Biotechnological process to prepare selenium nanoparticles

Abstract

The present invention refers to a biotechnological process for obtaining suspensions of biogenic nanoparticles of zerovalent selenium, which have a high stability, and which have agronomic functions specific to plant biostimulants, that are amplified as a result of the formation of a biocorona which includes bioactive molecules from the biotechnological system wherein these selenium nanoparticles were formed. The biotechnological process involves the biosynthesis of selenium nanoparticles by the consortium of microalgae, Nannochloris sp. 424-1, deposited under CCAP number 251 / 10 in the Culture Collection of Algae and Protozoa (CCAP), and yeasts, Rhodotorula mucilaginosa TazRr, deposited under DSM number 34804, at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, to promote the formation of suspensions of stable selenium nanoparticles. The process also includes separation and concentration steps by micro- and ultrafiltration on ceramic membranes.

Description

[0001] BIOTECHNOLOGICAL PROCESS TO PREPARE SELENIUM NANOPARTICLES

[0002] The present invention refers to a biotechnological process for obtaining suspensions of biogenic nanoparticles of zerovalent selenium, which have a high stability, and which have agronomic functions specific to plant biostimulants, that are amplified as a result of the formation of a biocorona which includes bioactive molecules from the biotechnological system wherein these selenium nanoparticles were formed.

[0003] Different processes for obtaining biogenic selenium nanoparticles are known. Elemental (zerovalent) selenium nanoparticles, due to their high surface-to-volume ratio, function as a reservoir for the gradual release of bioactive chemical species, selenide ions, selenite, and / or selenate (Skalickova et al. 2017, Nutrition, 33: 83-90), Selenium species released from selenium nanoparticles as a result of their disproportionate reaction act on crop plants as inorganic biostimulants (Medrano- Macias, et al. in Selenium and Nano-Selenium in Environmental Stress Management and Crop Quality Improvement. Springer International Publishing - Cham, 2022, pp. 41-54), with all the functions specific to this class of products intended for the treatment of cultivated plants: enhance the tolerance of cultivated plants to abiotic stress, increase / benefits the nutrients uptake and improve the quality of the harvest (du Jardin, 2015, Scientia Horticulturae, 196: 3-14). Selenium nanoparticles also have a high efficiency in the biofortification of the food chain (El-Ramady et al. 2020, Soil Systems, 4(3), 57).

[0004] Although selenium is not considered to be an essential element for plants, selenium has been shown to stimulate plant growth in recent decades (Hartikainen and Xue, 1999, Journal of Environmental Quality, 28: 1372-1375; Xue et al. 2001 , Plant and Soil, 237: 55-61), plays a role in plant protection against biotic stressors, phytopathogens (Hanson et al. 2003, New Phytologist, 159: 461-469) and pests (Mechora, 2019, Plants, 8, 262) and from abiotic stress, including drought (Ahmad et al. 2016, Journal of the Science of Food and Agriculture 96: 372-380) and enhance nitrogen assimilation and metabolism of nitrogenous compounds (Rios et al. 2010, Journal of the Science of Food and Agriculture, 90(11), 1914-1919).

[0005] Inorganic selenium compounds are generally used to treat cultivated plants. The toxicity of selenium compounds is extremely high. Inorganic selenium species have decreasing toxicity in the order of selenate > selenite > selenide > elemental / zerovalent selenium (Nuttall, 2006, Annals of Clinical & Laboratory Science 36:409-420). While selenate has a higher acute toxicity to rats than that of potassium cyanide, zerovalent selenium has a toxicity two orders of magnitude lower (Olson, 1986, Journal of the American College of Toxicology 5:45-70). Zerovalent selenium nanoparticles (SeNPs) have shown even lower toxicity than elemental selenium (Shakibaie et al. 2013, Pharmaceutical Biology 51 :58-63). At the same time, the efficacy of selenium nanoparticles (SeNPs) in inducing selenoenzymes is comparable to that of the most efficient form of organic selenium, seleno-methyl-seleno-cysteine (SeMeSeCys) (Zhang et al. 2008, Toxicological Sciences 101 :22-31). However, SeMeSeCys is a compound with similar toxicity to selenate and obtained through difficult, low-yield organic synthesis (Iwaoka et al. 2016, Proceedings of the National Academy of Sciences, Section A. 86: 499-509). Selenium nanoparticles have, as already mentioned, a toxicity similar (or even lower) to zerovalent selenium.

