High-temperature Thin-layer Drying Kinetic of Cultivated and Wild Algerian Olive Leaves Modeling and Effect on Oleuropein and Chlorophyll Contents

Olive leaves (OLs) are well known for being rich in oleuropein, their main bioactive molecule which has recently been attracting great interest from the scientific community due to its antiviral properties, including Covid-19 disease. Furthermore, the high-temperature/ short-time drying process has found applications for various plants and food processing, which might be also implemented for the drying of OLs. This study focuses on: 1. the mathematical modeling of thin-layer high-temperature-drying (HTD) kinetic of olive ( var. Chemlal and Oleaster ) leaves, and 2. the determination of HTD effect on some physicochemical properties (oleuropein, chlorophylls, and CIELab color parameters) of the dried olive leaves (DOLs). For this, four drying temperatures (100, 120, 140, and 160 °C) were applied. For comparison purposes, low-temperature DOL samples were also prepared. The obtained data have shown that among the tens tested mathematical models, the Midilli et al. model describes more correctly experimental data for all drying temperatures and for both olive leaf varieties ( R 2 = 0.9960, SEE = 0.0085, RMSE = 0.0165 and χ 2 = 0.0006). Moreover, the results show that the HTD at 120 and 160 °C does not differ from freeze-drying in terms of oleuropein retention ( p < 0.05), highlighting the technological interest in the high-temperature/short-time drying process. Considering the biological value of oleuropein, in particular its antiviral activity, the study deserves further investigation in order to elucidate certain questions such as the storability of DOLs, and their valorization as fortification ingredient in food and pharmaceutical formulations, evaluation in vitro of their biological activities, etc.


Introduction
Olive trees (Olea europaea) are widespread in the countries of the Mediterranean basin: Algeria, Tunisia, Spain, Italy, Greece, and others. Depending on the variety, their fruits (olives) can be consumed as it is after debittering [1][2][3], and/or used for oil extraction [4,5]. Concerning olive oil, it is well known to be the backbone of the Mediterranean diet [6][7][8].
The olive leaves (OLs) represent an important biomass generally used for animal feeding or burnt periodically on the fields. However, many scientific studies reveal their high richness in bioactive compounds known for their antioxidant, antimicrobial and antiviral properties, among others: oleuropein and derivatives (hydroxytyrosol, dimethyl-oleuropein, dihydro-oleuropéine, etc.), verbascoside, catechin, and rutin. All these considerations make the use of OLs very promising for food, pharmaceutical, and cosmetic applications [9][10][11][12]. In traditional medicine, they have been used since antiquity for the treatment of various physiological dysfunctions of the human body, such as fever, hypertension, malaria, diabetes, etc. [13][14][15][16].
With the advent of the Covid-19 pandemic, consumers, as well as scientists, have shown an unprecedented interest in medicinal plants, especially those with antiviral properties. In this context, oleuropein (oleuropeosides), the main compound of the olive leaves (but also present in the fruit and oil but in smaller quantities) with a concentration of up to 200 mg/g dry OL powder in some cases [17], has received special attention due to its anti-Covid-19 activity [18][19][20][21][22][23][24].
To facilitate the conservation and preparation of herbal teas and infusions, the dry form is preferred. In fact, the drying techniques for medicinal plants dehydration are numerous, but usually, solar drying (with or without direct exposure to solar radiation) and at low temperature (<100 °C) are the most commonly used conservation methods, especially on a small scale. However, although this method is not expensive, it has the disadvantage of being time-consuming and induces considerable losses of bioactive compounds, oleuropein in particular [12,25]. This is mainly related to enzymatic alteration reactions (oxidation and/or hydrolysis) [12,17]. This is what we have also observed in the case of black olives hot-air-dried in the temperature range of 25-75 °C [3]. Moreover, several authors have reported that drying at high temperatures (>100 °C) and for a short time maximizes the content of bioactive compounds in OLs and increases the antioxidant activity of the final product [14,25].
From a technological point of view, there are a few differences between high-temperature-drying and roasting processes. The purpose of drying is to remove water, while the main objective of roasting is to improve the sensory properties (color, aroma, and taste) [26,27]. In the present manuscript, the two terminologies (high-temperature-drying and roasting) are used interchangeably.
According to some previous studies, the high-temperature-drying of OLs allows both rapid reduction of the water activity and the inhibition of the enzymes present in the plant matrix [12,25,28]. Add to that, this treatment is very effective in preserving bioactive compounds. However, as far as we know, the kinetic of high-temperature-drying of OLs is not reported in the literature.
This present study focuses on: 1. the mathematical modeling of thin-layer high-temperature-drying (HTD) of olive (var. Chemlal and Oleaster) leaves, and 2. the determination of HTD effect on some physicochemical properties (oleuropein, chlorophylls, and CIELab color parameters).
For this, four drying temperatures (100, 120, 140, and 160 °C) were applied. For comparison purposes, low-temperature dried olive leave samples were also prepared.

