Biologically active beads of cellulose acetate and thymol, process for their production, their use and application process

Abstract

The invention relates to cellulose acetate beads impregnated with thymol (CAB-T), which have the property of long-term and controlled thymol release, lasting at least 40 days and even beyond 3.5 months, in air, the process for their production, as well as their use as biologically active beads With antimicrobial, insecticidal, and repellent properties, along with a stimulating effect on seed vitality and germination.

Description

[0001] BIOLOGICALLY ACTIVE BEADS OF CELLULOSE ACETATE AND THYMOL, PROCESS FOR THEIR PRODUCTION, THEIR USE AND APPLICATION PROCESS

[0002] Description

[0003] Field of invention

[0004] The invention relates to biologically active preparations of thymol, a process for obtaining the same, and their application in phytomedicine, agronomy, and the food industry.

[0005] Technical problem

[0006] The technical problem solved by the present invention is a biodegradable preparation based on cellulose acetate beads impregnated with thymol (CAB-T), produced by green technology without the use of organic solvents and without the generation of waste water, providing a prolonged release of thymol in air, and thus a long-lasting antimicrobial, insecticidal and repellent action, and at the same time a biostimulating effect on vitality and germination of seed of various plant species, and not being toxic to vertebrates.

[0007] Another problem solved by this patent application is obtaining cellulose acetate beads with the desired properties mentioned above, whereas conventional methods of impregnation of porous materials imply the use of large amounts of organic solvents, which subsequently need to be removed and disposed of. In addition, materials obtained through conventional methods do not have a homogeneous distribution of active substances, with the largest amounts deposited on the surface, resulting in their quick release. However, by using supercritical CO2and specific conditions of the supercritical impregnation process, all mentioned shortcomings are overcome, and the clamed material with specific properties is obtained.

[0008] State of the art

[0009] The US Patent US 5888930 (Smith et al., Asymmetric microporous beads for controlled release. Application date: 13.03.1997) describes asymmetric microporous particles for controlled release of pheromone gossyflur and synthetic insecticide diazinon for protection from agricultural pests. However, the particles of this invention exhibit asymmetric microporosity (without specifying porosity and pore size) and are obtained by dissolving a polymer in a suitable organic solvent, spraying the resulting solution into water, and then drying the obtained particles. After that, the particles are impregnated with the active component from the solution. Additionally, their proposed active component, the insecticide diazinon, is toxic to vertebrates.

[0010] However, the supercritical impregnation method, by which CAB-T was obtained, enabled the even distribution of thymol and a uniform distribution of pores with the size of about 0.4 pm, which resulted in a material capable of releasing thymol continuously for a period of more than 40 days, ensuring a prolonged insecticidal effect on the bean weevil. The Patent Application US 20100272818 Al (Franklin et al. Compositions and methods comprising terpenes or terpene mixtures selected from thymol, eugenol, geraniol, citral, and L-carvqne, Application date: 30.11.2005) describes a material based on thymol and other terpenes encapsulated in yeast cell wall glucan particles that can be used for plant protection as a fungicide and nematicide, but also in medical and veterinary practice. Thymol was already released completely from these particles after 48 h, and with later refinement of this encapsulation method the release time; was extended to 16 days (Soto et al. 2021, Yeast particle encapsulation of scaffolded terpene compounds for controlled terpene release. Foods, 10(6), 1207).

[0011] In the prior art, the effects of thyme essential oil and its dominant component thymol on the life history traits, behavior and physiology of the bean weevil (Acanthoscelides obtectus Say), a major insect pest of beans, are known. Given that these natural products, when applied directly to bean grains (Phaseolus vulgaris L., c.v. "gradistanac"), have been shown to reduce the survival and lifespan of adults, have an oviposition deterrent effect, induce oxidative stress and inhibit the in vivo activity of enzymes important for the transmission of nerve impulses (acetylcholine esterase) and antioxidative protection (glutathione S-transferase) (Lazarevic et al., 2020, Toxic, oviposition deterrent and oxidative stress effects of Thymus vulgaris essential oil against Acanthoscelides obtectus. Insects, 11, 563; Lazarevic et aL, 2022, Assessment of sex-specific toxicity and physiological responses to thymol in a common bean pest Acanthoscelides obtectus Say. Frontiers in Physiology, 13, 842314), it was concluded that thymol, a monoterpene phenol, has the potential for the development of contact insecticide / repellent that could be used to protect bean seeds in storage. However, preliminary research indicated that thymol quickly loses its insecticidal activity (< 12 h) due to its high volatility.