[0006] The chemical production of selenium nanoparticles involves the use of harsh conditions and concentrated mineral acids (Stroyuk et al. 2008, Colloids and Surfaces A: Physicochemical and Engineering Aspects 320: 169-174) or toxic reagents such as hydrazine (Mishra et al. 2005, The Journal of Physical Chemistry B 109: 12718-12723) or ionic liquids (Langi et al. 2010, Materials Research Bulletin 45: 668-671). Biosynthesis of selenium nanoparticles occurs under mild conditions (Wadhwani et al. 2016, Applied Microbiology and Biotechnology 100: 2555-2566). In general, biologically synthesized SeNPs are up to ten times less toxic than chemically synthesized nanoparticles (Mai et al. 2017, Nanotoxicology 11:87-97).

[0007] Since biosynthesized selenium nanoparticles have obvious advantages, several biotechnological processes for obtaining selenium nanoparticles have been patented. These are illustrated here by only a few processes. Patent application CN116200308A protect the strain of Lysinibacillus fusiformis KBD-5, deposited in China General Microbiological Culture Collection Center (CGMCC) under CGMCC number 26146, which stimulates plant growth and development and can also be used for the production of selenium nanoparticles. The RU2717997 patent describes a process using bifidobacteria Bifidobacterium adolescentis DSM 20083 or propionic bacteria Propionibacterium freudenreichii Sh85 to produce selenium nanoparticles. The cultivation medium is clarified whey, and nanoparticle synthesis occurs after adding 1 to 2 mg / l sodium selenite. Patent application CN 107881127 A protects a strain of Bacillus amyloliquefaciens, Lxz-41 , deposited under the number M2016578 at the China Center for Type Culture Collection (CCTCC), and a method for preparing nano- selenium using that strain. The selenium nanoparticle biosynthesis process involves the following steps: cultivation on a glucose and peptone-based medium that also contains a surfactant, addition of a selenium salt, from the beginning or by continuous addition, culture harvesting, ultrasonication of bacteria and separation of nanoparticles formed inside bacterial cells. Patent application CN105199979 A reveals a strain of Bacillus thuringiensis YLX-4, isolated from leaching waters from a selenium mine (Enshi, Hubei province, China), deposited under number M2013674 at the China Center for Type Culture Collection (CCTCC). The strain has the typical characteristics of seleno-bacteria and is also claimed for the production of nano-selenium. The CA2723655 A1 patent application describes selenium nanoparticle synthesis the synthesis of selenium nanoparticles by various photosynthesizing microorganisms, including Chlamydomonas reinhardtii UTEX 90 and Synechococcus leopoliensis UTEX 2434.

[0008] Nanoparticles produced by (bio)synthesis in biological environments can form a biocorona with higher stability already in the (bio)synthesis phase. This corona, formed during the (bio)synthesis phase, amplifies the properties of nanoparticles. For example, nano-selenium synthesized in the culture medium of fungi of the genus Trichoderma has better antifungal effects and causes a more pronounced inhibition of mycotoxin production, from Altemaria (83% TeA and 79% AOH), fumonisin B1 (63% FB1) and deoxynivalenol - 76% DON, respectively. (Hu et al. 2019, Food Control, 106, 106748). Also, biogenic nano-selenium produced by Trichoderma strains culture filtrates enhance the biocontrol activity of Trichoderma strains against Sclerospora graminicola in pearl millet (Nandini et al. 2017, Scientific reports 7: 2612).