Material and methods 2.1 Plant material
The OLs from cultivated olive trees (Chemlal variety) (COL) and wild olive trees (Oleaster) (WOL) were handpicked during the period of March-April 2015 from an olive grove in the Bouira region (Northern Algeria). After sorting, samples were maintained at 4 °C until use. In all cases, the samples were processed in less than three days.

Physical properties
The determination of different dimensions of studied OLs was performed on 20 randomly selected fresh leaves of each variety. Their linear dimensions (length, width, and thickness) and weight were determined by using a caliper (accuracy of 0.01 mm) and electronic balance (accuracy of 0.0001 g), respectively.
The water content of the fresh OLs was gravimetrically determined at 105 °C according to the procedure described by Idoui and Bouchefra [29].

Drying kinetics
The Thin-layer drying experiments were conducted at different temperatures (100, 120, 140, and 160 °C) using a laboratory static-oven dryer (Memmert, Germany). During the drying process, the samples were weighed periodically until the difference between two successive weighings is lower than 0.001 g (equilibrium state). There were three replications of each temperature for both COL and WOL.
To study the drying kinetics, the variation of moisture ratio (MR) versus time was analyzed by following the weight loss (water loss) moisture ratio (MR) was calculated using Eq. (1): where M 0 , M t , and M eq were the initial, at time t and equilibrium water content of OL sample, respectively, W 0 , W t , and W eq were the initial, at time t and equilibrium weight of OL sample, respectively. The water content at any time ( M t ) can be deduced as follows: The experimental data were analyzed by applying ten mathematical models widely applied in food drying operations (Table 1) [30][31][32][33][34][35][36]. Data analysis was performed by non-linear regression method with statistical software, Statistica 6.0. The goodness of fit of the tested models to the experimental data was evaluated by five error parameters [37]: R 2 (coefficient of determination) and SEE (Standard Error of Estimate) were obtained directly from the Statistica 6.0 software. RMSE (root means squared error), SSE (sum squared error), and χ 2 (reduced Chisquare) were calculated using the following formula: where MR pre,i : predicted by the model MR, MR exp,i : experimental MR, N: number of data points, and z: number of model constants.
A model is considered more adequate when R 2 values are higher (closer to 1) and SEE, RMSE, SSE, and X 2 values are lower (closer to 0).

Effective diffusivity coefficient and activation energy
The diffusivity coefficient governs the drying rate in a homogeneous and isotropic solid. This coefficient is affected by several parameters, in particular the water content, temperature, and physical properties of the matrix [36]. In a real product, the diffusivity coefficient takes into account various water delivery mechanisms (molecular diffusion, capillary flow, etc.) and it is then called the "effective diffusion coefficient". Effective diffusivity coefficients for each drying temperature were determined from the slope of the straight line, obtained by plotting ln(MR) against drying time [38].
An analytical solution of Fick's second law equation for an infinite slab (Eq. (6)) was used to estimate the apparent moisture diffusivity of the OLs from the high-temperature-drying kinetics [39].
The activation energy for the water diffusion during high-temperature-drying of OLs was determined based on Arrhenius-type equation [38]: By plotting lnD eff against 1/T a , a straight line is obtained and the activation energy is calculated as where D 0 , E a , R g , and T a are the constant equivalent to the diffusivity at infinitely high temperature (m 2 /s) pre-exponential factor (or Arrhenius constant), activation energy (kJ/mol), universal gas constant (8.314 J/mol K) and absolute temperature (K), respectively.

