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Drying Characteristics of Dracocephalum moldavica Leaves: Drying Kinetics and Physicochemical Properties

The aim of the study was to determine the effect of the method and temperature of the sublimation, vacuum and convective drying process on the kinetics of dehydration of the Moldovan dragonhead leaves, as well as on the physical and chemical properties, such as color coordinates, total content of phenolic compounds, antioxidant capacity, total monomeric anthocyanins content, total flavonoids content and content of essential oils. It was shown that the kinetics of the sublimation and vacuum drying process are best described by the logarithmic and Page models. Sublimation and vacuum drying were characterized by comparable process times. An increase in drying temperature caused a decrease in the content of phenolic compounds and antioxidant activity of the dried material and a significant change in the composition of essential oils. The lowest brightness of the dried material and the lowest color saturation was found after sublimation drying at 20 °C. The highest dried material quality was found in case of convective and sublimation drying at 20 °C. Record Type: Published Article Submitted To: LAPSE (Living Archive for Process Systems Engineering) Citation (overall record, always the latest version): LAPSE:2020.0776 Citation (this specific file, latest version): LAPSE:2020.0776-1 Citation (this specific file, this version): LAPSE:2020.0776-1v1 DOI of Published Version: https://doi.org/10.3390/pr8050509 License: Creative Commons Attribution 4.0 International (CC BY 4.0) Powered by TCPDF (www.tcpdf.org)

Introduction
The Moldavian dragonhead (Dracocephalum moldavica) is an annual plant growing up to about 80 cm. There are two varieties of this plant, with white and blue flowers, and it belongs to the Lamiaceae family. Under natural conditions, the plant is found in China and Mongolia, central Asia and central and eastern Europe [1,2].
The dried leaves of the Moldavian dragonhead have a lemon aroma and are used in natural medicine to prepare infusions due to their antiseptic, antibacterial, antioxidant, calming and appetite-stimulating properties [3]. The herb of the Moldavian dragonhead contains mainly terpenoids: neral, geraniol, geranyl acetate and flavonoids, iridoids as well as tannins [4,5]. The composition of the essential oil depends mainly on the climate in which the Moldavian dragonhead was grown. The oil obtained from cultivation in temperate climates contains higher amounts of alcoholic monoterpene derivatives, and from cultivation in warmer climates it contains higher amounts of aldehyde and ester monoterpene derivatives [6]. The oil and extracts from the dried dragonhead leaves are used drying and vacuum drying.The leaves were dried whole, and before sublimation drying they were frozen at −30 • C for 24 h. The moisture content of the fresh leaves of the Moldavian dragonhead was determined by weight method, drying them under vacuum at 95 • C ± 0.5 • C to obtain a constant weight. The moisture content of the leaves of the Moldavian dragonhead was 83.24% ± 0.35% (wet basis). Each method of drying was performed individually for leaves from each plot. In order to standardize the sample, dried leaves from each plot were mixed.

Drying Method
The convection drying process was carried out in a laboratory convection dryer (Promis Tech, Wrocław, Poland). Drying was carried out at a drying air temperature of 40 • C and 60 • C and a constant air flow velocity of 0.5 m·s −1 . Registration of changes in the mass of the material during drying was provided by a laboratory electronic scale, type B3 (AXIS, Gdańsk, Poland) with an accuracy of ±0.1 g, connected to a computer recording its indications. The drying process led to a final material moisture content of 10%.
The sublimation and vacuum drying processes were carried out in an ALPHA 1-4 (Martin Christ Company, Osterode am Harz, Germany) laboratory vacuum dryer. The device consists of a drying chamber, vacuum pump, ice capacitor, heating system consisting of 5 shelves placed on a rack and a control and measurement system. The device is equipped with a weighing system that records, in a continuous manner, changes in weight during the process (accuracy of weight ±0.1 g). The heat required for the phase transition is delivered to the material by contact. The heating panel temperature control is performed by means of a temperature sensor placed inside one of the lyophilizer panels [26]. The sublimation drying process was carried out for heating panel temperatures of 20 • C, 40 • C and 60 • C with a constant pressure value of 63 Pa. Vacuum drying was carried out for heating panel temperatures of 20 • C, 40 • C and 60 • C with a constant pressure value of 2000 Pa (in case of vacuum drying, the material was not previously frozen). Sublimation and vacuum drying were carried out to obtain a constant dried material humidity of 10%.We did not carry out convection drying at 20 • C, because this kind of drying at such low temperature is ineffective, takes a long time, can lead to the development of microorganisms and often does not allow to achieve an adequate level of moisture content after drying. The drying temperature was selected on the basis of the literature data mentioned in the introduction [21][22][23][24][25].

Modeling of Drying Curves
The drying kinetics is presented as a change in reduced water content (MR) as a function of drying time: where u t is the water content in the course of drying (kg H 2 O/kg DM), u 0 is the initial water content (kg H 2 O·kg DM −1 )and u r is the equilibrium water content (kg H 2 O·kg DM −1 ). The equilibrium water content after sublimation and vacuum drying is very low, so the equilibrium water content (u r ) is assumed to be 0 over the entire measuring range. Such simplification has little effect on drying kinetics [27][28][29].
The most frequent models in the literature were used to describe the curves of sublimation, vacuum and convective drying. The model equations are presented in Table 1.

Measurement of Color Coordinates
Color measurement was performed using the reflection method with an X-Rite 8200 spherical spectrophotometer with a measuring hole of 12.7 mm in diameter. A D 65 light source and a standard colorimetric observer with a 10 • field of view were used. Before each measurement, the instrument was calibrated using a white standard.
The color coordinates were determined in the CIEL*a*b* system. The measurement of color in this system is based on the numerical determination of the three coordinates L*, a*, b*, where L* stands for color brightness and ranges from 0 for a perfectly black body to 100 for a perfectly white body. The coordinate a* indicates the color change from green (−a*) to red (+a*), and b* indicates the color change from blue (−b*) to yellow (+b*).
On the basis of the designated color coordinates, the total change in color (∆E) in relation to the raw material was determined, as recorded in the cylindrical coordinates for the color saturation (C) and color shade (HU) of the dried material [35].
where L 0 *, a 0 * and b 0 * are the color parameters of the fresh sample.

Total Phenolic Compounds Content
The content of total phenolic compounds (TPC) was determined by the Folin-Ciocalteu method [36]. A 0.5 mL extract sample was mixed with 0.5 mL H 2 O, 2 mL Folin reagent (1:5 H 2 O) and, after 3 min, with 10 mL 10% Na 2 CO 3 . After 30 min, the absorbance of the mixed samples was measured at 725 nm. The measurement was carried out on a UV spectrophotometer Mini 1240 (Shimadzu, Japan). The content of the total phenolic compounds was expressed as gallic acid equivalent per gram of dry matter.

Antiradical Activity
The ability to neutralize free radicals against ABTS (2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate)) was carried out by the method developed by Re et al. [37]. The ability to neutralize free radicals against DPPH (2,2-diphenyl-1picyclohydrazil) was carried out according to Brand-Williams et al. [38]. The decrease in absorbance was measured quantitatively on the spectrophotometer at 734 nm for ABTS and 517 nm for DPPH.
The ability to neutralize free radicals of ABTS and DPPH was expressed as the EC 50 index-the dry matter concentration (mg·mL −1 ) causing a 50% decrease in the initial ABTS or DPPH radical concentration.

Total Flavonoids Content
Total flavonoids were estimated according to the method described by Bahorun et al. [39]. One milliliter of sample was mixed with 1 mL 2% AlCl 3 ·6H 2 O. After 10 min, absorbance at 430 nm was measured. The total flavonoids content was expresses as quercetin equivalent (QE) in milligrams per dry weight (DW).

Total Monomeric Anthocyanins
Total monomeric anthocyanins (TAC) were quantified by the pH-differential method according to Giusti and Wrolstad [40]. Extracts of cranberry powders were diluted with two buffer solutions at pH 1 and 4.5. Cy-3-G (MW = 449.2 g·mol −1 ) was used as a standard with a molar absorptivity coefficient of 26900. Results were expressed as mg of cyanidin-3-galactoside equivalent as mg of Cy-3-G per g DM.

Quantity and Composition of Essential Oils
The volume of essential oil was determined by steam distillation in the Dering apparatus. The 20 g of crushed material was placed in a distillation flask containing 400 mL of distilled water. The distillation process was carried out for 3 h from the start of boiling. The resulting volume of oil was converted into 100 g of dry matter. The qualitative composition and percentage content of the essential oil components was determined by GC/MS method, using the Varian Chrompack CP-3800 gas chromatograph with Varian 4000 MS/MS mass detector and flame ionization detector (FID). The VF-5 ms column (equivalent to DB-5) was used. The carrier gas was helium with a constant flow through the column of 0.5 mL·min −1 . The column temperature program was as follows: 50 • C for 1 min, increased to 250 • C at 4 • Cmin −1 , and then 250 • C for 10 min. Doser: 250 • C, split 1:50. A total of 1 µL of solution was dosed (10 µL of sample in 1000 µL of hexane). The recorded range was 40-1000 m·z −1 , and the scanning rate 0.8 sec·skan −1 . Kovats retention indices (non-isothermal Kovats retention index) were determined on the basis of the C10-C40 alkanes series, using the NIST Library and HP Chemstation spectral libraries.

Statistical Analysis of the Results
One-and two-factor analysis of variance was performed. Tukey's test was used to determine the significance of differences between the means. Each drying test was performed in triplicate and all analyses were performed in 5 repetitions. The analysis of drying kinetics regression was performed by non-linear estimation using the least squares method, determining the determination factor and the root mean square error (RMSE) as well as chi-squared test values (χ 2 ). The mean root square error (RMSE) and chi-squared test values (χ 2 ) were determined from the relationship where MR i,p -predicted value of reduced water content; MR i,e -experimental value of reduced water content; N-number of measurements; and n-number of parameters in the model equation. Statistica 13 from StatSoft was used for statistical analysis. All calculations were performed with the significance level α = 0.05. This level of α is used in many biological studies.