[0009] An additional advantage of nanoparticle (bio)synthesis processes in biological media is that these processes are "green" processes, which do not involve elevated temperatures or extreme pH. However, due to the toxic effects of selenium, the formation of zerovalent selenium nanoparticles occurs mainly with metabolites from culture media of microorganisms and not through direct biosynthesis by micro- organisms. Unlike nanoparticles synthesized assisted by metabolites in the culture medium of microorganisms, which can have different shapes and size distributions over a wide range of 20-550 nm (Vetchinkina et al. 2019, Industrial & Engineering Chemistry Research, 58, 17207-17218), nanoparticles biosynthesized by microorganisms are predominantly spherical in shape, with a narrower nanoparticle size distribution (Diko et al. 2020. Materials Chemistry and Physics, 246, 122583). The amphiphilic biopolymer biocorona of biogenic zerovalent selenium nanoparticles synthesized assisted by metabolites in the culture medium of microorganisms is obviously less developed than that of biogenic selenium nanoparticles biosynthesized by microorganisms (Constantinescu-Aruxandei et al., 2018, Nutrients, 10(10), 1466). However, a biocorona of amphiphilic (bio)polymers results in superior stability of suspensions of hydrophobic zerovalent selenium nanoparticles in aqueous media, due to the formation of a protective layer that stabilizes steric and electrostatic repulsive forces between nanoparticles (Zhang et al. 2019, Biomaterials Science, 7, 5112-5123, Tang et al. 2020. Journal of Food Engineering, 275, 109878).

[0010] Although nanoparticles biosynthesized by microorganisms are superior to those synthesized assisted by metabolites from the culture medium of microorganisms, yield is low due to selenium toxicity and selenium nanoparticles tend to aggregate during concentration. The solution of growing microorganisms on media into which limited concentrations of selenium salts are gradually introduced, by continuous addition of small amounts of soluble selenium salts (US patent 4530846) or by gradual release, as selenium is consumed, from insoluble salts (patent RO 112117 B1) or from complexes (patent RO 116770 B1) is not applicable for the synthesis of selenium nanoparticles. Microorganisms synthesize selenium nanoparticle to detoxify high selenium concentrations in their growth medium (Wadhwani et al. 2016, Applied Microbiology and Biotechnology, 100, 2555-2566), so low concentrations would be assimilated and not reduced to zerovalent selenium.

[0011] The physiologically active window of selenium for plants is very narrow, with doses with phytotoxic effects being about 2-3 times higher than those with beneficial effects (Moulick et al., 2024, Ecotoxicology and Environmental Safety, 270: 115832). To avoid crop treatment accidents, selenium nanoparticles need to have high stability.

[0012] The patent application RO135350 A2 refers to a process for obtaining stable suspensions of selenium nanoparticles, which includes the following steps: realization of a mixotrophic culture medium for algae including 2% baker's yeast vinasse, aseptic addition of a 10 mM sodium selenite solution, inoculation of the medium with a suspension of photosynthetic microorganisms, incubation until an optical density corresponding to the accumulation of 5 g per liter of medium is obtained, ultrasonication of the culture of photosynthetic microorganisms-selenium nanoparticles and centrifugal separation of cell debris, homogenization of the supernatant followed by concentration by tangential ultrafiltration of the suspension, up to a concentration of selenium nanoparticles resulting in an optical density at 600 nm D0600 of 0,4...0,8. The disadvantage of this process is the use of energy-intensive steps, such as centrifugation and ultrasonication, which significantly increase production costs due to energy consumption. Also, selenium toxicity reduces the rate of microalgae development and decreases the yield of selenium nanoparticle formation.