[0012] Furthermore, a porous material (CAB - cellulose acetate beads), which enables the controlled release of thymol in a liquid medium (water, hydrochloric acid solution, and phosphate buffer solution), is known in the state of the art. However, the application itself involves dissolving the particles, treating with a solution, which limits the application and results in reduced efficacy (Milovanovic et al. Supercritical impregnation of cellulose acetate with thymol. The Journal of Supercritical Fluids 97 (2015), 107-115).

[0013] In the present invention, conditions for impregnation of CAB with a controlled amount of thymol were unexpectedly found, so as to obtain cellulose acetate beads impregnated with thymol (CAB-T), which have the property of prolonged thymol release for at least 40 days, but in air, significantly increasing the possibility of application in phytomedicine and agronomy, and making the application itself more effective. The controlled, uniformly impregnated amount of thymol in newly obtained CAB-T enabled its prolonged and controlled effect on the bean weevil and bean seeds.

[0014] Summary of the invention

[0015] The invention relates to cellulose acetate beads impregnated with thymol (CAB-T) with the property of long-term (prolonged) and controlled release of thymol (at least 40 days and even longer than 3.5 months) in air, the process of obtaining the same, as well as their use as biologically active preparations with antimicrobial, insecticidal, repellent properties, as well as a biostimulating effect on seed vitality and germination. Brief description of the drawings

[0016] Figures 1 A-C show the appearance of cellulose acetate beads (CAB) before impregnation (Figure 11A), as well as their surface (Figure IB) and cross-sectional (Figure 1C) images, obtained using scanning electron microscopy.

[0017] Figures 2A-C show the appearance of cellulose acetate beads after the impregnation with thymol T) (Figure 2A), as well as their surface (Figure 2B) and cross-sectional (Figure 2C) images, obtained using scanning electron microscopy.

[0018] Figure 3 shows the pore size distribution for CAB and CAB-T.

[0019] Figure 4 shows the infrared spectrum (FTIR) of thymol, CAB and CAB-T.

[0020] Figure 5 shows the evaporation of pure thymol (♦) and the release of thymol from CAB-T (•) in air, at a temperature of 27°C.

[0021] Figure 6 shows CAB and CAB-T pore size distribution curves for Analysis I and Analysis II.

[0022] Figure 7 shows the thermal stability of thymol, CAB and CAB-T.

[0023] Figure 8 shows the powderX-ray diffraction (PXRD) of thymol, CAB and CAB-T samples.

[0024] Figure 9 shows the persistence of insecticidal activity of CAB-T.

[0025] Figure 10 shows the results of the double choice test showing the repellent effects of pure thymol (A.) and thymol impregnated into cellulose acetate beads (B.).

[0026] Detailed description of the invention

[0027] The invention relates to cellulose acetate beads impregnated with thymol (CAB-T), wherein the content of impregnated thymol is approximately 25-27%, preferably 26%, and the pore size is 0.028- 0.846 pm, the average pore size is 0.407 pm, the specific surface area is 31-32 m2 / g, the porosity is 57-58%, and the thermal stability exceeds 125°C. CAB-T has the property of long-term thymol release in air for at least 40 days, with approximately 60% being released, and preferably around 58% of the impregnated thymol. Even after this time, thymol is released steadily, and thus, for example, after 55 days, approximately 65% of thymol is released, and after 103 days, approximately 67.02% of thymol is released.

[0028] Furthermore, the invention relates to the process for supercritical impregnation (SCI) of cellulose acetate beads with thymol, comprising the steps of: a) placing 0.5 g-1.5 g of cellulose acetate beads above 0.5 g-3.0 g of thymol in a closed high- pressure vessel; b) exposing the vessel of step a) to a pressure of 290-310 bar and a temperature of 48-51°C; during a period from 2 h 50 min to 3 h 10 min, with a CO2 consumption of 20-24 g; c) decompression of the vessel of step b), which is carried out at 4.5-5.5 bar.

[0029] In a preferred embodiment of the SCI method of the invention, the used initial mass of cellulose acetate beads is 0,8 g, and of thymol is 2,0 g. Preferably, the process is carried out at a pressure of 300 bar and a temperature of 50°C, for 3 h, with a CO2 consumption of 22 g, and decompression is performed at 5 bar / min. The invention also relates to antimicrobial, insecticidal, repellent and biostimulating agents comprising CAB-T of the invention.

[0030] The invention also relates to the use of cellulose acetate beads impregnated with thymol with a longterm thymol release, as well as agents for protection from insects (insecticidal and / or repellpnt agents), antimicrobial agents and biostimulating agents to improve seed vitality and germination;

[0031] When using CAB-T according to the invention as an agent against insects, which enables a long-term exposure of plant material to the effect of thymol, in addition to a prolonged insecticidal effect, its prolonged, but also enhanced, repellent effect on insects is manifested, in comparison to thymol hot impregnated in beads.