Determination of physical chemical properties of DOLs
For comparison purposes, the OLs of both studied varieties were processed by two other drying methods: 1. blanching followed by drying at 60 °C and 2. freeze-drying.
Briefly, the blanched OLs were pretreated by soaking in boiling water for 2.5 min and then dried using a Memmert static oven at 60 °C until a constant weight was reached. Furthermore, the freeze-dried samples were prepared by using Cryodos-50 Telstar laboratory freeze dryer (temperature/vacuum pressure of −45 °C/0.44 mbars) without any pretreatment. The obtained samples were taken here as controls.
The prepared dried olive leaves (DOLs) samples are coded according to Table 2. They were analyzed for oleuropein content, chlorophylls content, and CIELab color indices. The leaf samples resulting from the different drying methods were powdered using a commercial grinder. Then, the obtained powders were stored at −20 °C until further analysis.

Oleuropein content
The oleuropein content was determined by spectrophotometry according to Amiot et al. [40] method as described by Hurtado et al. [41] (with some minor modifications). About 0.1 g (W) of leaf powder (DOL) was macerated in 20 mL of distilled water for 3 hours, at room temperature (~25 °C) and sheltered from light. After filtration, using Whatman filter paper grade 4, the absorbance (A) of the extract was measured, using a UV-V is spectrophotometer (Jasco) at two wavelengths of 280 nm (oleuropein + verbascoside) and 330 nm (verbascoside).

Chlorophylls content
Determination of chlorophyll a and chlorophyll b content of DOL was carried out according to Huang et al. [42]. Briefly, about 0.1-0.2 g (W) of DOL was extracted by 50 mL (V) of 80% (v/v) acetone for 2 min and filtered.
The absorption values of the filtrate were determined spectrophotometrically at two wavelengths of 663 nm (chlorophyll a) and 645 nm (chlorophyll b). The chlorophyll a and the chlorophyll b content in olive leave samples are calculated according to the following formulas: .
. , The total chlorophylls (a + b) is given by the formula:

Color determination
The color measurements were quantified by using a Minolta color reader (CR10, Japan). The L*, a*, and b* values were determined for each sample (DOL): lyophilized, blanched/dried, and dried (100, 120, 140, and 160 °C).

Statistical analysis
All analyses were done in triplicate and results were expressed as mean ±SD. Data were analyzed for differences between means using one-way analysis of variance (ANOVA) and Tukey's post-hoc test, with statistical significance when p < 0,05, using Xlstat 2014 software. In addition, a multivariate statistical analysis focused on principal component analysis (PCA) and hierarchical analysis clustering (HAC) was performed using the same software, to extract linear relationships among the variables studied, and to compare obtained DL samples based on their dissimilarities. In order to facilitate the reading of the graphs, the parameters taken into account in this study were codified as shown in Table 3.

Uncertainty analysis
Experimental error and uncertainty can be caused by several factors (instrument specification, instrument calibration, measurement condition, etc.) [43]. In the present study, the designated operating range and uncertainties of used instruments based on the manufacturer's data are given in Table 4. Uncertainty analysis is an important tool to provide the quality of measurements. In the present study, uncertainty analysis was performed according to Monte-Carlo method using LNE-MCM software (version 2017) from the French National Metrology laboratory (LNE). The uncertainties of the principal determined parameters were: ±0.58% (moisture ratio), ±2.75% (oleuropein content) and ±0.51% (chlorophylls tot).

Physical properties and water content of fresh olive leaves
The physical properties (length, width, thickness, and weight) as well as the water content of fresh leaves of COL and WOL are reported in Table 5.
From Table 5, it is easy to see the differences in lengths, thicknesses and water contents between COL and WOL leaves (p < 0.05). This result confirms the possibility of using the physical properties of the leaves to distinguish the oleaster from the olive varieties [44].
Because of its relatively high-water content, OLs cannot be stored for a long period, which makes necessary a conservation treatment, drying at high temperatures, among others.

