A method obtaining a preparation mixture based on purified PGA, particularly

Abstract

A method for obtaining a preparation - mixture based on purified PGA, especially for use in preventing reduced moisture in the soil for plants, including during drought using at least one bacterium for obtaining PGA, characterized in that in the first stage, one bacterial culture i.e. Bacillus licheniformis, preferably in liquid form with a CFU from 1*103 to 1*108, is placed in a sealed bioreactor, in the second stage, bacteria sterile growth medium is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH; and a temperature not higher than 41°C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours; and then the resulting cultivated mixture is then hygienically bottled. The subject of the invention is a preparation – mixture obtained with this method.

Description

[0001] A method obtaining a preparation mixture based on purified PGA, particularly for use in preventing reduced moisture in soil for plants, including during drought.

[0002] TECHNICAL FIELD

[0003] The object of the invention is a method of obtaining a preparation - mixture based on purified PGA, especially for use in preventing reduced moisture in the soil of plants, including during drought.

[0004] BACKGROUND ART

[0005] Polyglutamic acid (PGA) is a highly anionic, natural polymer produced by bacteria of the genus Bacillus spp as a fermentation product secreted into the growth medium. It is a water-soluble, biodegradable, non-toxic substance. PGA stimulates and improves immune activity, making it suitable for the selective delivery of chemotherapeutic agents (Tork et al., 2015; Chatteijee et al., 2018, 2019; Park et al., 2021). Due to its properties, PGA can be used both as a free polymer, as well as a hydrogel and / or polymer nanoparticles. Currently, it is widely used, among others: in sewage treatment, fertilizers, food products, skin care products, surgical adhesives, tissue engineering, drug delivery and other purposes. PGA also has a strong bactericidal effect, especially against gram-negative bacteria (Richard et al., 2001; Chatteijee et al., 2018, 2019; Park et al., 2021).

[0006] One of the model bacteria used in broadly understood microbiological and genetic research is E. coli (latin Escherichia coli). It is a gram-negative, relatively anaerobic bacterium belonging to the Enterobacteriaceae family. It is part of the physiological bacterial flora of the large intestine of a human and other warmblooded animals, participating as a symbiont in the breakdown of food, and contributing to the production of B and K vitamins. However, if the body's homeostasis is disturbed, E. coli may be pathogenic to humans and animals. Young organisms are particularly vulnerable to infection with this bacterium, and its various serotypes may also be present in the bodies of sick animals (Kaper et al., 2004; Jang et al., 2017). It is estimated that in the conditions of mass production of poultry and pigs, about half of the deaths of young animals are caused by colibacillosis (Cortes et al., 2010; Ozaki et al., 2017; Maertens et al., 2020; Newman et al., 2021) The resistance of Escherichia coli to environmental factors is relatively low. It dies after 20 minutes of heating at 60°C and is sensitive to all known disinfectants. However, its survival increases at lower temperatures and high humidity (Cho et al., 2010; Ganzle, 2015; Jang et al., 2017).

[0007] Drought is a natural phenomenon that occurs when a given area receives less rainfall than usual for an extended period of time. This can result in water scarcity, which can have a significant impact on agriculture and food production. Ways drought can lead to crop loss: Water stress: Drought can cause plants to lack sufficient water, leading to stunted growth, wilting leaves, and ultimately plant death. When water is scarce, plants may not be able to absorb enough water to meet their needs, resulting in physiological changes that affect their ability to produce crops. Reduced yields: Drought can also lead to reduced yields as plants may produce fewer or smaller fruits or seeds. This could have a significant impact on food production and could contribute to food shortages and price increases. Soil degradation: Drought can also contribute to land degradation, as a lack of water can cause soil to become compacted and more susceptible to erosion. This can lead to reduced soil fertility, which can further reduce yields over time. Increased susceptibility to pests and diseases: Plants exposed to drought may be more susceptible to pests and diseases, which can further reduce yields. This is because drought stress can weaken plants, making them more susceptible to attacks by pests and pathogens.

[0008] There are two ways to deal with water stress on plants: using biostimulants (hormone-like chemicals and peptides) and bioengineering using optogenetics or natural variation in genome-wide association studies.

[0009] Polyglutamic acid (PGA) is a natural component of biofilm, an optically active anionic polymer, occurring in two isoforms: a-PGA and y-PGA.