Drying Kinetics
Changes in the reduced water content (MR) as a function of the duration of sublimation, vacuum and convective drying of the dragonhead leaves is shown in Figures 1-3. On the basis of the conducted tests, it can be concluded that with the increase in drying temperature, the duration of the process is reduced-for all the analyzed drying methods. Increasing the temperature from 20 • C to 60 • C resulted in shortening the drying time by about 34% (sublimation drying) and by 32% (vacuum drying). An increase in temperature from 40 • C to 60 • C shortened the convective drying time by about 45%. At a given temperature level, vacuum drying was shorter than sublimation drying. However, the Processes 2020, 8, 509 6 of 17 differences were small and amounted to about 20 min. The shortest time was observed in the case of convective drying: at 60 • C, the time was 160 min.
conducted tests, it can be concluded that with the increase in drying temperature, the duration of the process is reduced-for all the analyzed drying methods. Increasing the temperature from 20 °C to 60°C resulted in shortening the drying time by about 34% (sublimation drying) and by 32% (vacuum drying). An increase in temperature from 40 °C to 60 °C shortened the convective drying time by about 45%. At a given temperature level, vacuum drying was shorter than sublimation drying. However, the differences were small and amounted to about 20 min. The shortest time was observed in the case of convective drying: at 60 °C, the time was 160 min.   conducted tests, it can be concluded that with the increase in drying temperature, the duration of the process is reduced-for all the analyzed drying methods. Increasing the temperature from 20 °C to 60°C resulted in shortening the drying time by about 34% (sublimation drying) and by 32% (vacuum drying). An increase in temperature from 40 °C to 60 °C shortened the convective drying time by about 45%. At a given temperature level, vacuum drying was shorter than sublimation drying. However, the differences were small and amounted to about 20 min. The shortest time was observed in the case of convective drying: at 60 °C, the time was 160 min.  The results of the regression analysis of the five analyzed models used to describe the kinetics of sublimation, vacuum and convective drying are presented in Tables 2-4. It can be seen that a good match of the experimental data was obtained for each of the analyzed models. The value of the coefficient of determination (R 2 ) of the equations ranged from 0.932 to 0.999. The square root from the mean square error (RMSE) and the value of the reduced test ( 2 ) had small values ranging from 0.0033-0.0678 and 0.00001-0.00481, respectively. In case of vacuum drying, the best match between the analyzed models and experimental data was obtained using a logarithmic model (R 2 -above 0.997;RMSE-below 0.0161); slightly worse results were obtained in case of the Page model. The highest value of the determination coefficient R 2 (0.996-0.999) and the lowest values of the RMSE The results of the regression analysis of the five analyzed models used to describe the kinetics of sublimation, vacuum and convective drying are presented in Tables 2-4. It can be seen that a good match of the experimental data was obtained for each of the analyzed models. The value of the coefficient of determination (R 2 ) of the equations ranged from 0.932 to 0.999. The square root from the mean square error (RMSE) and the value of the reduced test (χ 2 ) had small values ranging from 0.0033-0.0678 and 0.00001-0.00481, respectively. In case of vacuum drying, the best match between the analyzed models and experimental data was obtained using a logarithmic model (R 2 -above 0.997; RMSE-below 0.0161); slightly worse results were obtained in case of the Page model. The highest value of the determination coefficient R 2 (0.996-0.999) and the lowest values of the RMSE (0.005-0.0189) during sublimation and convective drying of dragonhead leaves were obtained for the Page model. The Page model very often gave the best match results among the most frequently used empirical models during leaves drying [31,41,42]. The logarithmic model was used to describe the kinetics of mint [43] and rosemary [44] leaves drying. The values of the coefficients occurring in the five analyzed regression equations are presented in Table 5 (sublimation drying), Table 6 (vacuum drying) and Table 7 (convective drying).

Color Assessment
In the whole measuring range (Table 8), dried Moldavian dragonhead leaf material was characterized by a higher brightness (L*) and color saturation (C) than the raw material. The value of the color shade (HU) was lower for the dried material in relation to the raw material. The value of the color coordinate L* of the dried material decreased with an increase in temperature for sublimation and vacuum drying and increased for the dried material obtained after convective drying. At a given temperature level, the greatest brightness was characteristic of the dried material obtained after sublimation drying, slightly lower values were observed for the dried material obtained as a result of vacuum drying, while the dried material obtained after convective drying had significantly lower values of the L* color coordinate.  The dried material obtained by sublimation drying at 20 • C and 40 • C was characterized by the highest brightness. The lowest value of this parameter was found in the dried material obtained after convective drying at 40 • C. In this case, the value of the L* coordinate was lower by about 12.7 units in comparison with the dried material obtained after sublimation drying (20 • C). The saturation of color (C) of the dried Moldavian dragonhead leaf material did not depend on the temperature of the sublimation drying. An increase in the temperature of the heating panels contributed to a slight increase in the value of color saturation of the dried material obtained after vacuum drying (these changes were statistically insignificant at 20 • C and 40 • C). Increasing the temperature of the drying air resulted in a decrease in the color saturation during convective drying of the Moldavian dragonhead leaves. The saturation of color closest to the raw material was obtained after sublimation drying (increase by about 8.5 units); the biggest differences occurred after convective drying at 40 • C (increase by about 19.6 units). The dried material from the Moldavian dragonhead, for all the analyzed drying methods, had lower values of color shade (HU) than the raw material. As the temperature of the heating panels increased (sublimation and vacuum drying), the value of the color shade decreased. The differences were small at 20 • C and 40 • C and deepened at 60 • C. At each temperature level, the dried material obtained after sublimation drying had higher values of color shade than that obtained by vacuum drying. The values of this color coordinate were lower by about 5 units at 20 • C, 4 units after drying at 40 • C and 2 units at 60 • C. Convective drying resulted in a significant reduction in the color shade values of the dried material, compared to the other two drying methods. Greater changes compared to the raw material were observed at 40 • C. Dried material from the Moldavian dragonhead leaves with the shade of color closest to that of the raw material was obtained after sublimation drying at 20 • C (about 7 units); the greatest difference between the shade of color of the raw material and that of the dried material occurred after convective drying at 40 • C (about 24 units). The total change in the color of the dried material from the Moldavian dragonhead ranged from 17.6 to 22.7. As the temperature increased, regardless of the drying method used, the value of the total change in color (∆E) decreased. The lowest ∆E values were obtained after sublimation drying at 60 • C, and the highest after convective drying at 40 • C. At a given temperature level, the lowest values of total color change were obtained after sublimation drying, and the highest after convective drying.
The main factors causing adverse changes in the color of the dried leaves are increased temperature and the presence of reactive oxygen in the drying agent. Undesirable changes of color, occurring in dried materials, are mainly due to enzymatic reactions and non-enzymatic browning [45,46]. The color change is also related to the breakdown of pigments, mainly anthocyanins, carotenoids and chlorophylls [47,48]; polyphenolic compounds contained in the raw material; and the breakdown of L-ascorbic acid [49]. The final color of the dried material is also affected by ions of certain metals and the pH of the dried material [50]. Adverse changes in the color of the dragonhead leaves were mainly from the degradation of the chlorophylls contained in them, which contributes to the browning of the dried material [51]. The browning process is accurately reflected by the change in color shade (HU) and lightness of the dried material (L*), the value of which decreases as the process temperature increases [52,53]. The chlorophyll content decreases with increasing temperature, process time [54] and the presence of oxygen, which causes the oxidation of unsaturated color compounds contained in the material [55], contributing to unfavorable changes in the color determinants of the dried material.

Phenolic Profile and Antioxidant Activity
The dried material obtained by the three drying methods studied was characterized by a decrease in total polyphenols (TPC), total monomeric anthocyanins (TAC) and total flavonoid content (TFC) ( Table 9). The dried material obtained after sublimation and vacuum drying at 20 • C and 40 • C did not differ significantly in TPC content. The total content of polyphenols at 60 • C was lower than at the other two drying temperatures, but the values obtained did not differ significantly in sublimation and vacuum drying. The drying temperature also did not influence the TPC content in case of convective drying. However, the TPC content obtained after convective drying was significantly lower than that of the other two drying methods (about 9 mg GAE·g DM −1 at 40 • C and about 14.5 mg GAE·g DM −1 at 60 • C). Sublimation and vacuum drying contributed to about 21%-31% TPC losses in dried leaves of Moldavian dragonhead, while convective drying caused losses of about 40%-45% in relation to the raw material.  Increasing the temperature of the sublimation, vacuum and convective drying contributed to a decrease in the total content of anthocyanins in dried dragonhead leaves (except for 20 • C and 40 • C-sublimation drying-where these changes were statistically insignificant). At a given temperature level, the highest TAC content was noted for the dried material obtained as a result of sublimation drying, slightly lower after vacuum drying (at 20 • C the changes were statistically insignificant compared to sublimation drying) and the lowest for convective drying. Sublimation drying resulted in a decrease in TAC content in the dried dragonhead leaves by about 0.8-1.9 mg Cy-3-G·g DM −1 , as compared to the raw material. Vacuum drying resulted in a loss of TAC content by about 0.9-2.6 mg Cy-3-G·g DM −1 . The dried product obtained after convective drying at 60 • C contained about 2.5 times less TAC than the raw material. As the temperature of the sublimation, vacuum and convective drying increased, the total content of flavonoids in the dried dragonhead leaves decreased. These losses were small after sublimation and vacuum drying at 20 • C and 40 • C (about 2%-8%, depending on the temperature and drying method). Increasing the temperature to 60 • C resulted in a decrease in TFC content by 24.5 mg QE·g DM −1 -sublimation drying-and by 27.7 mg QE·g DM −1 -vacuum drying-compared to 20 • C. The dried material obtained by convective drying was characterized by a significant reduction in TFC content, in relation to that obtained by the other two methods. The lowest TFC content was obtained after convective drying at 60 • C. The loss of this component was then about 40.7% compared to the raw material. In a similar way as described above, the drying method influenced the total content of polyphenols, anthocyanins and flavonoids in dried Vitexagnus-castus leaf material [56]. The highest content of these components was found in the dried material obtained after sublimation and vacuum drying. Convective dried material had significantly lower contents of TPC, TAC and TFC [56,57].
The drying of Moldavian dragonhead leaves, regardless of the method used and the drying temperature, resulted in a decrease in the antioxidant capacity of the dried material, compared to the raw material (against ABTS and DPPH radicals). In the whole measuring range, the EC 50 had a higher value for DPPH radical than for ABTS. As the temperature increases, for all drying methods, the value of EC 50 of dried material from Moldavian dragonhead leaves increases against ABTS and DPPH (decreasing antioxidant potential). Similarly, the influence of temperature on the DPPH radical scavenging rate in mulberry leaves was described by Ma et al. [58]. The increase in EC 50 value is small for the dried material obtained at all temperatures used for sublimation drying and vacuum drying at 20 • C and 40 • C, especially for the DPPH radical. The antioxidant potential of the dried material obtained after vacuum drying at 60 • C is slightly higher than that of the convective drying obtained at 40 • C. The lowest antioxidant potential was that of the dried product obtained after convective drying at 60 • C. The value of EC 50 increased by 1.8 times (ABTS) and 1.4 times (DPPH), respectively. Vuong et al. [56] have shown that sublimation and vacuum drying have little effect on the antioxidant capacity of the dried material; the ABTS and DPPH radical scavenging capacity is significantly reduced in the dried material obtained from convective drying. Table 10 lists the 13 main components of the essential oil contained in the raw material and dried leaves of Moldavian dragonhead. The percentage content of these components is from 98.5% to 99.5% of the total amount of obtained essential oil. The main components of the essential oil from Moldavian dragonhead leaves were geranial, geraniol, geranyl acetate, neral and neryl acetate. They belong to the group of non-ring-like monoterpenes (alcohols, ester aldehydes) with a smell similar to lemon and rose. The drying of Moldavian dragonhead leaves resulted in an increase in the content of geraniol, neryl acetate, linalool and, in most cases, geranyl acetate, but a decrease in the content of neral, geranial and β-pinene.