Drying kinetic
The thin-layer drying kinetic curves of COL ( Fig. 1 (a)) and WOL ( Fig. 1 (b)) at different temperatures are shown in Fig. 1. From these data, a higher drying temperature resulted in a significantly lower drying time. Thus, passing from 100 to 160 °C may reduce the drying time by ~3.5 times.
On the other hand, it should be noted that although the water content of the WOL is the lowest, compared to that of COL, the time required to reach equilibrium is longer. For his part, Hata [12] reported a drying time of 72 h when OLs are dried at 25 °C or freeze-dried. It is 18 h for temperatures between 50 and 65 °C, and 3 h for 70 °C.
The results of the thin-layer modeling of OLs are reported in Table 6. As can be seen, except Wang and Sing model, all other tested models seem to be appropriate for describing the thin-layer drying curves of COL and WOL. However, the Midilli et al. model provides the best fit to the experimental data (with mean values of R 2 = 0.996, SEE = 0.0085, RMSE = 0.0165, SSE = 0.0006, and χ 2 = 0.0697). This finding is in concordance with previous studies: thin-layer infrared drying of wet olive husk at temperatures between 80 and 140 °C [34], thin-layer microwave drying of celery leaves [45], thin layer drying of sour cherry in a solar dryer [46], etc.
The obtained parameter values of the Midilli et al. model are presented in Table 7.

Effective moisture diffusivity and activation energy
The values of effective moisture diffusivity ( D eff ) and activation energy ( E a ) deduced from the linearized Arrhenius equation-type of OLs are recapitulated in Table 8.      Effective moisture diffusivity of the OLs varies, respectively, from 6.36 × 10 −12 to 2.418 × 10 −11 and from 6.162 × 10 −12 to 2.52 × 10 −11 m 2 /s for COL and WOL. Globally, increasing the temperature from 100 to 160 °C increases the effective moisture diffusivity by three times for both varieties, confirming the considerable temperature effect on drying kinetics.
The graph giving ln( D eff ) as a function of 1/T a is a straight line in the range of studied temperatures of both varieties, indicating Arrhenius type dependence between D eff and T a (Fig. 2). The obtained activation energy values are 440.11 kJ/mol (COL) and 444.89 kJ/mol (WOL).