[0010] A method for improving stress resistance and yield of plants through heterologous synthesis of gamma-polyglutamic acid in plants is known from the Chinese patent description No. CN115725519A. The invention discloses a method for improving plant stress resistance and yield by heterologous synthesis of gammapolyglutamic acid in the plant, which includes the following steps: cloning and synthesis of three key enzyme genes Pgs A, PgsB and PgsC gamma-PGA from the plant strain to produce gamma-PGA and re- synthesis of codon-optimized PgsA, PgsB and PgsC gene sequences according to the codon preferences in the plant. The method includes the following steps: inoculating the transgenic plant with the plant expression vector PGA001, combining the plant expression vector PGA001 with the pU130-bar vector comprising the bar gene selection marker to obtain the plant expression vector PGA001, and transferring the plant expression vector PGA001 to the plant via Agrobacterium tumefaciens to obtain a new transgenic plant strain for gamma production. Experiments prove that the new line of transgenic corn obtained by this method can not only improve corn's drought resistance, but also can improve salt resistance, and can obviously improve corn's biomass under drought stress and normal growth conditions.

[0011] The results indicate that the method provided by the invention is an ecological, efficient and sustainable effective way to solve the problem of stable or reduced crop yields under the influence of drought, water scarcity and salt and alkali, and can be widely used in the treatment of drought-resistant and salt-resistant molecular breeding and cultivation of new plant varieties.

[0012] Highly effective PGA urea for environmental protection and its production technology are known from the Chinese patent description No. CN102515956A.

[0013] The invention discloses a high-performance environmental protection PGA urea and its production technology and belongs to the field of environmental protection. The production technology includes the following steps: adding gammapolyglutamic acid, trace elements and several rooting powder to ordinary urea, mixing evenly and forming a new high-efficiency environmental protection granulated urea in the granulation tower. The urea produced according to the invention binds sand, saves water, promotes and establishes roots, supports plant growth thanks to many nutrients, increases fertilizer efficiency, minimizes water and fertilizer losses and protects the ecological environment.

[0014] A mixed microbial agent suitable for saline-alkaline soils in arid and semi- arid regions and the use of a combined microbial agent are known from Chinese patent description No. CN114304187A. The invention provides a composite microbial agent suitable for saline-alkaline soil in arid and semi-arid regions and the use of a composite microbial agent. The composite bacterial agent disclosed in the invention includes alfalfa rhizobium, functional water retention peptide and drought-tol erant bacteria, wherein the dr ought-tol erant bacteria are endophytic Bacillus flexus CGMCC (China General Microbial Culture Collection Center) No. 21705 and functional water. The water retention peptide is produced by fermentation of Bacillus licheniformis CGMCC No. 8821. The microbial compound disclosed in the invention can be used to improve the activity of plant root systems and the resistance of plants to drought and resistance to salt and alkali, is particularly suitable for planting plants in regions with arid and semi-arid climates, obviously improves the nitrogen content ability binding of plants in dry conditions, obviously improves the adaptive capacity of plant root systems in conditions of water drought and saline-alkaline stress, improves plants' resistance to stress and improves yield.

[0015] The objectives of improving soil fertility and increasing crop yields were achieved, and the method has significant implications for improving the ecological environment of planting saline-alkaline soils in arid and semi -arid regions and the sustainable development of modem agriculture.

[0016] A method of controlling desertification by adopting a new biological material, gamma-polyglutamic acid (PGA), is known from Chinese patent description No. CN102383415 A. The invention relates to a method of controlling desertification by adopting a new biological material and discloses a method of controlling desertification by adopting a new biological material, gammapolyglutamic acid (PGA). The method includes the step of spraying an aqueous solution of gamma-PGA 1-10 times on the surface of the roots of desert plants, with the concentration of the aqueous PGA solution being 0.5-10%. The method has the following beneficial effects: on the one hand, the loose sand soil is adhered to the block structures by gamma-PGA, thus controlling the flowability of sandy soil in deserts and playing a good sand stabilizing role; on the other hand, the super strong water retention and absorption of gamma-PGA is used to spontaneously form hydrogel by absorbing hand-poured water and condensed dew formed at night, thus achieving the effect of slow moisture release and meeting the basic moisture requirements of plant seedlings with undeveloped root system ; the combination of both hands allows the transpiration and infiltration of moisture by plants to be reduced as much as possible and the soil environment in deserts to be improved, while at the same time supporting the growth of xerophytes in deserts, thus ultimately achieving the effect of converting sand into forests.