Essential Oils Content
The effect of the method and drying temperature on the content of other compounds present in the oil from the Moldavian dragonhead leaves was ambiguous. As the drying temperature increased, the geranial content decreased. Sublimation and vacuum drying, in the whole temperature range, caused slight losses of geranial content, not exceeding 18%, in relation to its content in the raw material. Convective dried material was characterized by higher losses of this component, which was about 35% at 40 • C and 62% at 60 • C, respectively. An increase in temperature from 20 • C to 60 • C resulted in a 3.4% decrease in neral content for sublimation drying and 4.5% decrease for vacuum drying. An increase in the convective drying temperature from 40 • C to 60 • C reduced the neral content by 5.1%. The geraniol content in the dried material increased with the temperature of sublimation, vacuum and convective drying. At a given temperature level, the highest content of geraniol was found in the dried material obtained after convective drying; the lowest after sublimation drying. The content of geraniol in the dried material obtained after convective drying at 60 • C was about 150% of its content in the raw material. The content of geranyl acetate in the dried material was stable in the range 22%-26% of the total amount of essential oil. The percentage of linalool content in the dried material essential oil increased with the temperature of vacuum and convective drying. In the dried material obtained at 60 • C, it was about seven times higher than in the raw material for vacuum drying, andalmost ten times higher in the case of convective drying. As the drying temperature increased, the content of neryl acetate in the dried material increased, regardless of the drying method used. This increase was the smallest in the dried material obtained after sublimation drying and the largest after convective drying. An increase in geraniol, neryl acetate and geranyl acetate content was also noted by Samadi et al. [59] in the Dracocephalum kotschyi Boiss dried material. At 70 • C, they noted significant decreases in the content of these essential oil components. In most cases, the content of essential oil components in the dried leaves was lower in the dried material than in the raw material and decreased as the drying temperature increased [60,61].
Analyzing the changes in the oil content in the dried material from the Moldavian dragonhead leaves, it can be stated that the aldehydes (geranialandneral) are subject to decomposition, leading to the formation of alcohols and esters (geraniol, linalool, neryl acetate and geranial acetate) during the drying process. The total content of essential oils in the dried dragonhead leaves was comparable with their amount in the raw material, after sublimation drying at 20 • C and 40 • C. For the other two drying methods, the total amount of essential oils was lower than in the raw material. As the temperature of vacuum and convective drying increased, the total amount of obtained oils decreased. The lowest content of essential oil was obtained after convective drying at 60 • C; it was 40% lower than in the raw material. In a similar way, the influence of temperature and drying method on the total content of essential oils was described by Arwgyropoulos and Muller [62] for dried material of lemon balm leaves, as well as by Figiel et al. [63] for dried oregano leaves.

Conclusions
The best match of the analyzed models describing the change in reduced water content as a function of drying time was obtained using a logarithmic model in case of vacuum drying, and a Page model for sublimation and vacuum drying. At a given temperature level, sublimation and vacuum drying were characterized by comparable process times. As the temperature increased, the determinants of the color of dried dragonhead leaves, the content of the phenolic compounds, the antioxidant capacity and the composition of essential oils differed to a greater extent from the values of these parameters for the raw material before drying. The differences were small for the dried material obtained after sublimation and vacuum drying, especially at 20 • C and 40 • C, and quite significant after convective drying. If the main emphasis is placed on the quality characteristics of the dried material, and the drying time plays a smaller role, it is recommended to carry out the process of drying Moldavian dragonhead leaves by sublimation or vacuum at a heating-panel temperature not exceeding 40 • C. If the drying time plays a major role, the drying heating process should be carried out by convection at 60 • C.


extfieldsofstudy: Medicine Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Nutritional, Functional, and Technological Characterization of a Novel Gluten- and Lactose-Free Yogurt-Style Snack Produced With Selected Lactic Acid Bacteria and Leguminosae Flours

Aiming at meeting consumers’ requirements for healthy foods, dietary needs (vegetarianism, lactose- and gluten-free), as well as the nutrition recommendations of the Health Authorities in terms of protein, fibers and bioactive compounds, the present study proposes a novel yogurt-style snack made with plant-derived ingredients. The biotechnological protocol includes the fermentation of a thermal-treated blend of cereal and legume flours by the selected lactic acid bacteria (LAB) Lactoplantibacillus plantarum DSM33326 and Levilactobacillus brevis DSM33325. The yogurt-style snack was characterized by protein and fiber concentration of 3 and 4%, respectively, and a low-fat content. Compared to the unfermented control, the yogurt-style snack was characterized by a significant higher concentration of free amino acids and lower contents of the antinutritional factors, i.e., phytic acid, condensed tannins, saponins and raffinose (up to 90%) mainly due to the LAB metabolic activity. Hence, an in-vitro protein digestibility of 79% and improvements of all the nutritional indexes related to the quality of the protein fraction (e.g., GABA) were achieved at the end of fermentation. According to the Harvard Medical School recommendations, the novel snack can be potentially classified as low-glycemic index food (53%). Antioxidant properties of the fermented snack were also improved by means of increased the total phenol content and radical scavenging activity. High survival rate of the starter LAB and a commercial probiotic (added to the snack) was found through 30 days storage under refrigerated conditions. The biotechnological protocol to make the novel snack here proposed is suitable for the large-scale application in food industry, giving a platform product with a peculiar and appreciated sensory profile.

INTRODUCTION
The concept of functional food perfectly meets the current consumer awareness regarding the correlation between diet and well-being; indeed, the healthiness is one of the main factors driving the consumer food choices (Granato et al., 2010). In this framework, research is investing in the development of novel foods which have a positive effect on the human organism through the modulation of a specific physiological function .
Although yogurt and fermented milks are the functional foods mainly requested by consumers, there is a growing demand for beverages with a yogurt-like formulation, but not milkbased, defined as "yogurt-like drinks". The vegetarianism, lactose intolerance and hypercholesterolemia are some of the reasons driving the increasing demand for milk-alternatives (Prado et al., 2008). Moreover, beverages turned out to be the most promising food category to better satisfy consumer demands, as they can be easily distributed, stored in refrigerated conditions and allow the easy incorporation of nutrients, bioactive compounds and probiotics (Corbo et al., 2014). To date, several beverages, mainly based on cereals, have been developed (Kandylis et al., 2016). Cereals are a good source of carbohydrates, proteins, vitamins, minerals and fibers (Blandino et al., 2003) and may determine excellent rheological properties (following a starch gelatinization process) when used as a basic ingredient yogurt-like products (Blandino et al., 2003;Coda et al., 2012).
Nevertheless, the increase of the global prevalence of celiac disease (circa 1-2% of the entire population) and the nutrition recommendations of the Health Authorities (e.g., the inclusion of high biological value protein, fibers and bioactive compounds in the diet) make the legumes promising candidates to produce novel functional foods and beverages (Maphosa and Jideani, 2017). Nevertheless, the choice of the optimal technological process to produce legumes-based foods needs to consider the presence of antinutritional factors (ANF) (phytic acid, condensed tannins, saponins, α-galactosides, etc.) which reduce the bio-availability of nutrients (mainly proteins and minerals) and negatively affect the sensory profile of the products (Curiel et al., 2015).
Several technologies have been proposed to produce cerealbased yoghurt-like beverages; nevertheless, fermentation appears to be the most widely used since, along with the technological properties, naturally enhances the nutritional and sensory profiles as well as prolongs the shelf life perspective (Hugenholtz, 2013). Overall, at industrial scale, the fermentation is carried out using selected starter cultures, which, once inoculated drive the process making it controllable and reproducible (Kandasamy et al., 2018).
Lactic acid bacteria (LAB) are often used for this purpose in various food matrices such as milk, meat, vegetables and cereals due to beneficial contributions of their metabolic traits on the product quality (Gobbetti et al., 2019a). The intense proteolytic (Savijoki et al., 2006) and specific enzyme activities (e.g., tannase, β-glucosidase, etc.) as well as the acidification (lactic and acetic acids) occurring during fermentation of cerealderived and other vegetable matrices lead to several improvement of the food matrices. Indeed, increased contents of bioactive compounds (amino acids, phenols, peptides) (Filannino et al., 2018), lower concentrations of the ANF, enhanced nutritional features (in-vitro protein digestibility, starch hydrolysis and lowering glycemic index) (De Angelis et al., 2009;Peyer et al., 2016), improved sensorial profiles (Peyer et al., 2016) and extended shelf-life (antimicrobial activity) (Angelov et al., 2006) often characterized the fermented foods.
In this study, a biotechnological protocol to produce a novel gluten-free, lactose-free and vegan yogurt-style snack is described. A blend of rice, lentil and chickpea flours was used as raw matrix for the snack production, then subjected to mild physical treatments and fermentation by using the selected starters Lactobacillus plantarum DSM33326 and Levilactobacillus brevis DSM33325 (formerly known as Lactobacillus plantarum and Lactoplantibacillus brevis, respectively) (Zheng et al., 2020). The main nutritional, functional and sensory properties of the fermented snack have been characterized. Aiming at defining the role of the selected starters, the features of the novel yogurtstyle snack were compared to those of the unfermented matrix. Starters survival, suitability to be used as a probiotic carrier and shelf-life of the novel snack in refrigerated conditions were also investigated.
L. plantarum DSM33326 and Le. brevis DSM33325 (Rizzello et al., 2020 patent) belonging to the culture collection of the Celery Srl (Polignano a Mare, Italy) and deposited in the DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) culture collection were used as starter for fermentation. The potential probiotic strain Lacticaseibacillus rhamnosus SP1 (formerly known as Lactobacillus rhamnosus) (supplied by Sacco Srl, Cadorago, CO, Italy), one of the best studied and documented probiotic strains, with more than 300 clinical studies that have proven its efficacy and safety (Segura-Badilla et al., 2020), was added to the novel snack to assess the survival under refrigerated conditions. Strains were routinely propagated in De Man, Rogosa and Sharpe (MRS, Oxoid, Basingstoke, Hampshire, United Kingdom) at 30 • C for 24 h. When used as starters for fermentation, LAB were cultivated until the late exponential phase of growth was reached (circa 16 h), harvested by centrifugation at 9,000 × g at 4 • C for 10 min, washed twice in 50 mM phosphate buffer (4 • C, pH 7.0) and resuspended in the tap water (final cell density of circa 7 log10 cfu/g) used for making the snack. When used, La. rhamnosus SP1 cells cultivated and washed as described above, were resuspended in the snack at the end of fermentation.
Biotechnological Protocol to Make the Novel Yogurt-Style Snack