[0013] The CN117025402A patent application protects the strain of Euglena gracilis RZTEg-1 , deposited at China Center for Type Culture Collection, under CCTCC deposit number M 2023563, producing stable selenium nanoparticle suspensions. The resulting selenium nanoparticles are intended for use as a pesticide or fertilizer. However, the process of using this strain of E. gracilis to produce selenium nanoparticles is an expensive process to scale up because it involves biomass centrifugation, crushing and grinding in a mortar with liquid nitrogen, and repeated washing followed by centrifugation to purify selenium nanoparticles. Another disadvantage of using this strain is the fact that it does not have biological activity on crop plants that can be transferred to selenium nanoparticles by transferring molecules into the biocorona. As mentioned, active molecules in a culture medium transfer their bioactivity through biocorona formation - see, for example, the recent review Ciobanu et al. 2024, Agronomy, 14(1), 190.

[0014] The technical problem solved by the invention is to develop a biotechnological process for obtaining selenium nanoparticles, reducing the metabolic toxicity of selenium salts, which do not include separation processes with high energy consumption and promote the biosynthesis of selenium nanoparticles with a biocorona of molecules with high biological activity.

[0015] The technical solution is to achieve biosynthesis of suspensions of zero-valent selenium nanoparticles by a strain of microalgae that has a proven biostimulant effect for plants, in an cultivation media where they are co-cultivated together with a yeast strain that reduces the prooxidant effect of selenium salts and promote the formation of stable suspensions of selenium nanoparticles. The process also includes alternative steps to those with high energy consumption, such as centrifugation.

[0016] The process according to the invention consists of the following steps: Preparing a culture medium with the following composition: 10 g / L whey powder with min. 12% protein and min. 72% lactose; NaHCO316.80 g / L; K2HPO4O.5O g / L; NaNO31.875 g / L; K2SO41.00 g / L; NaC1 1 .00 g / L; MgSO4· 7H2O 0.20 g / L; CaCI2• 2H2O 0.04 g / L, microelement solution 1 mL / L; Fe chelated solution 5 mL / L.

[0017] Aseptic addition of a 0.2 pm ultrafiltration-sterilized solution of 10 mM sodium selenite on a microbiological filter, in the ratio of 10 ml selenite solution to 90 ml medium.

[0018] Inoculation of medium with a suspension of the microalgae Nannochloris sp. 424-1 , deposited under CCAP number 251 / 10 in the Culture Collection of Algae and Protozoa (CCAP), containing between 108 and 109 viable micro-organisms per mL, at the ratio of 1 mL inoculant suspension at 8 mL medium and with a suspension of Rhodotorula mucilaginosa TazRr, deposited under DSM number 34804, at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, containing between 108and 109viable microorganisms per mL, in the ratio of 1 mL inoculant suspension at 10 mL medium;

[0019] Incubation of the algae-yeast consortium at a temperature of 28-30°C; illumination 250 μEm-2s-1, with a photoperiod / alternating illumination cycle - darkness of 12:12 hours; administration of synthetic gas mixture with composition: 7% CO2, 14% O2 and 79% N2at a flow rate of 30 mL / min, corresponding to aeration with 1 liter of gaseous mixture with 7% CO2per min per 100 liters of medium, until an optical density corresponding to the accumulation of 5 g per liter of medium is reached;

[0020] Flocculation of the algal-yeast culture by addition to the culture medium of a strigolactone mimic, 3-(4-Methyl-5-oxo-2,5-dihydrofuran-2-yl)-3H-benzothiazol-2-one, SL-F3, up to a final concentration of 10-8M;

[0021] Ultrasonication of culture of photosynthetic microorganisms - selenium nanoparticles for 10 min at a frequency of 20 kHz and an energy of 80 J;

[0022] Separation by microfiltration on ceramic membranes with pore sizes of 5 pm of cell debris, followed by separation of selenium nanoparticles by ultrafiltration on membranes of 50 nM and their concentration by ultrafiltration on membranes of 10 nM.