[0032] A long-term and extended exposure of plant material to the effect of thymol from cellulose acetate beads impregnated with thymol, according to the invention, unexpectedly showed their biostimulating effect on plant material, especially seed material. Thus, a special aspect of the invention is the process of applying cellulose acetate beads impregnated with thymol to seeds to enhance seed vitality and germination.

[0033] The process of applying cellulose acetate beads impregnated with thymol, according to the invention, is carried out by mixing an effective amount with plant material or placing it in close proximity to plant material and keeping it in a closed space, as CAB-T of the invention has the property of long-term thymol release in air. If necessary, depending on the size of the closed space, plant material and the type of pests, the plant material should be covered for faster and more efficient action. The closed space according to this invention would be warehouses, greenhouses, nurseries, containers, silos, and bags, in addition to laboratory vessels. The "plant material" here refers to all parts of a plant (root, stem, leaf, flower, fruit) as well as plants at all stages of development, such as seed, seedling, plant tissue in culture, developing plant, and adult plant.

[0034] This application process is acceptable for plant materials of various plant species, especially legumes and cereals.

[0035] Additionally, the CAB-T application process, according to the invention, simultaneously provides protection for plant material of different plant species from pests and has a biostimulating effect on plant material.

[0036] Examples of insects and plant material to which the CAB-T application processes of the disclosed invention may be applied, but without being limited solely to them, are provided in Table 1. Table 1. A review of storage insects sensitive to thymol and the plant materials they infest

[0037] In addition to protection from insects, due to its antimicrobial effect, and especially fungicidal effect, CAB-T according to the invention can be used in the process of protecting plant material from fungi, which are common pests in the field, glasshouses and storage areas for plant material. Examples of thymol-sensitive fungi include, but are not limited to, Alternaria alternate / , Alternaria solani, Botrytis sp., Fusarium acuminatum. Fusarium culmorum, Fusarium equiseti, Fusarium nivale, Fusarium oxysporum, Fusarium sambucinum, Fusarium semitectum, Fusarium solani, Monilinia sp., Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, Sclerotinia minor i Verticillium dahliae. In addition, fungi of the genus Penicillium {P. digitatum, P. citrinum, P. chrysogenum, P. brevicompactum, P. griseofulvum, P. verrucosum) i Aspergillus (A. niger, A. flavus, A. fumigatus, A. versicolor, A. sulphurous, A. awamori, A. parasiticus), which are more resistant to the low humidity prevailing in warehouses, are also sensitive to thymol.

[0038] The term "pest" refers to any bacteria, fungus, insect, mite, virus endangering the plant material.

[0039] The terms "insecticide agent" and "insecticide effect " refer to a substance and its ability to suppress harmful insects.

[0040] The terms "repellent agent" and "repellent effect" refer to a substance and its property of repelling harmful insects.

[0041] The terms "antimicrobial agent" and "antimicrobial action" refer to a substance and its property to suppress harmful microorganisms.

[0042] The terms "fungicide" and "fungicidal action" refer to a substance and its property to suppress fungi.

[0043] The term "biostimulating agent" or "biostimulator" refers to a substance that is applied to plants to improve nutrition and growth efficiency, abiotic stress tolerance, and / or crop quality characteristics, regardless of the nutrient content. The "biostimulating effect" of biostimulators is reflected in the stimulation and facilitation of the most sensitive phases of plant growth, development, and maturation.

[0044] The term "effective amount" in the context of this invention refers to the amount of CAB-T used under specific conditions (temperature and volume of the space in which it is applied) to manifest the desired biological effect (insecticidal, repellent, antimicrobial, biostimulating effect on seed vitality and germination).

[0045] The terms "about" and "approximately" are used synonymously and, in the context of the present invention, indicate that, when referring to a numerical value, the included values range from 5% less to 5% greater than the stated value. Exemplary embodiments

[0046] Materials

[0047] Cellulose acetate beads (CAB, CA-320SNF / EP, Eastman, Tennessee, USA), CO2 (purity 99.9%, Messer Tehnogas, Serbia), and thymol (> 99%, Sigma-Aldrich, Germany) were used for preparation of the material. Absolute ethanol (> 99.8%, Honeywell, Germany) was used in determining the amount of thymol in the impregnated CAB.