Processing
The biotechnological protocol to make the yogurt-style snack (YS) is represented in Figure 1. Rice, lentil and chickpea flours were mixed (ratio 2:1:1) prior use. The mixture of flours was mixed (1:4) with tap water and homogenized with an Oster 6805 (Jarden Consumer Solutions Ltd., Cheadle, United Kingdom) mixer. Aiming at starch gelatinization, as described by Lindeboom et al. (2005), the mixture was treated at 80 • C for circa 15 min under stirring conditions (70 rpm). Then, the mixture was first cooled down to circa 4 • C in 2 min and then warmed up to 30 • C prior the inoculum of L. plantarum DSM33326 and Le. brevis DSM33325 (ratio 1:1). The initial cell density of each strain was circa 7.2 log10 cfu/ml. The fermentation was carried out at 30 • C for 16 h. At the end of fermentation, the mixture was cooled down to 4 • C in 5 min, packaged in a glass jar and analyzed within 2 h after FIGURE 1 | Flow-chart of the biotechnological protocol to make the novel yogurt-style snack. fermentation. A YS aliquot added with the potential probiotic La. rhamnosus SP1 (Coda et al., 2011) at the cell density of 9.7 log10 cfu/ml (pYS) was packaged and stored in the same conditions. The apparent viscosity of YS and cYS (yogurt-style snack prior the fermentation) was measured on about 35 ml of the snack, using the A & D SV-10 sine wave viscosimeter (A & D Company Ltd., Japan). Viscosity measurements were carried out on snacks previously maintained at 25 • C for 30 min. Three independent productions were carried out and monitored. Samples were analyzed in triplicate.

Fermentation Monitoring
The pH of the snacks was determined by a M.507 pHmeter (Crison, Milan, Italy) equipped with a food penetration probe. Total titratable acidity (TTA) was determined on 10 g of snack homogenized with 90 ml of distilled water and expressed as the amount (ml) of 0.1 M NaOH to reach pH of 8.3. To determine the cell density of LAB, ten grams of sample were suspended in 90 ml of sterile sodium chloride (0.9%, wt/vol) solution and homogenized in a Bag Mixer 400P (Interscience, St Nom, France) at room temperature. Serial 10-fold dilutions were then plated into MRS (Oxoid) supplemented with cycloheximide (0.1 g/l) and incubated at 30 • C for 48 h.
Kinetics of growth were determined and modeled in agreement with the Gompertz equation, as modified by Zwietering et al. (1990): y = k + A exp{-exp[(µmax or Vmax e/A) (λ-t) + 1]}; where y is the growth expressed as Log cfu/g/h at the time t; k is the initial level of the dependent variable to be modeled (Log cfu/g); A is the cell density (between inoculation and the stationary phase); µmax is the maximum growth rate expressed as Log cfu/g/h; λ is the length of the lag phase measured in hours. Kinetics of acidification were modeled through a negative Gompertz equation. The fitting parameters were modified as follows: negative value of µmax was assumed as the maximal acidification rate (d max, pH/h); λ, the lag phase, as the time (h) before the beginning of the decrease of pH value (α; metabolic adaptation time), and A as the maximum decrease of pH ( pH) (Bevilacqua et al., 2008). The experimental data were modeled by the non-linear regression procedure of the Statistica 12.0 software (Statsoft, Tulsa, United States).

Chemical and Nutritional Characterization
Major components and moisture of the snack were determined as reported for raw materials. Water/salt-soluble extract (WSE) from samples was prepared and used for the determination of organic acids, free amino acids (FAA) and γ-aminobutyric acid (GABA) concentrations. Organic acids in the WSE were determined by High Performance Liquid Chromatography (HPLC), using an ÄKTA Purifier system (GE Healthcare, Buckinghamshire, United Kingdom) equipped with an Aminex HPX-87H column (ion exclusion, Bio-Rad, Richmond, CA), and an UV detector operating at 210 nm. FAA and GABA were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) with a Na-cation-exchange column (20 by 0.46 cm internal diameter), as described by Rizzello et al. (2010).

In-vitro Protein Digestibility and Nutritional Indexes
The in-vitro protein digestibility (IVPD) was determined by the method of Akeson and Stahmann (1964), as reported by Rizzello et al. (2016). The IVPD was expressed as the percentage of the total protein, which was solubilized after enzyme hydrolysis.
Chemical Score (CS) estimates the amount of protein required to provide the minimal essential amino acids (EAA) pattern for adults, which was recently re-defined by FAO (Food and Agriculture Organization) in 2007 (Millward, 2012). It was calculated using the equation of Block and Mitchel (1946), which compares the content of EAA of the YSS for the same amino acid of the reference. The sequence of limiting essential amino acids corresponds to the list of EAA, having the lowest chemical score (Block and Mitchel, 1946). The protein score indicates the chemical score of the most limiting EAA that is present in the test protein (Block and Mitchel, 1946). Essential Amino Acids Index (EAAI) estimates the quality of the test protein, using its EAA content as the criterion. EAAI was calculated according to the procedure of Oser (1959). It considers the ratio between EAA of the test protein and EAA of the reference protein, according to the following equation: The Biological Value (BV) indicates the utilizable fraction of the test protein. BV was calculated using the equation of Oser (1959): BV = ([1,09 × EAAI] -11,70). The Protein Efficiency Ratio (PER) estimates the protein nutritional quality based on the amino acid profile after hydrolysis. PER was determined using the model developed by Ihekoronye (1981): PER =−0,468 + (0,454 × [Leucine]) -(0,105 * [Tyrosine]). The Nutritional Index (NI) normalizes the qualitative and quantitative variations of the test protein compared to its nutritional status. NI was calculated using the equation of Crisan and Sands (1978), which considers all the factors with an equal importance: NI = (EAAI × Protein (%)/100).

Starch Hydrolysis and Predicted Glycemic Index
The analysis of starch hydrolysis (HI) was carried out on YS and cYS. The procedure mimicked the in-vivo digestion of starch (De Angelis et al., 2009). Aliquots of YS and cYS, containing 1 g of starch, were subjected to enzymatic process and the released glucose content was measured with D-Fructose/D-Glucose Assay Kit (Megazyme International Ireland Limited, Bray, Ireland). The degree of starch digestion was expressed as the percentage of potentially available starch hydrolyzed after 180 min. Wheat flour bread (WB) leavened with baker's yeast was used as the control to estimate the hydrolysis index (HI = 100). The predicted GI was calculated using the equation: pGI = 0.549 × HI + 39.71 (Capriles and Areas, 2013).

Total Phenols and Antioxidant Activity
Total phenols were determined on the methanolic extract (ME) of samples prior and after the fermentation. Five grams of each sample were mixed with 50 ml of 80% methanol to get ME. The mixture was purged with nitrogen stream for 30 min, under stirring condition, and centrifuged at 4,600 × g for 20 min. MEs were transferred into test tubes, purged with nitrogen stream and stored at circa 4 • C before analysis. The concentration was determined as described by Slinkard and Singleton (1977) and expressed as gallic acid equivalent. The radical DPPH% was used for determining the free radical scavenging activity (Rizzello et al., 2010). The synthetic antioxidant butylated hydroxytoluene (BHT) was included in the analysis as the reference (75 ppm).