[0017] A fertilizer assistant / enhancer and its application are known from the Chinese patent description No. CN105198560A. The fertilizer assistant is prepared from 20-80 parts by weight of the polybasic sugar alcohol composition and 20-80 parts of the polymer amino acid composition. The polybasic sugar alcohol composition is prepared from 5-10 parts of mannitol, 10-20 parts of sorbitol, 5-10 parts of xylitol, 20-30 parts of citric acid and 30-50 parts of zinc sulfate. The polymeric amino acid composition is prepared from 30-40 parts of gamma-PGA, 20-30 parts of PASP and 30-50 parts of calcium nitrate. The fertilizer assistant can be used for solid or liquid fertilizer in commercial crops, the weight ratio of fertilizer assistant to solid fertilizer is 10-30%, and the weight ratio of fertilizer assistant to liquid fertilizer is 20-50%. Small molecular weight sugar alcohol and amino acids integrated with biological sources have a beneficial effect on the absorption of plant roots / beyond the roots, improve the use efficiency of traditional fertilizer, do not pollute the environment or cause potential safety hazards, and can extend the quality guarantee period of cultivated fruits.

[0018] An innovative, effective fertilizer improving plant immunity is known from the Chinese patent description No. CN112194529A. The invention relates to the field of agricultural fertilizer technology, in particular to a new, effective fertilizer capable of improving the immunity of plants. The method includes the following steps: in the production process of compound fertilizer, the addition of three functional substances, namely potassium phosphite, polyglutamic acid and polypeptide urea, to finally produce a special pesticide with high utilization rate, and the interaction of potassium phosphite, polyglutamic acid and polypeptide urea to implementing the mutual functions of storage and absorption, thus obtaining a special fertilizer with a high utilization rate. The absorption and utilization rate of nitrogen fertilizer is improved, and the immunocompetence of plants is improved through the effect of systemic conduction, so that plants have a broad-spectrum antiviral ability.

[0019] Bacillus amyloliquefaciens and a method for producing gammapolyglutamic acid fertilizer synergist by adapting Bacillus amyloliquefaciens are known from the Chinese patent description No. CN110791441 A. The invention discloses the Bacillus amyloliquefaciens strain JX8-2. The strain is kept in the China General Microbial Culture Collection Center (CGMCC), the center address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing, 100101, China, the security number is CGMCC No. 14775, and the protection date is October 9, 2017. The invention also discloses the use of a strain for producing a gamma-polyglutamic acid fertilizer synergist and a method for producing a gamma-polyglutamic acid fertilizer synergist using the strain and Paenibacillus mucilaginosus.

[0020] From the American patent description No. US2022110326A1, a combination of pyroglutamic acid and a urease inhibitor is known to improve the health effects of plants. The subject matter described herein relates generally to compositions and formulations and methods of using combinations of pyroglutamic acid and NBPT to improve plant health characteristics such as crude protein yield and increasing percentage of nitrogen in plant tissue.

[0021] A biodegradable agent for conditioning and supplying saline-alkaline soils is known from the Chinese patent description No. CN102241986 A. Concerning soil salinity control technologies, the invention particularly relates to a remedial agent for saline soil. Biodegradable conditioner and conditioner for saline-alkaline soils is a solution prepared by mixing a mixture consisting of 50-70% of a high-polymer PGA product derived from microbial metabolism, 25-35% of urea and 5-15% of a root growth stimulator by weight with water in a ratio of 1 : 15. Without any harmful effect on the soil, the agent according to the invention can be adapted to different areas.

[0022] DISCLOSURE OF THE INVENTION

[0023] The aim of the invention is to develop a new method of obtaining and a new mixture of bacterial metabolites with properties that stimulate plant growth under drought stress and activate soil bacteria with reduced water content.

[0024] In soil that reaches the point of permanent wilting, the plant continues to maintain physiological processes (photosynthesis) and secretes antioxidant enzymes that prevent the destruction of plant tissues. Under natural conditions, it stimulates the growth of the root zone and intensifies the process of cation uptake (mainly potassium and calcium) from the soil.

[0025] A method of obtaining a preparation - mixture based on purified PGA, especially for use in preventing reduced moisture in the soil for plants, including during drought, using at least one bacterium for obtaining PGA according to the invention is characterized in that in the first stage, one bacterial culture i.e. Bacillus licheniformis, preferably in liquid form with a CFU from 1 * 10’ to 1 * 10i0preferably 1 *108, is placed in a sealed bioreactor, in the second stage, bacteria sterile growth medium is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH, and a temperature not higher than 41°C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above- mentioned conditions in the tank for at least 24 hours; and then the obtained cultivated mixture - the preparation is hygienically bottled.