[0048] Methods

[0049] Impregnation of CAB with thymol

[0050] The impregnation begins by placing CAB (0.8 g in a metal porous basket) above thymol (2 g in an open glass tub) in a closed 25 mL high pressure vessel. The vessel is then subjected to pressure and temperature conditions that are supercritical for CO2 (300 bar and 50°C), previously optimized. The CO2 consumption was 22 g. Under these conditions, supercritical CO2 (sc-CC ) acts as a solvent and fluid carrier for thymol, enabling its diffusion into the solid CAB matrix. After 3 hours of SCI (supercritical impregnation), the vessel was decompressed at a rate of 5 bar / min, returning CO2 to gaseous form. This causes CO2 to evaporate, leaving thymol within the solid CAB matrix. The impregnated material is stored in a closed glass container in the refrigerator until use. SCI is performed in replicates.

[0051] Characterization of CAB using Field Emission Scanning Electron Microscopy (FE-SEM)

[0052] The morphology of CAB before and after impregnation with thymol was determined using field emission scanning electron microscopy (FE-SEM, Mira3, Tescan, Czech Republic). The surface and sections of the samples were coated with a thin layer of Au / Pd (85 / 15) using a sputter coater (Polaron SC502, Fisons Instruments, UK) before the analysis. The image processing software ImageJ (version IJ 1.46r) was used to estimate the pore size distribution as well as the average pore diameter values for CAB and CAB-T.

[0053] Characterization of CAB using Fourier Transform Infrared Spectroscopy (FT1R)

[0054] The Fourier transform infrared spectra for thymol, CAB and CAB-T were obtained using an AFTR-FTIR spectrometer (Nicolet iSlO, Thermo Fisher Scientific Inc., Wisconsin, USA) in the range from 4000 cnrr1to 500 cm’1.

[0055] Characterization of CAB by examining thymol release

[0056] The release rate of thymol from impregnated CAB was tested as described previously (Pajnik, J et al. Functionalization of Polypropylene, Polyamide and Cellulose Acetate Materials with Pyrethrum Extract as a Natural Repellent in Supercritical Carbon Dioxide. Journal of Supercritical Fluids 2018, 136, 70-81. i Zhang, Y et al. Encapsulation of Thymol in Biodegradable Nanofiber via Coaxial Electrospinning and Applications in Fruit Preservation. Journal of Agricultural and Food Chemistry 2019, 67, 1736-17*41) with minor variations.

[0057] Impregnated samples (215 mg) exposed to air were placed in an oven (OV-11, Jeio Tech, Korea) at 27°C. For comparison, the evaporation of pure thymol (58 mg) was performed. The amount of remaining thymol was estimated gravimetrically. This test lasted more than 40 days and was done in triplicate. The obtained results were confirmed by UV spectroscopy.

[0058] CAB-T were placed in absolute ethanol (50 mL) and after several days at room temperature, the absorbance at 277 nm was measured using a spectrophotometer (Shimadzu Spectrophotometer UV- 1800, Japan). A calibration curve for thymol was obtained using concentrations ranging from 9.56 to 114.75 ppm. The fit is linear in the operating range with the linear correlation coefficient R2= 0.9996.

[0059] Characterization of CAB using Mercury Intrusion Porosimetry (MIP)

[0060] The measurements were performed on a fully automated conventional apparatus Carlo Erba Porosimeter 2000 (pressure range: 0.1-200 MPa; pore diameter between 7.5 and 15000 nm). The data analysis acquisition was performed using Milestone Software 200. Two subsequent intrusion-extrusion analyzes were also performed (Analysis I and Analysis II). Before the actual analysis, samples were vacuumed for 2 h in a dilatometer placed in Macropores Unit 120.

[0061] Characterization of CAB using thermogravimetric analysis (TGA)

[0062] The thermogravimetric analysis was performed using SDT Q600, TA Instruments. The experiments were performed in a nitrogen flow (100 mL min-1), with the temperature range of 600°C at a heating rate of 10°C min-1.

[0063] Characterization of CAB using X-Ray Diffraction (XRD)

[0064] The samples were examined on an Ital Structure APD 2000 powder X-ray diffractometer (GNR Instruments). The samples were exposed to radiation with a wavelength of 1.5418 A Cu Ka and analyzed in the 20 range from 10 to 70° (step width 0.02 °20, time per step 1 s).

[0065] RESULTS

[0066] Characterization of CAB and CAB-T samples

[0067] SCIs at chosen conditions enabled thymol content in CAB of 26.1 ± 0.5%. Figures 1A-C show the appearance, surface and cross-section of CAB before the impregnation process, while Figures 2A-C show the appearance, surface and cross-section of CAB-T after the impregnation with thymol. I|t is noticeable that SCI does not affect the appearance of CAB. However, scanning electron microscopy images reveal minor changes in pore size, which are confirmed by pore size distribution measurements, shown in Figure 3.