Antinutritional Factors
Raffinose and phytic acid were determined using Megazyme kit K-PHYT 05/07 and Raffinose/D-Galactose Assay Kit K-RAFGA (Megazyme), respectively, following the manufacturer's instructions. Total saponins were determined as reported by Lai et al. (2013) with minor modifications. Briefly, the freezedried samples (0.5g) were mixed with 10ml of petroleum ether by shaking for 4h. The residues (20 mg) were then extracted with 5 ml of 80% (vol/vol) aqueous methanol with shaking for 4h. The extracts were kept at 4 • C in the dark until they were subjected to analysis. Total saponin content (TSC) was determined using the spectrophotometric method (Lai et al., 2013). All data are expressed on dry weight basis. Condensed tannins were determined using the acid butanol assay, as described by Hagerman (2002).

Sensory Analysis
Sensory analysis of YS and cYS was carried out by 10 trained panelists (5 male and 5 females, mean age: 35 years, range: 18-54 years). After a roundtable discussion about the attributes, 16 attributes (Supplementary Table S1) were selected as the most frequently recognized by all the members of the panel. These were included in a score sheet for the quantitative evaluation with a scale from 0 to 10, with 10 the highest score. According to the IFST Guidelines for Ethical and Professional Practices for the Sensory Analysis of Foods, assessors gave informed consent to tests and could withdraw from the panel at any time, without penalty or having to give a reason.

Shelf-Life, Starters and Probiotic Survival
Aiming at investigating the microbiological quality of the yogurtstyle snack and the survival of the inoculated strains under refrigerated conditions, YS, pYS, and cYS were stored at 4 • C for 30 days. The cell density of presumptive LAB, yeasts, molds and Enterobacteriaceae during storage (each 10 days) was determined as reported above. Yeasts were plated on Sabouraud Dextrose Agar (SDA, Oxoid), supplemented with chloramphenicol (0.1 g/l) at 25 • C for 48 h. Molds were enumerated on Potato Dextrose Agar (PDA, Oxoid) at 25 • C for 48 h. Total Enterobacteria were determined on Violet Red Bile Glucose Agar (VRBGA, Oxoid) at 37 • C for 24 h.
To confirm the identity of the starter strains, LAB were isolated from YS, pYS and cYS after 10 (t1), 20 (t2) and 30 (t3) days of storage. At least 15 colonies of presumptive LAB were randomly selected from MRS plates containing the three highest sample dilutions. Gram-positive, catalase-negative, nonmotile rods isolates were cultivated into the same media at 30 • C for 24 h and re-streaked onto the same agar medium. All isolates considered for further analyses were able to acidify the culture medium. Genomic DNA from pure cultures of bacterial strains was extracted using a Bacterial Genomic DNA Isolation Kit 50 preps (Norgen Biotek Corp, Thorold, Canada), according to the manufacturer's instructions. Three oligonucleotides, P4 5 -CCGCAGCGTT-3 , P7 5 -AGCAGCGTGG-3 , and M13 5 -GAGGGTGGCGGTTCT-3 , with arbitrarily chosen sequences, were used for bio-typing LAB isolates as reported by Nionelli et al. (2014b). RAPD-PCR (Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction) profiles were acquired by the MCE-202 MultiNA microchip electrophoresis system (Shimadzu Italia S.R.L., Milan, Italy), using the DNA-2500 reagent kit (100 to 2500 bp) and the 2-logDNAladder (0.1 to 10.0 kb) (Promega S.R.L., Padua, Italy) according to the manufacturer's instructions. The identity of L. plantarum DSM33326, L. brevis DSM33325, and L. rhamnosus SP1 were determined by comparing the RAPD-PCR profile of the inoculated strains with those of the LAB isolated during storage.

Yogurt-Style Snack Production
The proximal chemical composition of flours used in this study is reported in Supplementary Table S2. Rice flour was characterized by a carbohydrates content circa 40% higher than the legumes. On the contrary, these latter contained circa 4-times higher protein than the rice. Chickpea was characterized by the highest content of dietary fiber (up to 4-times) and fat (up to 2-times) (Supplementary Table S2).
After 16 h of fermentation LAB reached a cell density of 9.8 ± 0.1 log10 cfu/g. LAB strains showed a lag phase of circa 2.4 h and µmax of 0.24 log10 cfu/g/h. Along with the microbial growth, acidification was also monitored. Indeed, the pH values decreased from 6.50 ± 0.16 (cYS) to 4.04 ± 0.09 (YS). The decrease occurred with a Vmax of 0.3 dpH/h. TTA increased as results of the fermentation, indeed value of 4 ml NaOH 0.1 M higher than cYS were found in YS. Lactic and acetic acids had concentrations of 11.9 ± 0.2 and 5.4 ± 0.2 mmol/l, respectively, in YS, while they were not detectable in cYS ( Table 1). The fermentation led to an increase of the apparent viscosity which was circa 14% higher than in cYS.

Nutritional Characterization
The nutritional label of the novel yogurt-style snack is reported in Table 2. Overall, it was characterized by low fat (circa 0.57%) and high fiber (circa 4%) concentrations. Protein content was circa 3.2%.
The fermentation led to an improvement of the nutritional profile of the matrix. Total FAA (TFAA) were circa 67% higher in YS as compared to cYS, as the consequence of the proteolysis operated by the selected starters on the native proteins. Significantly higher concentration of all the single amino acids were found in YS as compared to cYS (Figure 2), with a median increase value of circa 1.8-fold. The highest increases were observed for cysteic acid, Met, Thr and Gly concentrations (circa 2-fold compared to cYS), while the highest concentrations (>100 mg/l) were found for Cys and Arg (142.4 and 130.1 mg/l, respectively) (Figure 2).
The intense proteolysis well reflected on the IVPD. A multistep protocol, which mimics the in-vivo gastrointestinal digestion, was used to estimate the snack protein digestibility, that resulted circa 18% higher for YS compared to unfermented cYS ( Table 2). The digestible protein fraction was further characterized. In particular, the amino acid composition and the related CS were calculated, using the FAO reference. Based on CS, the sequence of limiting amino acids included Met, Cys and Trp, although the scores were significantly higher in YS compared to cYS. Indeed, the highest protein score was observed for YS ( Table 2). EAAI and BV, which are commonly used to estimate the quality of food proteins, were higher for YS (circa 13 and 16%, respectively) compared to cYS ( Table 2). The PER of YS was slightly higher than cYS. The NI, the only index calculated on the basis of the amount of digestible protein besides the amino acid composition, resulted circa 63% higher in YS than cYS ( Table 2).
Values of HI, considered to be a presumptive measure of the GI in healthy subjects (Åkerberg et al., 1998), have been found to be circa 30% lower in YS than cYS. Accordingly, the pGI of YS was 53% (circa 13% lower than that of cYS) ( Table 2).
The improvement of the nutritional profile was achieved also through the decreases of the ANF (Table 3). Indeed, the concentration of condensed tannins was circa 90% lower in YS as compared to cYS. Moreover, from 30 to 50% lower values were found for raffinose, phytic acid and saponins in YS compared to cYS ( Table 3).

GABA and Antioxidant Activity
GABA, a functional non-proteic amino acid, was found in cYS at the concentration of 90 mg/ml. Fermentation led to a significant increase to the final content of 110.9 mg/l (Figure 2).  Fermentation also caused the increase of the total phenol's concentration (Figure 3). Overall, such compounds are the main responsible for the antioxidant activity of the food matrices. In particular, the concentration of total phenols in YS was circa 50% higher than cYS. Accordingly, the radical scavenging activity of YS was found to be circa 40% higher than that of cYS (Figure 3).

Sensory Analysis
The sensory profile of the cYS and YS was assessed. Overall, YS was characterized by sensory attributes (acidic and creamy odor and taste as well as the viscosity) considered optimal in yogurt products. Indeed, YS received high scores of pungent and creamy odors (mean value of 8/10) strongly affected by the presence of bacterial metabolites (fermentation) in the final products. Some of these, mainly organic acids, affected also the taste of YS which was found to be more acidic than the cYS (Figure 4). The viscosity and the degree of spoon adhesion were also enhanced through the biological acidification which affected the gel formation and maintenance. Indeed, higher values were found for YS as compared to cYS (Figure 4).

Shelf Life and Probiotic Survival
The microbiological shelf-life of the yogurt-style snacks was assessed by monitoring the survival of molds, yeasts, Enterobacteria and LAB for 30 days storage (every 10 days) under refrigeration conditions. Supplementary Table S3 shows the trends on the microbial groups investigated. LAB in YS remained stable over time, with a cell density of circa 9 log10 cfu/l. The analysis of the RAPD-PCR confirmed the dominance of the two starters. No other LAB strains were present in YS at the end of fermentation and during storage at cell density higher than 4 log10 cfu/ml. An increase of the LAB density of circa 2.5 log10 cfu/l was found in cYS during storage. Increases of the cell density of yeasts and molds were also found in cYS, reaching value of 5.40 ± 0.67 and 5.06 ± 0.21 log10 cfu/l, respectively, after 30 days of storage. In YS, yeasts number reached value of 3.15 ± 0.23 log10 cfu/l while no growth was found for molds. Enterobacteria were found to be <10 cfu/10 ml in both cYS and YS (Supplementary Table S3).
Aiming at investigating the suitability of the YS to be used as carrier for probiotics, the survival of La. rhamnosus SP1 was also investigated. The cell density of the probiotic strain in pYS slightly decreased from circa 9 to 8 log10 ufc/l (Supplementary Table S3).