[0026] Preferably the bacteria sterile growth medium comprises per 300 liters of bioreactor - monosodium glutamate in an amount from 12 to 18 kg, preferably 15 kg, glucose in an amount from 18 to 24 kg, preferably 21 kg, sodium citrate in an amount from 1, 7 kg to 2.5 kg, preferably 2.1 kg, ammonium sulfate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, sodium nitrate in an amount from 1.7 kg to 2.5 kg, preferably 2. 1 kg, yeast extract in amount from 4.3 kg to 5.7 kg, preferably 5 kg, peptone in an amount from 4.1 kg to 5.9 kg, preferably 5 kg, zinc sulfate in an amount from 0.18 kg to 0.24 kg, preferably 0.21 kg, dipotassium phosphate in an amount from 0.17 kg to 0.25 kg, preferably 0.21 kg, hydrated iron sulfate in an amount from 0.17 kg to 0.25 kg, preferably 0.21 kg.

[0027] A mixture based on purified PGA, especially for use in preventing reduced moisture in the soil of plants according to the invention is characterized in that it comprises by volume: volumetric mixture of bacterial cell bodies Bacillus licheniformisin the amount 20%, high molecular weight glutamic acid homopolypeptides (>lMDa) - in the amount 4%, medium molecular weight glutamic acid homopolypeptides in the amount of 20%, low molecular weight glutamic acid homopolypeptides - in the amount of 5%, low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - up to 5% by volume and the rest is water.

[0028] The essence of the solution according to the invention is the development of a mixture of metabolites having a high concentration (up to 30% of the solution) of gamma-polyglutamic acid.

[0029] The preparation-mixture comprises approximately 400 different (active) chemical compounds, including: metabolites of the transformations of the above- mentioned bacteria (approximate results from the mass spectrometer).

[0030] Properties of the preparation - mixture according to the invention (which are also its additional advantages):

[0031] 1. Stimulating plant growth in drought conditions (permanent wilting point)

[0032] 2. Stimulating the physiological processes of plants to prevent the effects of drought stress (accumulation of cations in tissues, antioxidant enzymes, increase in proline production, increase in the production of simple sugars) 3. Stimulating an increase in the metabolism of soil bacteria and an increase in the activity of soil enzymes, including urease.

[0033] Polyglutamic acid (PGA) is a naturally occurring polymer composed of glutamic acid residues that may play a role in plant drought resistance. PGA is produced by several species of bacteria that colonize the rhizosphere of plants and has been shown to have various beneficial effects on plant growth and stress tolerance. Here are some ways PGA can contribute to plant drought resistance: Water Retention: PGA has a high water-holding capacity and can absorb and retain large amounts of water in the soil. This can help maintain soil moisture levels and reduce plant water stress. Osmotic regulation: PGA can also help regulate the osmotic potential of plant cells and maintain turgor pressure under drought conditions. This may help prevent water loss and dehydration in plant tissues. ROS scavenging: PGA has antioxidant properties and can scavenge ROS generated during drought stress. This may help protect plant tissues from oxidative damage. Nutrient Availability: PGA can bind to soil minerals and nutrients and release them slowly over time, making them available to plants. This may be particularly important under drought conditions when nutrient uptake may be limited. Overall, the use of PGA as a soil amendment or plant growth-promoting bacteria (PGPB) inoculant has been shown to improve plant growth and drought tolerance in several crop species.

[0034] In plant cultivation, y-PGA can also be used to remove toxic metal ions from the soil. This polymer can form chelating compounds with toxic metal ions, such as Ni2+, Cu2+, Mn2+, A13+ and Cr3+, which facilitates their removal from the substrate. Thanks to its ability to bind y-PGA ions, it can be used as a fertilizer supplying Ca2+, Fe2+, Fe3+, Zn2+ and Mn2+ cations to plant rhizospheres.

[0035] Polyglutamic acid (PGA) has been studied for its potential to increase plant growth and reduce water stress, but its effectiveness as a superabsorbent or drought growth stimulator may depend on various factors such as specific application, concentration and environmental conditions. As a superabsorbent, PGA can absorb and retain large amounts of water, which can help improve soil moisture retention and reduce irrigation needs. In this way, it can potentially reduce water stress and improve plant growth. However, the effectiveness of PGA as a superabsorbent may depend on factors such as soil type, application rate and irrigation frequency. As a drought growth stimulator, PGA has been shown to improve plant growth and yields under water scarcity conditions. It may help maintain cell turgor and protect plants from oxidative stress, which may improve their drought resistance. In this way, PGA can potentially stimulate plant growth under drought conditions. Overall, PGA has a potential drought growth stimulator, but its effectiveness may depend on various factors and further research is needed to fully understand its potential applications.

