c	0.494 ± 0.049 c
Control-rested	VD	652	0.97	0.05	0.049 ± 0.006 c	0.509 ± 0.043 c
PEF-rested	VD	592	0.88	0.05	0.054 ± 0.005 c	0.491 ± 0.046 c

* In each column, a different letter superscript represents statistically significant differences (p < 0.05).

reports the experimental MR and water activity of Thai basil leaf samples that were dried to the target MR of 0.2, 0.1, and 0.05. Regardless of the pre-drying treatment, the water activity of the samples dried to the same MR were similar. For convective drying (CD), control-rested samples took longer to dry than control samples (relative drying time higher than 1), whereas PEF-rested samples took less time to dry than control samples (relative drying time less than 1) in all MR levels. Rested samples (both control and PEF-treated) required less vacuum drying (VR) time than control samples at all MR levels. In both convective and vacuum drying, the PEF-rested samples had a faster drying time than controls. The experimental MR of different samples in each target MR levels was not statistically different (p < 0.05).

3.2. Rehydration Capacity

Table 2. The maximum rehydration capacity (RC) obtained after 18 h rehydration of Thai basil leaf samples subjected to different pretreatments and dried by convective drying (CD) and vacuum drying (VD) to MR of 0.2, 0.1, and 0.05. Data are shown as average ± standard deviation of the mean for n = 3.

Pretreatments	Drying Methods	RC (kg Water/kg Dry Matter) *
		MR = 0.20	MR = 0.10	MR = 0.05
Control	CD	8.05 ± 0.33 b	4.50 ± 0.20 a	2.81 ± 0.39 a
Control-rested	CD	9.17 ± 1.99 bc	5.35 ± 0.05 b	3.52 ± 0.51 a
PEF-rested	CD	11.6 ± 0.78 c	6.28 ±0.29 c	3.59 ± 0.57 a
Control	VD	6.33 ± 0.07 a	4.22 ± 0.22 a	2.79 ± 0.61 a
Control-rested	VD	7.59 ± 0.99 b	4.21 ± 0.07 a	3.58 ± 0.39 a
PEF-rested	VD	7.61 ± 0.19 b	4.24 ± 0.12 a	3.43 ± 0.58 a

* In each column, a different letter superscript indicates statistically significant differences (p < 0.05).

reports the maximum rehydration capacity (RC) of leaf samples dried to the moisture ratios of 0.2, 0.1, and 0.05 using CD and VD. At MR of 0.2 and 0.1, the convective dried, rested samples (control and PEF-treated) show the highest RC. The rehydration capacity of PEF-rested samples convective dried to the MR of 0.2 was significantly higher than control and control-rested samples (p < 0.05). The effect of pretreatments on RC was lost upon complete dehydration (MR = 0.05). Among the vacuum-dried samples, resting shows to influence RC only at MR of 0.2. Figure 3 shows the rehydration curves of PEF-rested samples (treatment with the highest RC) dried to MR levels of 0.2, 0.1, and 0.05. At the MR of 0.2 and 0.1, CD samples reached the maximum rehydration capacity faster than VD samples. There were no differences in the rehydration time to the maximum rehydration capacity of the samples at MR of 0.05 for either drying procedures.

3.3. Ion Release during Rehydration

Ions released during the rehydration process increases the electrical conductivity of the rehydration water. Conductivity results for each pretreatment, drying method, and moisture ratios are shown in Figure 4A–C. The electrical conductivity of samples that were frozen and thawed was 128.70 ± 3.29 µS/cm. Samples that were convectively dried had lower electrical conductivity than those that were vacuum dried for moisture ratios of 0.2 and 0.1 (p < 0.05). At these levels of MR, the PEF-rested, convective dried samples showed the lowest values of conductivity (p < 0.05) (50.8 ± 3.3 and 84.9 ± 5.4 for the MR of 0.2 and 0.1, respectively), whereas the conductivity of the vacuum dried samples (113.5 ± 3.1 and 134.40 ± 2.6 for the MR of 0.2 and 0.1, respectively) were equal or similar to that of the sample that was frozen and thawed. There was no difference in conductivity between the samples after drying to an MR level of 0.05 (Figure 4C).