[0023] The micronutrient stock solution contains the following amounts, expressed in g / L: H3BO3, 2.860; MnSO4-4H2O, 2.030; ZnSO4-7H2O 0.222; MoO3(85%) 0.018; CuSO4• 5H2O, 0.079; CO(NO3)26H2O 0.494.

[0024] The chelated Fe stock solution is prepared as follows: 0.69 g FeSO4· 7H2O are dissolved in 80 ml distilled water and 0.93g Na2EDTA are added. The resulted solution is heated, boiled for 5 min , cooled to room temperature and brought to a final volume of 100 ml.

[0025] The microalgae strain Nannochloris sp. 424-1 , deposited under CCAP number 251 / 10 in the Culture Collection of Algae and Protozoa (CCAP), SAM Research Services, Scottish Marine Institute, Aryll, UK, produce a biomass that generates bioactive compounds acting as plant biostimulants, including polysaccharides that function as elicitors of plant defense systems.

[0026] The strain Rhodotorula mucilaginosa TazRr, deposited under number DSM 34804, at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, produces phytohormones that stimulate algae growth, glutathione that reduces the pro-oxidant effect of sodium selenite, and a significant amount of amphiphilic mannoproteins, which stabilize suspensions of selenium nanoparticles and promote penetration through the cuticle of plants.

[0027] The process according to the invention has the following advantages:

[0028] · Reduces metabolic toxicity of sodium selenite to microalgae due to reduced glutathione production by associated yeasts in the consortium;

[0029] · Reduces energy consumption due to separation by micro- and nano-filtration;

[0030] · Increases the stability of selenium nanoparticle suspensions due to the inclusion of yeast amphiphilic mannoproteins in the selenium nanoparticle biocorona;

[0031] · It potentiates the plant biostimulant effect on cultivated plants of foliar applied nanoselenium suspensions, due to the association in the selenium nanoparticles biocorona of the biopolymers acting as elicitors of the plant defense system and those that promote penetration through the plants cuticles.

[0032] Further examples of invention embodiments are presented below, illustrating it without limiting it.

[0033] Example 1. Prepare 2.5 liters of medium with the following composition: 10 g / L whey powder with min. 12 % protein and min. 72 % lactose; NaHCO316.80 g / L; K2HPO40.50 g / L; NaNO3 1.875 g / L; K2SO41 .00 g / L; NaCI 1 .00 g / L; MgSO4· 7H2O 0.20 g / L; CaCI2• 2H2O 0.04 g / L, microelement solution 1 mL / L; Fe chelated solution 5 mL / L. The micronutrient stock solution contains the following amounts, expressed in g / L: H3BO3, 2.860; MnSO4·4H2O, 2.030; ZnSO4-7H2O 0.222; MoO3(85%) 0.018; CuSO4• 5H2O, 0.079; Co(NO3)2- 6H2O 0.494.

[0034] The chelated Fe stock solution is prepared as follows: 0.69 g FeSO4• 7H2O are dissolved in 80 ml distilled water and 0.93g Na2EDTA are added. The resulted solution is heated, boiled for 5 min, cooled to room temperature and brough to a final volume of 100 ml. Sterilize the medium at 121 °C for 30 min. A 0,2 pm filtration-sterilized solution of sodium selenite is added aseptically to the sterile medium at the ratio of 10 ml selenite solution to 90 ml supernatant and 250 mL, respectively. The 2.5 liters of sterile medium are placed in a PBR 2S Biostat photobioreactor (Sartorius Stedim Biotech).

[0035] The growth medium is inoculated with 250 ml of inoculum from photosynthesizing microorganisms culture, Nannochloris sp. strain 424-1, deposited under CCAP number 251 / 10 in the Algal and Protozoan Culture Collection (CCAP), containing between 108and 109cfu / mL and 250 mL suspension of Rhodotorula mucilaginosa TazRr, deposited under DSM number 34804, at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, containing between 108and 109cfu / mL.