[0068] Namely, the scanning electron microscopy images of CAB cross-section reveal pronounced porosity. The pore size ranges from 0.104 to 0.988 pm resulting in an average pore size of 0.491 pm. On the other hand, the pore size of CAB-T ranges from 0.028 to 0.846 pm resulting in a slightly smaller average pore size of 0.407 pm. This observation is due to thymol deposition on pore walls during SCI, which increases their diameter and decreases the free pore volume, as expected.

[0069] The FTIR analysis was performed to determine the interaction potential of thymol and CAB. The obtained spectra for thymol, CAB and CAB-T are shown in Figure 4. The broad absorption band at 3468 cm'1, seen in the spectrum of the control sample, corresponds to OH stretching of the hydroxyl group. The bands at the wave number range of 2941-2878 cm"1are attributed to of C-H bond stretching. The intense peak at 1735 cm"1is attributed to strong C-0 group stretching. The bands at 1433, 1369 and 1225 cm"1come from C-H bending, and rocking and waving vibrations, respectively. The strongest peak at 1035 cm"1refers to C-O-C (ether linkage) of glycosidic unit, followed by the peak at 904 cm"1, which originates from saccharides. The observed bands are characteristic of cellulose acetate and correspond to literature data. In addition to the bands characteristic of cellulose acetate, the FT-IR spectra of CAB samples impregnated with thymol exhibit additional absorption bands characteristic for thymol. The maxima in the range of 1620 and 1458 cm"1correspond to the phenolic ring of thymol, while the new maximum detected at 804 cm"1refers to the out-of-plane shift waging vibration of aromatic C-H.

[0070] Study on thymol evaporation and release

[0071] The results of pure thymol evaporation and thymol release from CAB into the air over a period longer than 40 days are shown in Figure 5. It is noticeable that the initial release during the first 5 days was very fast. After 10 days, thymol is released more slowly, reaching the value of about 58% after 40 days, and the maximum value of about 65% after 55 days.

[0072] On the other hand, the entire amount of pure thymol evaporates already after 18 days. This confirms that the CAB impregnation enables a prolonged release time and controlled release of thymol over a period of up to 3.5 months. Characterization of CAB using mercury porosimetry

[0073] Mercury porosimetry results (total pore volume (Vtot), specific zone area (SHg), average pore diameter (Dav), total density, porosity (P) and pore size distribution (PSD) of the analyzed sample are showrji in Table 2 and Figure 6.

[0074] Table 2. Structural characteristics of CAB and CAB-T c Samp Ile A Anallys-is , t ,ot $Hg DavvP

[0075] (cm’ g'1) (m2g-1) (nm) (%)

[0076] The total pore volume of the sample from the first analysis (Analysis I) was 0.94 cm3g'1, while the total pore volume of the sample from the second analysis (Analysis II) was reduced by about 55%. The pore size distribution (PSD) curve (Figure 6) of the CAB sample shows a unimodal PSD curve with the mean pore diameter centered at 313.9 nm and 248.7 nm for Analysis I and Analysis II, respectively. The measurements performed in two successive analyses were intended to remove potential doubts about the presence of interparticle (voids) and intraparticle space, but also to determine which types of pores dominate. In this case, the analyzed sample is in the form of granules, where the presence of interparticle and intraparticle porosity is minimized. However, it is obvious that the total pore volume and porosity are significantly reduced. This can be attributed to the specific shape and types of pores that are formed during synthesis or formed by the addition of different active components. These formed pores cannot be emptied during the extrusion phase, where they remain occupied by mercury and are not available for the next intrusion cycle (Analysis II). The pore morphology can be better seen using scanning electron microscopy (SEM).

[0077] Characterization of CAB using thermogravimetric analysis (TGA)

[0078] The thermal stability of all samples was evaluated using TGA (Figure 7). The TGA curve of pure thymol shows that its complete degradation occurs in one step at approximately 175°C. On the other hand, the initial mass loss is approximately 4 mass % up to 115°C, visible in the TGA curve of cellulose acetate in relation to adsorbed water, indicating that the sample is hygroscopic in nature. Upon further heating, the sample remains stable up to 300°C before degradation of approximately 81 mass % occurs.

[0079] A temperature of 365°C can be considered as the degradation temperature. The TGA curve of the composite also shows an initial mass loss due to adsorbed water, though, as expected, in the slightly smaller range of 3 mass %. The degradation of thymol in the composite occurs in two steps, at 125°C (6 mass %) and 215°C (17 mass %), while the degradation of cellulose occurs in one step, at 360°C (64 mass %). Since the degradation of thymol was prolonged and its degradation changed in relation to that of pure thymol, it can be concluded that certain intermolecular interactions formed between thymol and cellulose acetate. Characterization of CAB using Powder X-Ray Diffraction (PXRD)

[0080] The results of this analysis can be seen in Figure 8. The crystallinity of thymol was confirmed by PXRD, as was the amorphous state of cellulose acetate. However, the nature of the composite is dual. Although the broad "hallo" pattern characteristic of amorphous substances is dominant, visible reflections at 16.5; 19; and 25 °20 indicate the presence of crystalline thymol. Moreover, since he thymol content is 26.1%, these reflections could be stronger. This means that amorphization of thyrjnol has occurred within a certain range, but it is not fully completed.