DISCUSSION
To date, the "healthy living" has been defined as the most impactful trend influencing the food industry (Gelski, 2019); indeed, consumers show preferences for minimally processed, closer-to-nature foods. Moreover, veganism and free-from foods trends are gaining popularity forcing industries to the production of novel foods (Costa and Johnson, 2019). Naturally nutrientrich whole or minimally refined grains perfectly meet these needs and the industrial success in the commercialization of plantbased beverages suggest that novel uses of cereals, legumes and pseudocereals can provide a suitable alternative for consumer demands (Coda et al., 2017;Costa and Johnson, 2019).
Nevertheless, the protein in grains is not always comparable to traditional sources (mainly animal) and they often contain ANF which impair the nutritional properties as well as the palatability of these products. Fermentation, mainly with LAB, has been shown to be one of the most suitable unit operations used for processing grains since the onset of agriculture having a positive impact on the nutritional and sensory profiles of the grains-derived foods (Ganzle, 2019).
Here, a protocol to produce a milk-alternative yogurtstyle snack made using gluten-free flours is described. The formulation, composed of rice, lentil and chickpea flours (2:1:1) and water (1:4) as well as the thermal protocol (80 • C for 15 min) have been previously selected (Savijoki et al., 2006 patent) according to the optimal textural (e.g., viscosity) and sensorial properties of the products (data not shown). Moreover, the choice of the most suitable microbial starters, L. plantarum  DSM33326 and Le. brevis DSM33325, was made according to their metabolic traits influencing the nutritional, chemical and sensory features of the final products (high acidification and proteolytic activity, capability to cause the degradation of ANF as observed in different legume-derived matrices) (Rizzello et al., 2020 patent). The use of selected LAB in making novel plant-based beverages has already been recognized as the most efficient tool to drive and standardize the fermentation, to confer specific nutritional properties, such as the pre-and probiotic benefits, and to improve the sensory profile of the raw ingredients (Coda et al., 2017;Gobbetti et al., 2019a).
In this work, aiming at highlighting the effects of the fermentation, the nutritional, functional and technological features of a novel yogurt-style snack were compared to those of an unfermented matrix, however, obtained with the same formulation and technological processes.
Overall, the formulation allowed the obtainment of a lowcalories and low-fat snack. Indeed, the energy value of the snack was 67.7 kcal on 100 g with circa 0.6% of fat. Rice flour was mainly used to confer a creamy structure, thanks to the abundance of gelatinizable starch. The use of lentil and chickpea flours allowed the enrichment in dietary fibers, that resulted markedly higher compared to the conventional milk-derived products, and the increase of protein concentration and quality compared to other cereal-derived beverages (Coda et al., 2017).
Dietary fibers were circa 4%, corresponding to 6 g on 100 kcal, while proteins were circa 3%, which covered more than 20% of the energy value of the product. According to European Regulation EC No. 1924/2006, 2006, on nutrition and health claims on food products, the novel yogurt-style here proposed snack can be defined as low in fat, high in fiber and protein.
Along with the common characteristics associated to the lactic acid fermentation, i.e., decrease of pH due to the synthesis of lactic and acetic acids, the YS was characterized by enhanced FIGURE 3 | Total phenols (bars) and radical scavenging activity (stars) of the novel yogurt-style snack (YS) and the unfermented matrix (cYS). Fermentation was carried out in triplicate at 30 • C for 16 h.
The dependence of the lactic acid bacteria on a complex proteolytic system to successfully ensure the fermentation processes, has widely been exploited for the improvement of the nutritional quality and bioavailability (peptides and free amino acids) of the protein in plant-derived matrices (Gobbetti et al., 2019a). In particular, the intense proteolysis operated by L. plantarum DSM33326 and Le. brevis DSM33325 during fermentation of the cereal/legume substrate led to a very high content of free amino acids (circa 1.1 g/l), that resulted more than 50-fold higher compared to a conventional milk yogurt (Tamime and Robinson, 1999), and 5-fold higher than a cereal yogurt-like beverage made with oat flakes (Nionelli et al., 2014a).
GABA, a functional amino acid present at relatively high concentration in legume flours (Nikmaram et al., 2017) and released from LAB through to the glutamate decarboxylase (GAD) activity (Renes et al., 2017), was at concentration higher than 100 mg/l in YS. Such concentration is potentially able to confer functional effects (Inoue et al., 2003). GABA is the major inhibitory neurotransmitter of the central nervous system and has several beneficial properties such as antihypertensive, prevention of diabetes, diuretic and tranquilizer effects (Diana et al., 2014).
As previously observed , proteolysis occurring during fermentation affected level of protein digestibility, that resulted, in the novel yogurt-style snack, circa 18% higher than the unfermented matrix. Aiming at investigating the protein quality of the novel yogurt-style snack, the digestible protein fraction was used for the determination of the protein quality indexes (Rizzello et al., 2014).
Thanks to the complementarity of the essential amino acid composition of the proteins from cereal (rice) and legume (lentil/chickpea) ingredients (Stone et al., 2019), YS had a very high EAAI. It resulted circa 54% higher than that found for a similar yogurt-like snacks made with quinoa, a pseudocereal overall known for the high quality of the protein fraction (Lorusso et al., 2018). Accordingly, all the indexes elaborated based on the comparison of the essential amino acid ratio to the FAO reference protein (Millward, 2012) resulted higher than those found for food products obtained from a single cereal, legume or pseudocereal matrix (Lorusso et al., 2018;Rizzello et al., 2019;Sozer et al., 2019). Within the indexes that are used to evaluate the nutritional value of foods, the NI combines qualitative and quantitative factors and is considered a global predictor of the protein quality. Since the higher protein bioavailability due to the extensive proteolysis caused by the selected starters during fermentation, the NI value of the yogurt-style snack here described was markedly higher than the unfermented control.
Besides the proteolysis caused by LAB, the higher value of the IVPD in the yogurt-style snack may also depend on the decrease of ANF, also occurring during fermentation. Indeed, legumes flours (lentil and chickpea), are overall characterized by the presence of ANF which may negatively affect the digestibility and the sensory properties of the derived products (Joye, 2019). Excluding trypsin inhibitors, that are thermolabile, condensed tannins and phytic acid are known to strongly complex the protein molecules, thus decreasing their bioavailability (Samtiya et al., 2020). Lactic acid bacteria fermentation has largely been proposed as tool to decrease such ANF; indeed, YS was characterized by 90% and 40% lower values of the condensed tannins and phytic acid, respectively, compared to the control. It was previously found that lactic acid bacteria can directly hydrolyze the complex tannins-protein through tannase activity (Pranoto et al., 2013) and can contribute to the phytic acid degradation providing microbial phytases (Karaman et al., 2018;Väkeväinen et al., 2018). Decreases of saponins and raffinose, due to the fermentation process have also been found. Although have some interesting biological properties (e.g., increase the permeability of the small intestinal mucosal cells, antifungal activity and lowering blood cholesterol) and industrial relevance (e.g., preparation of soaps, detergents, etc.), saponins are considered as antinutrient and flavor factors (Arendt and Zannini, 2013). Indeed, high concentration of saponins lead to decrease in mineral and vitamin bioavailability and astringent or bitter taste of the products (Cheeke, 2000;Tarade et al., 2006). YS contained circa 30% lower content of saponins compared to the control. Similar trend was found for raffinose, an α-galactoside not digested by pancreatic enzymes and metabolized by bacteria of the large intestine with gas production. Raffinose and similar galactosides present in legumes, like stachyose and verbascose, are subjected to enzymatic hydrolysis by LAB (Coda et al., 2017). Lower content (circa 50%) of raffinose in YS as compared to cYS may suggest an increased product digestibility and reduced digestive discomfort (Filannino et al., 2015).
Compared to the unfermented matrix, the concentration of total phenols in YS increased significantly. A similar phenomenon was already observed during legume fermentation by LAB (Gobbetti et al., 2019b), as the consequence of the combined effects of acidification, affecting solubility, and microbial hydrolytic enzymes that further promotes the enzymatic release of free phenolic compounds from glycosylated and more complex forms (e.g., trough feruloyl-esterase and β-glucosidase activities) (Filannino et al., 2015). As estimated toward DPPH radical, the increased concentration of total polyphenols observed after fermentation, corresponded to a proportional increase of the antioxidant activity.
The HI of YS was 30% lower than the unfermented matrix which led to a pGI of 53.4 ± 0.8. According to the Harvard Medical School recommendation which ranks foods with a GI ≤ 55 as low, between 56 and 69 as moderate and ≥ 70 as high GI foods, YS can be recognized as "low GI food". Although promising value of the pGI were collected, in-vivo GI testing as described by the Regulation ISO 26642 (International Organization for Standardization) defined by FAO (Food and Agriculture Organization of the United Nations) and WHO (World Health Organization) need to be carried out. Overall, the gelatinization leads to changes in starch structure resulting in rapid degradation with release of fermentable sugars (Poutanen et al., 2009) from which mainly depend the HI and pGI of the cYS. LAB, fermenting sugars may have lowered the pGI of the product.
The quality of food products relies also on its the sensory acceptability as well as the shelf-life (Peri, 2006). Evaluation of the sensory profile and microbiological quality during storage have been carried out. Overall, YS was more like yogurt mainly due to the better odor, taste and texture characteristics.
Yogurt typically possess a smooth, viscous gel with a sharp acidic taste mainly due to the lactic acid (Das et al., 2019). The role of the fermentation was highlighted by the high scores (circa 8/10) of pungent and creamy odors which mainly rely on presence of organic acids, diacetyl and acetaldehyde, respectively, produced by microbial metabolisms (Wang et al., 2019). However, the role of proteolysis in the releasing of volatile compounds and precursors cannot be overlooked (Das et al., 2019). Organic acids, mainly lactic, release by the selected strains, also contributed to higher score of acidic taste in YS. Although it is not typically associated with the aroma profile of yogurt, lactic acid plays a vital role in the overall flavor profile of yogurt (Das et al., 2019). Moreover, the low pH (circa 4) of the YS might have contributed to the improved viscosity and adherence to spoon. Here, texture characteristics depend on the starch gelatinization and it was previously observed that pH values lower than 4 allow the preservation of the viscoelastic properties of the starch gel during long-storage period (Hirashima et al., 2012). The YS sensory profile was judged stable during the storage (data not shown), in agreement with previous studies showing the high sensory stability during shelflife of vegetable beverages fermented with LAB (Peyer et al., 2016;Lorusso et al., 2018).
Moreover, the persistence of LAB at high cell density and the low presence of yeasts and molds in YS through the 30 daysstorage under refrigerated condition suggest the stability of the microbiological quality. Although the determination of the main flours contaminant, Bacillus cereus, was not performed, the synergic effect of the refrigeration conditions and acidic environment is already reported as effective toward its spore germination and growth (Valero et al., 2003;Franceschini et al., 2020). Contrary to what observed in YS, a relevant presence of yeasts was found in cYS thus potentially compromising the quality of the product. However, under the study conditions, no undesirable odor and taste have been perceived by the panelists. The use of milk alternatives products as carrier of functional ingredients represents an interesting and promising approach to produce novel functional foods (Sethi et al., 2016). In this framework and aiming at evaluating the preliminary suitability of YS to be used as probiotic carrier, La. rhamnosus SP1, a well-known commercial probiotic strain, has been included in the formulation and its survival (during storage) investigated. The high cell density (circa 8 log10 ufc/l) found in the pYS at the end of the storage suggest that the YS may be considered adequate as vehicle for probiotics (WHO/FAO, 2002). However, further investigations on the role of the probiotic strains on the properties of the yogurt-style snacks may be required.