Physical chemical properties of DOLs 3.4.1 Oleuropein content
To begin, it must be remembered that the Oleuropein (Fig. 3) is an ester (hydroxytyrosol + elenolic acid) belonging to the family of secoiridoid polyphenols [13]. This molecule and its derivatives are the main bioactive compounds of the olive tree products: leaves, fruits, twigs, and oil, and are responsible for therapeutic and preventive virtues, widely described in the scientific literature [47,48]. Oleuropein and some of its derivatives are the main compounds responsible for the bitterness of leaves, olives, and even oil. It should also be noted that this molecule is water-soluble and heat resistant [12].
The oleuropein content at the end of the processing of DOLs are shown in Table 9.
The oleuropein content in freeze-dried leaves of the COL (7.71 g/100 g) is comparable to that found in WOL (7.87 g/100 g) (p < 0.05). It should be recalled that the extraction rate could be sensibly enhanced if the extraction was performed with hydroalcoholic solutions [12]. In all cases, our results are comparable to those (6 to 9 g/100 g of dry matter) reported by Achat et al. [47] but lower than those (9-13% of Powdered Leaves/of dry matter) communicated by Savournin et al. [49] concerning the OLs of 14 Tunisian (Bid el Haman, Chemlali, and Meski) and French (Aglandau, Cailletier, Cayet Rouge, Cayon, Grossanne, Lucques, Picholine, Picholine Noire, Tanche, Verdale de l'Herault, Verdale Picholine hybrid) varieties, the latter authors having used an aqueous-methanolic extraction. Globally, the differences in the oleuropein composition can be attributed to various factors including extraction and quantifying method [25,50].
It is well known that freeze-drying can ensure better preservation of the raw material properties and that its applications on a large scale are limited due to its high cost. Therefore, it is more reserved for the drying of thermo-sensitive substances. Compared to freeze-dried DOLs, HTD procedures have induced a significant decrease in the oleuropein content of OLs (p < 0.05) whatever the variety and the preparation conditions, except for DOLs obtained at 160 °C, where the drying time is shorter indicating the technological interest of high-temperature/short-time drying process. For both olive varieties, the lowest contents are recorded during the longest drying time at low temperatures, in particular at 100 °C, and this for both varieties. This decrease is of the order of 36 to 50% compared to freeze-dried DOLs. On the other hand, except for the DOLs obtained at 120 and 160 °C (case of WOL), there is no significant difference between samples dried at the temperatures 120, 140 and 160 °C. In our opinion, the fluctuations observed in the effect of temperature on the oleuropein content could only be explained by taking into account the effect of the time-temperature couple, in particular the activation energy linked to the degradation reaction of this molecule. In addition, the much lower value (3.97 g/100 g DOL), obtained at 100 °C in the case of COL, is probably also related to the initial water content and the thickness of leaves which are relatively higher in this variety.
Indeed, several authors have already highlighted the thermoresistance of oleuropein and its vulnerability to hydrolytic and oxidative enzymes [12,51,52]. In the case of olive fruits of the Manzanilla variety (green-yellow color on the surface), Garcia et al. [52] have found good correlation between oleuropein content and enzymatic browning. In addition, the chemical and enzymatic degradation reactions of oleuropein lead principally to an improvement in content of hydroxytyrosol, whose virtues on the human body are widely also described in the scientific literature, namely antimicrobial, hypoglycemic, hypolipidemic, hypocholesterolic and antioxidant [13,28].
Regarding the effect of temperature on the intensity of oleuropein degradation, there are various opinions in the literature. Al Juhaimi et al. [50] highlighted the heat sensitivity of polyphenols of OLs, among which oleuropein. Ahmad-Qasem et al. [25] have underlined the interest of HTD in increasing the rate of water removal from the plant matrix. These same authors have recalled the direct and/or indirect (by reducing water activity) thermal inactivation of polyphenol oxidase. Mostly, the complete inactivation of this enzyme is observed at higher temperatures exceeding 70 °C [53].
Concerning the blanched DOLs, for which the raw leaves were pretreated in a boiling water bath before drying at 60 °C in order to denature the endogenous enzymes, the obtained results revealed a significant (p < 0.05) decrease in oleuropein content (25-30%). This diminution may be related to the release of oleuropein from the plant matrix into the soaking media during the blanching step.
Hata [12] has reported that drying of unblanched OLs at temperatures below 100 °C leads to significant losses of oleuropein up to 70%. This decrease is even more important when the drying is done at low temperatures as also highlighted by Boukhiar et al. [3] concerning the drying of black olive fruits at temperatures ranging from 25 to 75 °C.
For valorization purposes of OLs, as a potential source of oleuropein, recent scientific studies show that it is possible to use them, as-is or after extraction, to enrich various products for human consumption: olive oil [47], table olives [54], coffee [55], date powder tablets [56], etc.

Chlorophylls content
Numerous recent scientific studies show that chlorophylls contribute to the health benefits of medicinal plants [57]. However, their presence in the oil is undesirable because of its pro-oxidant effect in the presence of light [58].
The chlorophyll contents (a, b, and total) of processed DOLs are given in Table 10. From these results, the chlorophyll b content of all considered samples is about two times higher than that of chlorophyll a, regardless of variety and preparation method.
Moreover, as expected for both varieties, the chlorophyll a and b contents of the lyophilized DOLs were statistically higher (p < 0.05) than those of the other dried samples, indicating the degradation effect of hot-air drying on the pigments. Different superscripts in the same column indicate significant difference at p < 0.05.

Fig. 3 Oleuropein
In fact, the effect of temperature on the degradation of chemical and biochemical compounds, including chlorophylls, is well described by the Arrhenius equation but this dependence is a function of the nature of the molecule and also the surrounding environment. Theoretically, it is also well known that an increasing temperature of 10 °C implies an increase in the alteration rate of 2-3 times.
In addition, it should be remembered that the role played by enzymes (peroxidase and chlorophyllase) in the deterioration of chlorophylls is important. In this context, Khaushal et al. [59] recommended a chemical alkali blanching using sodium bicarbonate (0.1%) for 10 seconds in Colocasia Schott leaves.
As mentioned previously, the physical and chemical changes induced by pyrolysis reactions during roasting depend directly on the treatment conditions (temperature and duration in particular) and material characteristics [27]. Concerning the chlorophylls, these reactions induce their conversion into pheophytins and pyropheophytins [60].
In the case of blanched samples, the decrease in chlorophyll contents can be attributed, in addition to the effect of enzymes and pyrolysis reactions, to the release of pigments in the soaking media as previously explained for oleuropein.