[0036] PGA may help stabilize cell membranes, which may reduce cell damage caused by oxidative stress. Increased photosynthesis: PGA can increase photosynthesis in plants, which may help reduce oxidative stress by increasing the production of antioxidants. Overall, reducing oxidative stress in plants through the use of PGA can result in improved plant growth, yield and crop quality. Additionally, the use of PGA may bring potential benefits to human health by increasing the nutritional quality of crop plants.

[0037] CONCLUSIONS - use of the preparation according to the invention:

[0038] 1. Significantly increases plant growth even at the permanent wilting point.

[0039] 2. Improving the condition of plants, especially corn, wheat and grass, by limiting the effects of oxidative stress.

[0040] 3. Supports enzymatic activity in the soil.

[0041] 4. Stabilizes soil moisture in optimal water conditions.

[0042] 5. PGA effectively protects plants in drought conditions.

[0043] Data from field experiments that support the use of y-polyglutamic acid (y- PGA) under drought stress and propose its application in grassland management. Water treatment combined with PGA application on sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by:

[0044] 1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+ / 3+, Ca2+, Mg2+ and N-NH4+, N-N03-,

[0045] 2) elongation of plant roots,

[0046] 3) increasing aboveground biomass, 4) improving the regeneration capacity of the dominant grass Nardus stricta, and

[0047] 5) improving the regeneration of dicotyledonous plants.

[0048] In the case of meadows on sandy soils, the use of low concentrations of PGA (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and for improving the functional properties of plants. Irrigation had a greater effect than PGA application only for the dicotyledonous to monocot ratio. Again, it was most beneficial to use PGA at the lowest concentration, but in combination with watering. y-PGA can protect microorganisms from phagocyte attacks and high salt concentrations while providing extracellular nutrient storage and promoting biofilm formations. y-PGA is water-soluble, biodegradable, biocompatible, edible and nontoxic to humans and the environment. It has the ability to bind metals and absorb water, and its biotechnological production for agricultural applications is becoming more and more profitable. This polymer has many potential applications in the fields of pharmaceuticals, water and soil treatment, food, cosmetics and agriculture.

[0049] Due to its ability to bind ions, y-PGA can be used as a fertilizer supplying cations such as Ca2+, Fe2+, Fe3+, Zn2+, Mn2+ to plant rhizospheres.

[0050] PGA can also be used to reduce salt-induced plant growth inhibition. Under salt stress conditions, accumulation of toxic levels of cellular Na+ and limited K+ uptake were observed in plant cells.

[0051] Surprisingly, watering had no effect on soil parameters under drought stress conditions and only a minor effect on plant parameters. Sandy soils have a high filtration rate and even when watered with various concentrations of PGA, no positive effect was noted.

[0052] In our study, low PGA concentration increased MR. This parameter was also accelerated by irrigation conditions. These results indicate that PGA accelerates the growth of dicotyledonous plants, but watering is necessary during the growing season, and even providing 30% of the calculated water deficit for a given month in a multi-annual period increases the DMR.

[0053] PGA is a biopolymer that is produced by bacteria as a component of biofilm under artificial conditions, but should be treated as a natural element of ecosystems. Its presence in agrosystems is limited as a result of the elimination of many bacterial strains during agricultural work and soil changes. Artificial PGA supplementation to agroecosystems is beneficial to crops but has not been previously used in grasslands. We have shown that precise doses of PGA improve plant growth and shoot regeneration during periods of drought. We do not believe that it directly retains water as a hydrogel, but it can be considered an effective biostimulator that helps the plant grow and survive periods of drought. These data are preliminary and research should be continued in various soil conditions, as well as on intensively used and naturally valuable grasslands.