[0036] Conditions for cultivation in the photobioreactor of the algae-yeast consortium are the medium volume: 3 L; working temperature: 28°C; illumination 250 μEm- 2s-1, with a 12 hour photoperiod - alternation illumination cycle : darkness of 12:12 hours; administration of synthetic gas mixture with the composition: 7% CO2, 14% O2and 79% N2at a flow rate of 30 ml / min, corresponding to aeration with 1 liter gaseous mixture (similar to flue gases) with 7 % CO2per min per 100 liters of medium; speed of the peristaltic pump recirculation pump 70%, respectively a recirculation flow rate of 3500 ml / min; automatic measurement of the working parameters is programmed from the bioreactor software: pH, turbidity (OD), temperature, light, recirculation rate, CO2flow / aeration with synthetic gas mixture.

[0037] Incubate until an optical density corresponding to the accumulation of 5 g per liter of medium is reached. The algal-yeast culture is flocculated by adding 3.03 mL of 10-6mimic solution of strigolactone 3-(4-methyl-5-oxo-2,5-dihydrofuran-2-yl)-3H- benzothiazol-2-one, SL-F3, reaching a final concentration of 10-8M mimic of strigolactone. The formed flocks are harvested by filtration on the filter with gauze.

[0038] The filter-harvested wet biomass is ultrasonicated by inserting a probe with a diameter of 10 mm to a depth of 5 cm into the solution to be processed and applying ultrasound for 10 min at a frequency of 20 kHz and an energy of 80J. The resulting suspension is separated by microfiltration and ultrafiltration. An XLAB 5 micro / ultrafiltration unit (Pal Corporation, New-York, NY, USA) is used, provided with membranes of Membralox®. Separation occurs according to size, more precisely according to the hydrodynamic radius of particles in the feed stream. Microfiltration separation of cell debris is initially performed on ceramic membranes with pore sizes of 5 μm, followed by separation of selenium nanoparticles by ultrafiltration on 50 nM ceramic membranes. The suspension of selenium nanoparticles concentrates up to 0.8 ODeoo by tangential ultrafiltration by ultrafiltration on 10 nM ceramic membranes.

[0039] The nanoparticles in the resulting suspension are analyzed by dynamic light scattering (DLS, Amerigo, Cordouan, Pessac, France) and transmission electron microscopy (Tecnai™ G2 F20 TWIN, FEI - Thermo Fisher, Hillsboro, OR, USA). Spherical selenium nanoparticles are obtained with a mean size value of 124 ± 32 nm and a zeta potential value of -20,8 mV.

[0040] Example 2. Work as in Example 1 , with the following differences. The yeasts Rhodotorula mucilaginosa TazRr is no longer be introduced into the algal culture medium. Growth is significantly slower, the difference is 4 days in terms of reaching the density necessary for harvesting. Concentration by tangential ultrafiltration up to OD600of 0.8 cannot be achieved because selenium nanoparticles precipitate. Limit concentration to ODeoo of 0.3. Selenium nanoparticles are not spherical, but have an irregular cylindrical shape, and their exact size cannot be determined. The value of zeta potential is increased to -2.5 mV.

[0041] Example 3. The effect of foliar treatments with nanoparticles performed according to Example 1 on induction of specific defense response enzymes in wheat plants compared to separate application of sodium selenite and microalgae-yeast extract Nannochloris sp. 424-1 - Rhodotorula mucilaginosa TazRr was determined. The microalgae-yeast extract was obtained as in Example 1 , with the following differences: sodium selenite was no longer added, and lysate separation was achieved only by microfiltration on 5 gm ceramic filters, to eliminate insolubilized and / or aggregate cell debris.