[0081] Biological activity of CAB-T - effect on the bean weevil

[0082] Adult mortality

[0083] The effect of pure thymol (T) and thymol impregnated in cellulose acetate beads (CAB-T) on the survival of bean weevil adults was investigated by exposing the adults to different concentrations of thymol dissolved in ethanol (0.6-1.35 mg / 20 g beans) and CAB-T (10-50 mg beads containing 2.56- 12.8 mg thymol / 20 g beans). The dependence of adult mortality on the concentration of pure thymol was examined in Petri dishes (d = 10 cm) in which 20 g of bean grains, filter paper (4 x 1 cm2), with a total of 100 .L of ethanol or ethanol solution of thymol, and 10 adults with a 1:1 sex ratio, were introduced. To test the effect of CAB-T, different amounts of empty or thymol-impregnated beads (10— 50 mg) were introduced into Petri dishes. For each concentration of thymol, 5 replicates were tested. Mortality was determined after two days of treatment, and the lethal concentrations shown in Table 3 were determined by probit analysis.

[0084] Table 3. Toxicity of pure thymol (T) and thymol impregnated in cellulose acetate beads (CAB-T) in the bean weevil Acanthoscelides obtectus after48 h of exposure. Experimental groups exposed to different concentrations of thymol had the 1:1 sex ratio. Lethal concentrations leading to 10% (LCio), 50% (LC5o) and 99% adult mortality (LC99) are expressed in mg thymol / 20 g of bean grains / Petri dish (d = 10 cm). Confidence intervals are shown in parentheses.

[0085] Adult emergence in Fl generation

[0086] After determining mortality, the surviving adults were left in Petri dishes to form the next generation. The number of the emerged individuals was drastically reduced even at low lethal concentrations of both pure and impregnated thymol.

[0087] The investigation of the insecticidal activity of CAB-T is presented in Table 4, where it is evident that the number of emerged bean weevil adults decreases as the concentration of CAB-T increases. Table 4. The number of emerged bean weevil adults in the Fl generation in response to the treatment of the parental generation with different concentrations of pure thymol (T) and thymol impregnated into cellulose acetate beads (CAB-T).

[0088] TTCAB-T NCAB-T

[0089] (mg) (mg)

[0090] 0.00 72.0 ± 10.4 0 72.616.9

[0091] 0.60 9.615.7 * 2.56 30.8111.8 *

[0092] 0.75 1.61 1.6 * 5.12 0.410.4 ***

[0093] 0.90 0 7.68 0

[0094] 1.05 0 10.24 0

[0095] 1.20 0 12.80 0

[0096] 1.35 0

[0097] Comparison with the control: Dannet's test for NT and Welch's test for NCAB-T (* p < 0.05; *** p < 0.001)

[0098] Persistence of insecticidal activity

[0099] Figure 9 shows the persistence of insecticidal activity of thymol impregnated into cellulose acetate beads. After different time periods following the treatment of bean grains with LC99 concentration, the mortality of bean weevil adults was determined after 48 h. The persistence results of CAB-T, shown in Figure 9, indicate that mortality gradually decreases over time, but that even after 38 days, it does not completely lose its insecticidal activity. After 32 days, the adult mortality of bean weevil was about 14%. This mortality is close to the mortality caused by the concentration of thymol impregnated into cellulose acetate beads, which simultaneously completely inhibits the establishment of the Fl generation (20 mg CAB-T with 5.12 mg thymol / 20 g beans in a Petri dish d - 10 cm).

[0100] Repellency

[0101] The repellent activity of CAB-T was also tested, and the results are shown in Figure 10. The results of the two-choice test, which shows the repellent effects of pure thymol (Figure 10A) and thymol impregnated into cellulose acetate beads (Figure 10B), are presented. The number of adults that, after 2 hours, move to the side of the Petri dish (d = 10 cm) with the control treatment (1OO .1 of 96% ethanol on filter paper 1 x 4 cm or non-impregnated cellulose acetate beads - CAB) and the number of adults that move to the side of the Petri dish treated with thymol (100 .1 of ethanol solution of thymol or impregnated thymol - CAB-T) were determined. 10 adults with the 1:1 sex ratio were introduced into the center of each Petri dish.