Color measurements
As known, color is a key quality factor of numerous food and non-food materials. It is generally considered to be the most decisive parameter for consumer acceptability and a useful tool for monitoring many food processes (roasting, drying, baking, etc.).
CIELab color parameters (L*, a*, and b* values) of the prepared DOLs are presented in Table 11. As can be seen, there is a noticeable increase in browning degrees with increasing drying time and/or temperature.
Compared to the freeze-dried leaves whose color characteristics are closer to those of fresh OLs, drying induced significant decreases (p < 0.05) in L* and a* values for both leaf varieties indicating the effect of browning with a loss of green color. From our point of view, these changes could be associated with non-enzymatic browning reactions (also called Maillard reaction), caramelization, and degradation of chlorophylls (bright green color) into pheophytins and pyropheophytins (yellow-brown color) as already mentioned in the literature about OLs [60][61][62]. It is also worth noting that the products of the Maillard reaction would have antioxidant properties as reported by Lin et al. [62] for almond kernels. Regarding the b* values, its variations are rather difficult to explain, depending, however, on both the variety and the operating conditions of preparation.   Fig. 4, it is easy to see that Oleur, Chl a, Chl b, Chl tot, and L* are positively correlated with F1 but negatively with a*. On the other hand, b* and WL are correlated with F2, while Time is correlated with F3. Moreover, the calculated Pearson correlation coefficient shows the existence of strong correlations (R > 0.98) between Chl a, Chl b, and Chl tot. For the oleuropein content (Oleur), it is positively correlated with L* and negatively with Time and a*. Concerning the color parameters, a* is negatively correlated with Oleur, Chl a, Chl b, Chl tot, and L*. In addition, L* is positively correlated with Oleur, whereas b* is correlated only with WL. At last, the Time parameter is not correlated with WL (best shown by the biplot of F2 versus F3 components, not presented here).
From Fig. 5, it is clearly observed that blanched, dried at 120 and at 140 °C DOL, of both olive varieties, present relatively similar characteristics because they are clustered together. As a complement to PCA, the dissimilarity dendrogram that was determined by hierarchical ascending classification (HAC), with Euclidean distance and Ward criterion, allows good visualization of the links between the different DOLs studied (Fig. 6).
It should be noted here that the two variables Chl a and Chl b are not taken into account, as they are strongly correlated with the Chl tot. In addition to the latter parameter, the variables considered here were Oleur, L*, a*, b*, Time, and WL. Moreover, the missing values were replaced by the mean values (case of Time and WL of lyophilized and blanched DOLs).
It is clear at first sight that the varietal effect is negligible compared to the preparation method (Fig. 6).
These findings are in agreement with the PCA results presented above concerning: 1. the similarities of DOLs blanched, dried at 120 and at 140 °C, and 2. the neglecting of the varietal effect.

Conclusion
This study focuses on: 1. the mathematical modeling of high temperature thin layer drying (HTD) of olive (var. Chemlal and Oleaster) leaves, and 2. determination of HTD effect on some physicochemical properties (oleuropein, chlorophylls, and CIELab color parameters).
The obtained data have shown that among the tens tested mathematical models, that of Midilli et al. describes more correctly experimental data for all drying temperatures and for the both varieties. Moreover, the results show that the HTD at temperatures between 120 and 160 °C does not differ from freeze-drying in terms of oleuropein retention (p < 0.05), highlighting the technological interest of high-temperature/short-time drying process.
Considering the biological value of oleuropein, in particular its antiviral activity, the study deserves further investigation in order to elucidate certain questions such as the storability of DOL, their valorization as fortification ingredient in food and pharmaceutical formulations, evaluation in vitro of their biological activities, etc.