[0042] The wheat plants (Triticum aestivum L. cv. Pajura) were grown under climatic chamber conditions (Phytoclima, ArraLab, Albarraque, Portugal), in trays of 4 kg containing a growth substrate enriched with nutrients for the first weeks of growth (Canna Terra Professional Plus, Canna International BV, Oosterhout, Netherlands). The trays were maintained at 22±2°C during daylight and 17±2°C during dark with a 12-hour photoperiod with 360 mE.m-2s-1illumination from neon tubes. The substrate contained reserves of initial nutrients, so the plants were not fertilized. Humidity in trays was maintained by daily watering. After 21 days after germination, foliar treatments were applied with suspensions of selenium nanoparticles, diluted to DO600= 0.2 equivalent and applied at a dose equivalent to 40 mL / m2, as well as with sodium selenite 1 mM and microalgae-yeast extract Nannochloris sp. 424-1 - Rhodotorula mucilaginosa TazRr 0.1 %, applied in the same spraying dose. The application was done by spraying, with a glass atomizer with metal stopper and rubber pear (model 15- RD, DeVilbiss Helathcare, Somerset, PA, USA).

[0043] Two weeks after application, wheat plants were taken, in which the activity of enzymes specific to inducing the defense response by salicylic acid, SA (peroxidase, EC 1.11.1.7, polyphenol oxidase, EC 1.10.3.1) (Kang and Guo 2014, Acta Physiologiae Plantarum, 36: 2287-2297) and jasmonic acid pathway, JA (lipoxygenase E.C. 1.13.11) was determined (Motallebi et al. 2015, Acta Physiologiae Plantarum, 37:1-11).

[0044] The wheat leaves were ground in the presence of liquid nitrogen. To 0.2 g of ground leaves was added 10 ml phosphate buffer (0.1 mol, pH 6.1) After stirring for one hour in a refrigerator, the solution was centrifuged at 13,000 g for 15 minutes at 4°C. Supernatants (enzyme extracts) were then used to determine the activity of various oxidases, differentially induced by HS or JA. The activities of peroxidase, POx, and polyphenol oxidase, PPO, were determined spectrophotometrically by increasing optical density (OD), DO470, and DO420, respectively. The substrates used for POx and PPO were guiacol / H202 and catechol, respectively. 0.3 ml of extract were added over 1.7 ml of substrate 1 mM in 0.1 M Tris-HCI buffer, pH 7.8. (Gomes et al. 2005, Scientia Agricola, 62: 547-551). LOx activity was measured at 234 nm. The substrate solution containing linoleic acid (Sigma, Sigma Aldrich, St.Louis, MO, USA) was prepared according to the method of Bohland et al. 1997 (Plant Physiology, 114: 679- 685), purged with nitrogen and stored at -20°C in aliquot parts.

[0045] To determine LOx activity, 60 μl of extract was diluted to 1 ml of 0,1 M phosphate-citrate buffer, pH 6.2, 0,1 ml of substrate solution was added, and incubated for 15 min at 30 °C.

[0046] The optical densities readings for determining POx and PPO activity were taken in dynamic mode, every second, for 2 minutes on a CLARIOstar spectrophotometer (BMG Labtech, Ortenberg, Germany). Enzymatic activities were expressed in units per gram of fresh substance (U / g). One POx and PPO enzyme unit was defined as the amount of enzyme that causes an increase of 0.1 DO units per minute per ml of extract. One enzyme unit LOx is the amount of enzyme that determine an increase in optical density of 0.001 units per minute per ml of extract. The reagent controls used as reference for spectrophotometric readings were extraction and reaction buffers. The results are presented in Table 1 . Tab. 1. Activity of enzymes specific to the induction defense response via salicylic acid (peroxidase, POx, polyphenol oxidase, PPO) and jasmonic acid (lipoxygenase LOx) in wheat plants treated with selenium nanoparticles obtained according to Example 1 and with sodium selenite and microalgae-yeast extract Nannochloris sp. 424-1 - Rhodotorula mucilaginosa TazRr

[0047] The results demonstrate a balanced induction of the defense response in wheat plants, under the action of foliar treatment performed with selenium nanosuspensions made according to Example 1. Micro-algae extract acts predominantly as an activator of the salicylic acid defense response. Sodium selenite has limited action in inducing the plant defense response, most likely due to its pro-oxidant effect of inducing higher levels of reactive species in plant tissues.