CONCLUSION
Here, a novel gluten-and lactose-free yogurt-style snack was produced and characterized following an integrated approach including the evaluation of the main nutritional, functional, technological and sensory features. The novel product, conceived to be similar to conventional yogurt for the high density of viable LAB and the creamy structure (or texture), combines the advantages deriving by the use of a mixture of cereal and legume flours (high concentration of dietary fiber and an overall high biological value of the total proteins), and those deriving by the fermentation of the raw ingredients operated by selected strains of LAB (increase of the free amino acids and GABA content, increase of protein digestibility and nutritional indexes, enhancement of the antioxidant activity, decrease of the starch hydrolysis index and antinutritional compounds, and overall sensory appeal). The biotechnological protocol for making the novel snack here proposed is suitable for the large-scale application in food industry, giving a platform product largely customizable, for example with additional pro-and pre-biotics, and many other ingredients (e.g., fruit).

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/Supplementary material.

AUTHOR CONTRIBUTIONS
EP carried out the data elaboration, writing, and review and editing of the manuscript. SR and CD carried out the formal analysis and data curation. DC and VC were responsible for the development of the biotechnological protocol for making the snack. CR conceived the experimental design and was the scientific advisor. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
Celery SRL is gratefully thanked for supporting the development of the protocol to make the snack and the snack production at laboratory and pilot-plant scales.


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Morphological traits of Ferula foetida (Bunge) Regel. in the desert of Mangystau

The morphological traits of growth in aboveground and underground organs of Ferula foetida found in Mangystau, depending on the age and place habitat of the plant, have been studied for the first time. A very high variability (up to 54-98%) of the great majority of the morphological traits, except for sizes of leaves of the 1st rosette and length of the main root before branching (12-26%), is noted for all the studied populations, which is explained by almost proportional participation in formation of composition of populations with species of different age. Meanwhile, variation of isomers in both, aboveground and underground parts within each age-grade declines by several-fold (<10%) and remains stable regardless of the living environment.

Introduction
Ferula foetida Bunge (Regel) is a medicinal and edible plant [1,2]. The herb is used in medicine in the East, which is defined by its rich composition of bioactive substances accumulated mainly in the underground organs and in fruits. Composition of the gum resins include mainly resin (up to 50-70%), gum (up to 35-40%) and ether oils (up to 10%), as well as vanillin, free ferulic acid and other components. Ferula is a good forage plant and is edible for people as well -leaves and shoots of some types of F.foetida are consumed in fresh, cooked, fried, pickled forms and as a spice [3][4][5]. In arid Mangyshlak, asafetida is very common in the Eastern Mangyshlak geobotanical zone [6], in the southern part of the Mangyshlak peninsula, in the Northern Ustyurt on argillaceous plains, on fixed and semi-fixed sands of central and southern desert areas. F.foetida is confined to sandy and rubble-clayey soil in deserts. Commonly, this specimen forms large populations acting as a dominant. Over 20 populations of F.foetida overtake large areas and reproduce very well in natural conditions annually. Its morphological traits in different populations are diverse which is related with the growing environment.
The objective of this survey was the study of morphological traits of F.foetida in natural habitats in the Mangystau region.

Herbal raw material
During expeditions in 2015-2018, 6 multiple-aged (from 1 to 7 years) natural associations of F.foetida located in sandy massifs of Tuyesu and Karynzharyk, western and southern parts of Tynymbai Shoky upland, Karaadyr tract, on the northern shoulder of the Burma mountain, were selected for studying the morphological traits.

Study methods
The examined populations with participation of F.foetida were exposed to geobotanical descriptions in accordance with the standard practices [7][8][9], characteristics of soil coverage based on the selected soil and analyses of its salinity and aquatic-physical indicators [10]. During the studies of asafetida morphology, measurements of 6 aboveground and 10 underground growth indicators (Table 1) were made across the whole age interval -from one to seven years. The selected material was processed with the use of mathematical statistics and the application Stat Graphics Centurion XVI.I (2011).

Description of territories
Tuyesu sands are located in the southern part of Mangyshlak peninsula, where F.foetida participates in saxaul-ferula-sagebrush-psammophytic-shrubby associations. Haloxylon aphyllum acts as the dominant in the association, F.foetida is a co-dominant. The generic projective coverage (GPC) is 50-65%. The soil is sandy and loamy, mainly non-saline or sub-saline, supplied with shallow fresh water. The groundwater depth is 3.5(5.0)-41 m. Tynymbai Shoky upland is also assigned to the southern regions of Mangyshlak peninsula. The F.foetida population occurs within saxaul-ferula and sedge-white-ground-sagebrushferula associations. The soil significantly varies in its mechanical composition and salinity. It is sandy to the depth of 30 cm, light loamy from 30 to 50 cm, and middle loamy in the interval from 50 to 100 cm. The salinity of soil from the surface to the depth of 50 cm is low, average in the interval of 50-80 cm; and high on depth of 80-100 cm. Water content is extremely low; the plants draw water from precipitation and shallow ground water of different salinity. Karynzharyk sands are located in the southern-western part of the Karynzharyk hollow, in the southern-karynzharyk region of Mangystau. The soil is sandy and loamy, non-saline across the whole genetic profile. Karynzharyk sands are poorly flooded or arid; the plants draw water from precipitation and shallow ground water of different salinity. Ground water occurs in individual small areas at the depth available for desert plants. Thickets of F.foetida are widely common within kemrud-sagebrush-ferula-grasses-saxaul and sedgekemrud-sagebrush-thirst-quenching-saxaul associations. GPC is 50-65%. F.foetida has been identified on loamy massifs in kalligonum-shrubby associations in the vicinity of Karaadyr tract (8-10 km southward the Karynzharyk sands). The soil there is non-saline from surface down to 80 cm, with low salinity within the interval of 80-100 cm. Water supply to the natural populations is very low; the plants draw water only from precipitation. Water permeability of the soil is high. In the area of Burma hill, associations with participation of F.foetida were identified on low gullied slopes with sub-saline semi-consolidated clay-loamy soil. Thinned thirst-quenchinggurgan-sagebrush-grasses populations are specific for this area with participation of shrubs -goat's wheat and shrubby bindweed. The area has low water content. The soil is nonsaline from the surface down to 50 cm; with average salinity in the interval of 50-80 cm and low salinity in the interval of 80-100 cm. Water permeability of soil is average.

Results and discussion
F.foetida is a perennial monocarpic plant which annually generates bottom leaf rosette and increases the root volume. The plant develops most actively during its first 3-4 years. During the 5-7 years of vegetation, the plant enters the reproductive period; it flowers and bears only once. Nutriments accumulate in the root during the whole life cycle and then are used for formation of the stem with reproductive organs, after the root completely perishes [11].
The results of the surveys demonstrated vast range of variability: height of the plants (20-89 cm) and root depth (34-155 cm) in Tuyesu sands; 17-69 cm and 44-162 cm in Tynymbai Shoky uplands; 19-72 cm and 35-142 cm in Karynzharyk sands respectively. By the average height (Table 1), it is noted that the plant grows most intensively in the populations in Tuyesu sands (63.57 cm), slower in Karynzharyk sands (51.44 cm). The shortest plants grow in loamy massifs of Tynymbai Shoky uplands (49.20 cm). The ratio between the associations in the depth of roots is a bit different: Tynymbai Shoky upland (122.07 cm), Karynzharyk sands (102.83 cm) and Tuyesu sands (92.66 cm). The difference in height and root depth between the populations is statistically significant at 5% level of significance. It has been established that if the correlation ratio between the morphology data within one population is in average 0.8-0.9, then in whole for all the studied associations it decreases to 0.5-0.6 due to influence of a number of soil and reclamation factors: water availability, mechanical composition, salinity, alkalinity. The most intensive growth of roots in Tynynbai Shoky upland is due to adaptive reaction to deeper ground water and favorable impact of the ratio between the upper soft sand and lower middle loamy layers of the soil. Weight of the underground part of F.foetida takes up to до 40-85% of the total raw phytomass of the plants (Table 1), where its largest share is steady in the habitats with fixed sandy and loamy soil of mild rockiness and deep ground water -Tuyesu sands and southern part of Tynybai Shoky upland. Not always strong development of aboveground phytomass can guarantee more intensive formation of the root system. This is influenced by adaptive reaction of F.foetida to formation of a stronger succulent underground structure for accumulating moist and its rational use through a weaker assimilative mechanism of the plants according to the specific habitat conditions. During entering into the reproductive period (from 5-6 years), a gradual growth inhibition is observed regardless of soil and reclamation conditions in the natural associations. Just the opposite tendency has been identified for the weight of roots, whose formation is the most active from 4 years to the end of life cycle of the plant. The most actively F.foetida develops during the first 3-4 years in the majority of its morphological traits including height of the plant and depth of the roots. Starting from 5-6 years, gradual growth inhibition, and even growth arrest, occurs regardless of edaphic conditions in the natural populations. Just the opposite tendency has been identified for the weight of roots, whose formation is the most active from 4 years to the end of life cycle of the plant. It has been identified that regardless of the habitat conditions, F.foetida annually forms one bottom leaf rosette and one branching of the root. A very high variability (up to 54-98%) of the great majority of biometric parameters, except for sizes of leaves of the 1 st rosette and length of the main root before branching (12-26%), is noted for all the studied populations, which is explained by almost proportional participation in formation of composition of populations of different age.
Meanwhile, variation of isomers in both, aboveground and underground parts within each age-grade declines by several-fold (<10%) and remains stable regardless of the living environment. Just the opposite tendency has been identified for the weight of roots, whose formation is the most active from 4 years to the end of life cycle of the plant.