3.4. Photosynthesis and Respiration

In comparison to convective dried samples, vacuum dried samples at MR 0.2 had lower oxygen generation and consumption rates (p < 0.05) (Figure 5). The photosynthesis and respiration of fresh Thai basil leaves were 16.72 ± 1.72 and 6.69 ± 1.44 µmol O₂/min/cm², respectively. At the MR of 0.1, convective dried samples did not show any photosynthesis capability (p < 0.05), whereas vacuum dried, rested samples (both control and PEF) showed a slight photosynthesis activity at these MR levels (in the range of 0.48–1.61% of that of the fresh sample). At this level of MR, the oxygen consumption rate of the PEF-rested, convective dried samples was significantly higher than that of the vacuum dried samples (p < 0.05). At MR of 0.05, neither respiration nor photosynthesis were detectable, irrespective of the drying method used.

3.5. Vacuum Drying Increased Cell Death

When comparing the properties of the vacuum dried and convective dried samples dried to MR 0.2 and 0.1, it can be seen that the vacuum dried samples have a lower rehydration capacity (Table 2), higher release of ions to the rehydration water (Figure 4) and lower respiration and photosynthesis capacities than convective dried samples (Figure 5) for all pretreatments, demonstrating that vacuum drying caused more tissue collapse and damage to the cells, leading to increased cell death.

3.6. Leaf Color

The total color change of rehydrated Thai basil leaf samples for all pretreatments and drying methods are shown in Figure 6. The color change was significantly higher when convective drying was used at every level of MR (p < 0.05). Dehydrating from MR 0.2 to MR 0.1 did not have significant effect on color changes. However, complete dehydration to MR 0.05 significantly increased the color change for both drying methods (p < 0.05).

3.7. Trichome Structure

The number of trichomes on the leaf surface was between 5–7 trichomes/2 mm² area of the leaf surface (square area, Figure 7A). Figure 7B–H shows representative micrographs of trichomes on the surface of Thai basil leaf samples at a magnification of 300 times. Fresh samples (Figure 7B) showed a more intact structure than rehydrated samples (Figure 7B–H). PEF-rested samples had less collapsed surface in both convective (Figure 7E) and vacuum drying (Figure 7F) than the rehydrated control and control-rested treated samples (Figure 7G,H).

When examined under a magnification of 700 times, intact trichomes can be generally seen as expanded structures sticking out from the surface of the leaves (Figure 8A). Partially collapsed trichomes were also found on the fresh samples (Figure 8B), but in very small numbers (less than 5%). Rehydrated samples showed different kind of trichomes: intact (Figure 8C), partially collapsed (Figure 8D), and collapsed trichomes (Figure 8E). Intact trichomes were seen more in fresh samples than in the rehydrated samples. When the area covered by a trichome was measured (Figure 8F), the area in fresh samples and PEF-rested, convective dried samples were found to be the largest (

Table 3. Microscopic evaluation of trichome areas of Thai basil leaves subjected to different pretreatments prior to drying with convective air (CD) at 40 °C or under vacuum (VC). The samples were dried to an MR of 0.05. Data are shown as average ± standard deviation of the mean for n = 3.

Samples	Drying Method	Partially Collapsed Trichomes (%)	Collapsed Trichomes (%)	Area of Trichomes (µm2)
Fresh	CD	3 ± 2 a	0 ± 0 a	2267 ± 89 a
Control	CD	33 ± 5 b	18 ± 4 cd	1204 ± 133 cd
Control-rested	CD	27 ± 3 c	19 ± 5 cd	1001 ± 115 cde
PEF-rested	CD	20 ± 4 d	5 ± 3 b	2218 ± 65 a
Control	VD	32 ± 5 b	23 ± 3 d	727 ± 80 e
Control-rested	VD	29 ± 4 c	15 ± 3 c	827 ± 102 de
PEF-rested	VD	27 ± 5 c	7 ± 3 b	1785 ± 76 b

In each column, a different letter superscript indicates statistically significant differences (p < 0.05).

). In both convective and vacuum drying, the percentage of collapsed trichomes in PEF-rested samples was significantly lower than in control and control-rested samples (p < 0.05). In these samples, the area of partially collapsed trichomes were between 78–97% of the intact trichomes.