Claims

Claims1. Process according to the invention characterized in that consists of the following steps: making a culture medium with the following composition: 10 g / L whey powder with min. 12% protein and min. 72% lactose; NaHCO316.80 g / L; K2HPO40.50 g / L; NaNO31.875 g / L; K2SO41 .00 g / L; NaC1 1.00 g / L; MgSO47H2O 0.20 g / L; CaCI2• 2H2O 0.04 g / L, microelement solution 1 mL / L; Fe chelated solution 5 mL / L; aseptic administration of a 10 mM sodium selenite solution, sterilised by ultrafiltration on a 0,2 pm filter, in the ratio of 10 ml selenite solution to 90 ml medium; andnoculation of the medium with a suspension of the microalgae Nannochloris sp. 424-1 , deposited under CCAP number 251 / 10 in the Culture Collection of Algae and Protozoa (CCAP), containing between 108and 109viable micro-organisms per mL in the ratio of 1 mL inoculating suspension at 8 mL medium and with a suspension of Rhodotorula mucilaginosa TazRr, deposited under DSM number 34804, at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, containing between 108and 109viable micro- organisms per mL, at a ratio of 1 mL inoculating suspension at 10 mL medium; andincubation of the algae-yeast consortium at temperature of 28-30°C; illumination 250 pEm-2s-1, with a photoperiod / alternation illumination - dark cycle of 12:12 hours; administration of synthetic gas mixture with composition: 7% CO2, 14% O2 and 79% N2at a flow rate of 30 mL / min, corresponding to an aeration with 1 liter of gas mixture with 7% CO2per min per 100 litres of medium until an optical density corresponding to the accumulation of 5 g per litre of medium is reached; flocculation of the algal-yeast culture by addition to the culture medium of a strigolactone mimic, 3-(4-methyl-5-oxo-2,5- dihydrofuran-2-yl)-3H-benzothiazol-2-one, SL-F3, up to a final concentration of 10-8M; ultraconation of photosynthesizing microorganism culture - selenium nanoparticles for 10 min at a frequency of 20 kHz and an energy of 80 J; separation by microfiltration, on ceramic membranes with pore sizes of 5 pm of cell debris, followed by separation of selenium nanoparticles by ultrafiltration on 50 nM membranes and their concentration by ultrafiltration on 10 nM membranes.

2. Procedure according to claim 1 characterized in that the stock solution of micronutrients contains the following quantities, expressed in g / L: H3BO3, 2.860; MnSO4-4H2O, 2.030; ZnSO4-7H2O 0.222; MoO3(85%) 0.018; CuSO4· 5H2O, 0.079; CO(NO3)2- 6H2O 0.494.Procedure according to claim 1 characterized in that the chelated Fe stock solution is prepared as follows: as follows: 0.69 g FeSO4• 7H2O are dissolved in 80 ml distilled waterand 0.93g Na2EDTA are added and the resulted solution is heated, boiled for 5 min , cooled to room temperature and brought to a final volume of 100 ml.

3. Process according to claim 1 characterized in that the microalgae strain Nannochloris sp. 424-1 , deposited under CCAP number 251 / 10 in the Culture Collection of Algae and Protozoa (CCAP), SAM Research Services, Scottish Marine Institute, Aryll, UK, forms a biomass that generates bioactive compounds as biostimulants for plants, including polysaccharides which act as elicitors of plant defence systems.

4. Process according to claim 1 characterized in that the strain Rhodotorula mucilaginosa TazRr, deposited under DSM number 34804 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, produces phytohormones that stimulate algae growth, glutathione which reduces the pro- oxidant effect of sodium selenite, and a significant amount of amphiphilic mannoproteins, which stabilize suspensions of selenium nanoparticles and promotes penetration through the cuticle of plants.

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