Conslusion
Following the surveys conducted in the arid conditions of Mangystau, it has been established that in the majority of cases the difference among the main growth indicators of F.foetida is morphologically similar. It confirms the assignment to the strictly defined ecological niche with sandy and strongly stony soils. Specimen of different age take almost proportional participation in formation of the composition of F.foetida populations, which explains very high variability (up to 54-98%) of isomers of growth, although variability of morphological traits inside each age group significantly decreases (<10%) and maintains at constant level for the whole profile of habitat conditions in Mangystau region. It has been established that up to 40-85% of total phytomass falls on the weight of underground part of the most medicine-valuable F.foetida, and its maximal specific weight is observed in the habitat conditions with fixed sandy and loamy soil of average rockiness and with deep ground waters. Thereby, strong development of aboveground part of F.foetida does not always ensure intensive formation of root rhizosphere, since the adaptive reaction takes place through formation of a succulent root structure for accumulating moist and its rational evaporation via a weaker leaf system. This work was supported by a state grant from the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (AP05130892).


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Study of the Processes of Electroosmotic Cleaning of Soils from Oil Pollution on a Three-dimensional Physical Model

The methods and results of laboratory modeling of processes in clay soils contaminated with oily substances are reviewed: on samples, on one-dimensional and three-dimensional physical models. Laboratory facili- ties, the procedure for conducting experiments and processing their results are described. The possibility of a point and integral forecast of the degree of soil contamination by the results of measurements of its electrical re- sistance is confirmed. The dynamics of changes in porosity, humidity, and particle size distribution of oil-contaminated soils during electric treatment as a result of thermal and osmotic effects by comparing direct measure- ment data and electrophysical monitoring using a system of micro-sensors was revealed. A comparison of the results of the electric treatment of clean soil, contaminated with liquid and viscous oil products is given.

Introduction
One of the most common pollutants is oil and petroleum products. Accidents at oil producing enterprises during the transportation of oil lead to disastrous consequences. In the absence of emergency situations, oil pollution of soils occurs in mass volumes. So, in the mining and coal processing enterprises of Kuzbass, such events occur at stationary and mobile fuel and oil filling stations, fuel and lubricant warehouses, and a significant number of contaminations occur in the soil foundations of existing buildings and structures, which complicates or eliminates their mechanical removal for decontamination [1].
Methodological approaches to solving the problem of soil cleaning from contamination are considered in the fundamental monograph [2]. Modern studies conducted by various organizations are aimed at the development of physicochemical [3,4], biological [5,6,7] decontamination methods or their combination [8,9]. At the same time, it is emphasized that with the exposure to point (local) sources of pollutant emission the importance of monitoring methods increases (location of pollution zones, control of changes in the concentration of ecotoxicant) [10,11].
Promising is the application of the electrochemical method used in deactivating treatment of low-permeable soils, combining high-density current passing through soil, electroosmotic saturation of soils with an active liquid, dilution of viscous components or deactivation of the pollutant, and removal of solutions containing dissolved pollutant. The experimental and theoretical foundations of electroosmotic processes described in [12] have found practical application in land reclamation (drawdown and dewatering) and construc-tion (consolidation of soil with chemical solutions). Due to the complexity and insufficient knowledge of a number of physical and chemical processes that occur during electric treatment, as well as the energy intensity of this method, it has not yet become a frequent practice.
The idea of using the electrochemical method for cleaning soils from oil pollutants and other ecotoxicants is being developed at Moscow State University under the guidance of Professor V. A. Korolyov. The results of fundamental studies of electrosurface phenomena in clayey rock proved the practical significance of this research line [13][14][15].
Another unsolved problem is the control of processes of soil treatment at its various stages; this area of research is being developed in the Geocontrol Laboratory of KuzSTU.

Method of research
To identify patterns of changes in the physical properties of soil during electroosmotic cleaning up from oil pollution, the studies were conducted in the laboratory of KuzSTU on the experimental installation, the scheme of which is shown in Fig. 1.
The main objectives of the experimental studies were as follows: -Study the change patterns in the physical properties of soil during electroosmotic cleaning up from oil pollution; -Study the physical parameters of the soil being cleaned; -Study electrocoagulation processes that contribute to the transition of petroleum products into a solid state.
The physical model is a section of the electrochemical cleaning zone with the actual dimensions of the electrodes and the distance between them in a horizontal section. As the test medium, compacted loam mixed with a mixer with oil was used. The concentration of the oil contaminant corresponded to what was practically possible during gradual pollution or during an oil spill as a result of an accident. Thus, none of the physical parameters of the model differed from the corresponding parameters of nature. A bathtub of 1000x500x360mm in size is made of a non-conductive material -laminated, moisture-proof plywood, waterproofed with a plastic film and filled with loam. Electrodes made in the form of perforated steel electric-welded tubes with an inner diameter of 51 mm blanked off at the bottom with a wooden plug were immersed in the soil to a depth of 300 mm.
The experimental technique included the measurement of the electrical resistivity of contaminated soil with the help of probes made of four-core copper cable. Microprobe sensors with a contact spacing of AM = MN = NB = 10 mm are immersed in soil to a depth of 180 mm. The total number of sensors is 22. The measuring device is a KP-2 logging tool that implements direct current resistivity measurements in pulsed mode with automatic polarization compensation developed at KuzSTU. The remaining electrical characteristics of the soil and installation were measured with a MY64 electronic multimeter. The electric field in the model was created by a stabilized power supply unit with a voltage of U = 50-300 V, with a current strength of up to 0.6 A. Graphs of changes in the relative electrical resistivity along the main axis of the electric treatment at its various stages are shown in Figs. 2 and 3.
The physico-mechanical characteristics of the soil were determined using the following instruments and accessories of the PLL-2 field laboratory: a set of sample bottles for determining moisture, an electronic balance, an oven, a set of sieves.
At the first stage of the experiment, two baths were processed in parallel. The first was loaded with pure clay loam weighing 260 kg, mixed with water (52) kg, and the second was loaded with clay loam weighing 250 kg, mixed with water (47 kg) and waste oil as an oil contaminant (8.1 kg).

Results and Discussion
From the above experimental results, it follows that the electroosmotic processes in the near-electrode regions during the processing of clean and oil-contaminated soil are interrelated and have significant differences due to the difference in the electrically conductive properties of the aqueous solution (low resistivity) and machine oil (dielectric): -in pure soil (Fig. 2), a progressive movement of moisture from the cathode to the anode occurs, accompanied by a decrease in resistivity in the zone of moisture accumulation and an increase in resistivity in the electric drying zone, while the anode drainage zone exceeds the moisture saturation zone along the length of the water, and the formation of these zones occurs sufficiently quickly, 30-35 A·hours from the start of electric treatment; -in the contaminated soil (Fig. 3), the wave-like nature of the change in resistivity is recorded, due to the fact that the viscosity of the oil is much higher than that of the aqueous solution, while the positive peaks of resistivity, corresponding to the predominance in the pores of the oil product, and negative, corresponding to the predominance of the aqueous solution, become more contrasting and move to the anode zone; in the cathode region during the outflow of moisture, the electrical resistivity changes insignificantly, which indicates the predominant effect on the insulation of mineral soil grains with an oil film.  The following features of the formation of near-electrode volume zones are established: -around the anode in clean soil there is an asymmetry of resistivity, a zone of moisture accumulation with =0.6-0.7 corresponds to a circular sector in the range , from the lateral and reverse directions the outflow of fluid is recorded; a zone of thermal drainage of the soil is formed immediately near the cathode electrode (r = 90 mm), but as the distance from the axis of the electrode increases, the anisotropy of the electrical resistivity is significantly smoothed; -in a contaminated soil in the cathode zone, a pronounced layer of thermal drainage is not fixed, otherwise the structure of this zone does not differ from the clean soil; in the anode volume, the oil product accumulation sector reaches .   The results of studies of the physical properties of the soil before and after treatment are given in Table 1, and the particle size distribution of the soil is shown in  From the data in Table 1 it follows that in both soils during electrical treatment, chemical bonding of moisture and oil product occurs, which leads to a general decrease in soil moisture by 5-20% and a decrease in its density by 2-10% in both wet and dry conditions. Very significant changes are established in the particle size distribution of the soil: -in clean soil, only in the anode zone, a sharp decrease in the content of the fraction <0.1 mm and an increase in the fraction of 2.0 mm were established; -in contaminated soil throughout the treatment area there is an increase in particle size with the transition of the fraction <0.1 mm to the range 0.1-0.5 mm due to oil coagulation processes; sticking of small particles together, while the transition of the oil product from a liquid to a solid state is probable, which is considered less toxic and environmentally harmful.

Conclusion
An analysis of the results allowed us to draw the following conclusions: -when an electric current is passed and oil products in the soil pores are exposed to it, they turn into a solid cohesive state, which leads to an increase in resistivity of the soil throughout the treatment area, as well as to a decrease in humidity; -as a result of the electrolytic effect of direct current on the contaminated soil, the formation of solid particles and the adhesion of these particles occur, which leads to a change in the particle size distribution of clay soil in the direction of increasing the content of larger fractions; -when treating soil with electric current, soil moisture decreases, and an increase in the percentage of large fractions leads to a decrease in soil density, both in wet and in dry condition; -the process of phase transformation of an oily product in the soil pores due to electrical and thermal coagulation occurs throughout the inter-electrode space. The study was conducted as part of grant MK-1212.2020.5 "Geological and geophysical monitoring of the processes of electrochemical cleaning of soil bases of structures from oil pollution".