Remarkably, the PEF-rested samples dried with either method showed more intact trichomes than the control-rested samples and the area covered by each trichome was similar to that in fresh leaves (Table 3).

4. Discussion

Reversible electroporation can reduce the drying time of Thai basil leaves when used as pretreatment for both convective and vacuum drying (Table 1). The faster drying rate induced by the reversible electroporation and guard cells electroporation was previously reported by Kwao et al. [4], where the drying time for basil dried at 50 °C was reported to be reduced 37% in comparison with the control. Telfser and Gómez Galindo [12], drying basil at 40 °C, reduced the drying time by 57%. In this investigation, the PEF-treated samples were allowed to rest for 24 h in darkness before drying. In both convective and vacuum drying, the PEF-rested samples had a faster drying time than controls (21–77%). This result can be explained by the stomatal opening caused by guard cell electroporation, which persists during the 24 h resting period, as described by Thamkaew et al. [17].

Under the study conditions, the higher cell and tissue damage caused by vacuum drying in comparison with air drying can be explained by the different mechanisms by which water is removed from the tissue. In convective drying, a constant drying rate occurs as the food’s internal moisture migrates outward to the surface at the same mass transportation rate as the moisture evaporation at the food’s surface. This allows the internal moisture to remain liquid until the surface moisture has mostly evaporated [16]. As a result, the main damage to the cells may be caused by the heat of the drying process, in which the protective effect of resting may be able to protect the cells at certain levels of dehydration. Vacuum drying, on the other hand, exposes food tissues to a very low vapor pressure, resulting in an extreme vapor pressure difference between the food tissues and the atmosphere, which lowers the boiling point of the moisture in the food tissues [16]. As a result, the internal moisture of food tissues evaporates directly as vapor from the food structure.

However, the better preservation of color obtained by vacuum drying (Figure 6) underlines the damaging effect of heating on quality preservation, as it provokes the degradation of chlorophyl (for a review see Thamkaew et al. [22]) Therefore, vacuum dried samples show higher cell damage but better color preservation than air-dried samples.

Our results also show that the effect of resting after PEF treatment on protecting the cells upon certain levels of dehydration (Table 2, Figure 3 and Figure 4) is limited to high water activities and could not be seen upon complete dehydration (Figure 4). However, the more intact trichomes present on the PEF-rested samples (Table 3) is remarkable for the effect of reversible permeabilization on these tissue structures. This is a surprising result, as it might be expected that collapse and cell damage would occur at every level of the tissue upon complete dehydration. Our hypothesis is that resting after reversible PEF treatment still exerts a protective effect on the leaf surface, including the trichomes.

PEF-induced stress may act as an abiotic stress, inducing metabolic responses capable of protecting cells or tissue structures upon further stress [23,24,25], such as in this case of drying. Abiotic stress conditions, such as UV irradiation, have been reported to increase the concentration of polyphenols, including lignin deposition in sharp-headed trichomes [26]. Leaf trichomes play a crucial protective role against several biotic and abiotic plant stress factors [27] as they are directly exposed to the surroundings, often first encountering challenging environmental conditions [28]. Therefore, trichomes can act as a component of physical defense against stress, and their morphology and secretory properties may adapt accordingly. PEF-induced morphological or structural changes on trichomes is an interesting issue for further research as trichomes contain the most characteristic aroma compounds of basil leaves, which may also be better preserved, or even increased, during the time of resting as a consequence of PEF-induced tissue perturbation [29].

5. Conclusions

The following are our primary findings:

• Under the studied drying conditions, vacuum drying provoked more cell damage and tissue collapse than convective air drying at MR of 0.2 and 0.1. Under complete dehydration (aw = 0.05) damage seemed to be similar for both drying methods irrespective of if the leaves were PEF pretreated or not.
• The protective effect of resting after reversible PEF application on metabolic and ionic integrity was only detected at high water activities and suppressed upon full dehydration (aw = 0.5).
• Samples dried under vacuum showed less color degradation upon rehydration when compared to convective dried samples.
• When dried with either convective or vacuum drying, reversible electroporation followed by resting resulted in higher trichome preservation than in samples that were not PEF-treated. When the PEF-rested treatment was combined with convective drying, the area of the trichomes was found to be similar to that of the fresh leaf sample.