[0102] The results of the repellency test with the thymol solution showed (Figure 10A) that it is a weaker repellent than thymol impregnated into cellulose acetate beads (Figure 10B). Biological activity of CAB-T - effect on seed vitality and germination

[0103] The bean seeds were exposed to ethanol (control) and to the ethanol solutions of thymol (1.05%; 1.4%; 1.8%) at a temperature of 27°C for 90 days. Also, during the same period and under the same temperature conditions, the beans were exposed to non-impregnated cellulose acetate beads (control) and thymol-impregnated beads (50 mg, 100 mg and 200 mg CAB-T / 20 g beans in Petri dishes d =110 cm).

[0104] Bean germination was tested under laboratory conditions according to the instructions of the International Association for Seed Testing (ISTA International Rules for Seed Testing; International Seed Testing Assoc.: Zurich, Switzerland, 2011), with minor modifications, to determine seed quality after treatment with pure thymol and thymol impregnated into cellulose acetate beads. Twenty seeds per treatment were placed on three-layer Whatman 181 filter paper moistened with 20 mL of distilled water in Petri dishes (d = 20 cm). After 5 days, to maintain the constant humidity of the filter paper, 10 mL of distilled water was added to each Petri dish. Germination was monitored on 60 seeds in three replicates of 20, for each treatment. The experiment was carried out in a room for growing plants under constant conditions (temperature: 24 ± 2°C; light:dark photoperiod: 12 h:12 h). The number of germinated seeds was recorded on the twelfth day. As a measure of germination, seeds that had germinated at least 1 mm were taken. The obtained results showed that the ethanol solution of thymol did not affect the percentage of seed germination (Table 5), while increased concentrations of impregnated thymol (100 and 200 mg of CAB-T) had a stimulating effect on germination (Table 6).

[0105] Table 5. The effect of the thymol ethanolic solution (X ± SE) on seed germination percentage (G%), shoot length (SL), root length (RL), seedling dry mass (SDM), and vigor indices, monitored for 12 days in vitro.

[0106] Concentration G%aSLaRLbSDMaVigor 1aVigor 11a

[0107] (%) (cm) (cm) (g)

[0108] 0 76.67 + 1.90 + 4.44 ± 0.021 ± 471.1 ± 1.66 ±

[0109] 4.41 0.27 0.66 0.003 98.4 0.48

[0110] 1.05% 68.33 ± 3.24 ± 7.71 ± 0.042 ± 732.0 ± 2.92 ±

[0111] 4.41 0.26 1.02 ** 0.005 * 229.5 1.11

[0112] 1.4% 71.67 ± 2.14 ± 4.99 ± 0.036 ± 497.4 ± 2.57 ±

[0113] 1.67 0.29 0.74 0.006 60.0 0.67

[0114] 1.8% 78.33 ± 2.39 ± 5.51 ± 0.040 ± 613.8 ± 3.10 ±

[0115] 3.33 0.32 0.64 0.006 * 63.3 0.62

[0116] Comparison with the control: Dunnet's (a) and Welch's (b) test (* p < 0.05; **p < 0.01) Table 6. The effect of thymol impregnated into cellulose acetate beads (X ± SE) on seed germination percentage (G%), shoot length (SL), root length (RL), seedling dry mass (SDM), and vigor indices; monitored for 12 days in vitro. I

[0117] Concentration G%aSLbRLbSDMbVigor 1aVigor II lOOmg CAB 75.00 ± 1.32 ± 0.12 3.33 ± 0.35 0.014 ± 338 ± 54 1.04 ±

[0118] 2.89 0.001 0.10

[0119] CAB-T 95.00 ± 4.20 ± 0.51 13.55 ± 0.092 ± 1686 ± 8.71 ±

[0120] 0.00 ** *** 1.33 *** 0.012 *** 79 *** 0.68 **

[0121] 200mg CAB 66.67 ± 2.13 ± 0.27 3.90 ± 0.53 0.030 ± 383 ± 2.04 ±

[0122] 3.33 0.005 109 0.64

[0123] CAB-T 81.67 ± 3.36 ± 0.42 11.45 ± 0.082 ± 1163 ± 6.39 ±

[0124] 7 26 * *** 1.17 *** 0.011 *** 106 *** 1.12 **

[0125] Comparison with the control: contrast test (a) and Welch's test (b) (* p < 0.05; ** p < 0.01; *** p <

[0126] 0.001)

[0127] To provide additional information on seed quality and sowing potential, vigor tests were conducted to assess seedling vitality. The aim of these tests was to evaluate the effects of the treatment on the growth of bean seedlings after 12 days of germination. Seedlings were grown in Petri dishes (d = 20 cm) in a plant-growing room under constant conditions (temperature: 24 ± 2°C; light:d a rk photoperiod: 12 h:12 h). Shoot length, root length, and seedling dry mass (shoot + root) were measured on the twelfth day in 10 seedlings per replicate (30 seedlings per experimental group). According to Abdul- Baki & Anderson formulas (1973, Vigor determination in soybean seed by multiple criteria. Crop Science, 13, 630-633), two seedling vitality indices were calculated:

[0128] Vigor index I = Germination % x Total length of seedlings (root + shoot)

[0129] Vigor index II = Germination % x Total dry mass of seedlings (root + shoot)

[0130] The results indicated that the lowest concentration of the thymol solution had a stimulating effect on root growth and seedling dry mass, but there were no significant changes in the vitality (vigor) index (Table 5). In contrast, except for shoot growth at the lowest concentration of impregnated beads, all tested parameters showed an increase. Depending on the concentration, the vigor I index increased by 2-5 times, and the vigor II index by 2-8 times (Table 6).

[0131] Statistically significant correlation coefficients were obtained between germination percentage and vigor I (r = 0.846, p = 0.002), as well as between germination percentage and vigor II (r = 0.829, p = 0.003). Significant correlation coefficients, between germination and vigor I and II, predict a good fate for treated seed in the field. The confirmed biostimulatory effect of CAB-T (50 mg, 100 mg, 200 mg) on seed vitality and seedling growth makes it desirable for application in phytomedicine and agronomy.

Claims

Claims1. Cellulose acetate beads impregnated with thymol, characterized in that the content of impregnated thymol is approximately 25-27%, the pore size is 0,028-0,846 pm, the average pore size is 0,407 pm, and they have the property of long-term release of thymol in air.

2. Cellulose acetate beads impregnated with thymol according to claim 1, characterized in that the content of impregnated thymol is 26%.

3. Cellulose acetate beads impregnated with thymol according to claims 1 and 2, characterized in that the duration of long-term release is at least 40 days.

4. Cellulose acetate beads impregnated with thymol according to claim 3, characterized in that approximately 58% of thymol is released.

5. Cellulose acetate beads impregnated with thymol according to claims 1 and 2, characterized in that approximately 65% of thymol is released over a period of 55 days.

6. Cellulose acetate beads impregnated with thymol according to claims 1 and 2, characterized in that approximately 67% of thymol is released over a period of 103 days.

7. A process for supercritical impregnation of cellulose acetate beads with thymol, characterized in that it comprises the steps of: a) placing 0.5 g-1.5 g of cellulose acetate beads above 0.5 g-3 g of thymol in a closed high- pressure vessel; b) exposing the vessel of step a) to a pressure of 290-310 bar and a temperature of 48-51°C; during a period from 2 h 50 min to 3 h 10 min, with a CO2consumption of 20-24 g; c) decompression of the vessel of step b), which is carried out at 4.5-5.5 bar.

8. The process according to claim 7, characterized in that the pressure is 300 bar and the temperature is 50°C, for 3 hours, with a CO2consumption of 22 g, and decompression is performed at a rate of 5 bar / min.

9. An agent for protection from insects with an insecticidal and / or repellent effect, characterized in that it comprises cellulose acetate beads impregnated with thymol according to claims 1-6.

10. An antimicrobial agent, characterized in that it comprises cellulose acetate beads impregnated with thymol according to claims 1-6.

11. A biostimulating agent, characterized in that it comprises cellulose acetate beads impregnated with thymol according to claims 1-6.

12. The use of cellulose acetate beads impregnated with thymol according to claims 1-6, as an agent for protection from insects, an antimicrobial agent, and a biostimulating agent.

13. The use of cellulose acetate beads impregnated with thymol according to claim 12, characterized in that the agent for protection from insects is an insecticide and / or a repellent.

14. The use of cellulose acetate beads impregnated with thymol according to claim 13, characterized in that the insecticide and / or repellent suppresses bean weevil.

15. The use according to claim 12, characterized in that the antimicrobial agent is a fungicide.

16. The use according to claim 12, characterized in that the biostimulating agent is an agent for improving vitality and germination of seeds.

17. A process for applying cellulose acetate beads impregnated with thymol according to claims 1-6, characterized in that an effective amount thereof is mixed with plant material or placed in the immediate vicinity of the plant material, kept in a closed space, and, if necessary, the plant material is covered.

18. The application process according to claim 17, characterized in that the plant material is selected from seeds, seedlings, plant tissue in culture, developing plant, and adult plant.

19. The application process according to preceding claim 18, characterized in that the plant material is bean seeds.

20. The application process according to claim 16, characterized in that the closed space refers to warehouses, greenhouses, nurseries, laboratory vessels, silos, containers, and bags.

Images