[0054] The results of our field experiment combining watering and application of different concentrations of y-PGA on grasslands during drought clearly showed that PGA acts as a biostimulant, mitigating the effects of stress on plants. The biostimulating effect of PGA consisted in increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+ / 3+, Ca2+, Mg2+ and mineral forms of nitrogen (N-NH4+ and N-N03-), elongating plant roots, and increasing aboveground biomass, as well as improving the regeneration capacity of the dominant grass N. stricta and the regeneration of dicotyledonous plants. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial both for the availability of macro- and microelements and for the functional characteristics of plants. Irrigation had a greater effect than PGA application only for the dicotto monocot ratio (DMR). Again, it was most beneficial to use PGA at the lowest concentration, but in combination with watering. Based on the results obtained, we recommend using PGA concentrations in the range of 0.3-1% for managing grasslands on light soils. PGA in the given doses is a promising treatment for protecting grassland ecosystems against drought stress.

[0055] Thanks to the preparation-mixture according to the invention, it will be possible to offer on the market a substance / intermediate product based on purified gamma-polyglutamic acid (PGA).

[0056] The subject of the invention is explained in the example of the embodiment. EXAMPLE

[0057] Example (a method of obtaining and mixing)

[0058] A method of obtaining

[0059] In the first stage, one bacterial culture i.e. Bacillus licheniformis in liquid form with a CFU of l*108, is placed in a sealed bioreactor,. In the second stage, bacteria sterile growth medium is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at 8.5 pH; and a temperature not higher than 41 °C, optimally 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours.

[0060] Then the obtained cultivated mixture - the preparation is hygienically bottled.

[0061] The bacteria sterile growth medium was used having composition per 300 liters of bioreactor - monosodium glutamate (15kg), glucose (21kg), sodium citrate (2.1kg), ammonium sulfate (2.1kg), sodium nitrate (2.1kg), yeast extract (5kg), peptone (5kg), zinc sulfate (0.21kg), dipotassium phosphate (0.21kg), hydrated iron sulfate (0.21 kg).

[0062] The obtained mixture comprises by volume: volumetric mixture of bacterial cell bodies (B. licheniformis) - 20% by volume, high molecular weight glutamic acid homopolypeptides (>1 MDa) - 4% by volume, medium molecular weight glutamic acid homopolypeptides - 20% by volume, low molecular weight glutamic acid homopolypeptides -5% by volume, low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - up to 5% by volume and the rest is water.

[0063] Dosage - plants

[0064] The mixture according to the invention comprises a maximum of 3% PGA.

[0065] The best dosage is a preparation with a concentration of 0.5% PGA (10 ml PGA per m2of plants).

[0066] One-time spraying of plants with the preparation according to the invention - once every' 4-5 weeks (additionally as an emergency treatment just before drought).

[0067] An overdose has a negative effect on plant growth.

[0068] The effectiveness of the preparation for up to forty days (one spray).

[0069] Watering - preventively before a period of expected drought or during sowing (reducing seedlings mortality).

[0070] Apply at sowing and later as an intervention after the expected occurrence of drought events in the weather.

Claims

Patent claims1. A method for obtaining a preparation - mixture based on purified PGA, especially for use in preventing reduced moisture in the soil for plants, including during drought using at least one bacterium for obtaining PGA, characterized in that in the first stage, one bacterial culture i.e. Bacillus lichenifonnis, preferably in liquid form with a CFU from l* 103to l* 108, is placed in a sealed bioreactor, in the second stage, bacteria sterile growth medium is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH; and a temperature not higher than 41 °C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours; and then the resulting cultivated mixture is then hygienically bottled.

2. The method of obtaining the preparation-mixture according to claims 1, characterized in that the bacteria sterile growth medium comprises per 300 liters of bioreactor - monosodium glutamate in an amount from 12 to 18 kg, preferably 15 kg, glucose in an amount from 18 to 24 kg, preferably 21 kg, sodium citrate in an amount from 1, 7 kg to 2,5 kg, preferably 2.1 kg, ammonium sulfate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, sodium nitrate in an amount from 1.7 kg to 2,5 kg, preferably 2.1 kg, yeast extract in amount from 4.3 kg to 5.7 kg, preferably 5 kg, peptone in an amount from 4.1 kg to 5.9 kg, preferably 5 kg, zinc sulfate in an amount from 0,18 kgto 0.24 kg, preferably 0.21 kg, dipotassium phosphate in an amount from 0.17 kg to 0.25 kg, preferably 0.21 kg.

3. A mixture based on purified PGA, especially for use in preventing reduced moisture in the soil for plants, characterized in that it comprises by volume: volumetric mixture of bacterial cell bodies Bacillus licheniformis in the amount 20%, high molecular weight glutamic acid homopolypeptides (' > 1 MDa) - in the amount 4%, medium molecular weight glutamic acid homopolypeptides in the amount of 20%, low molecular weight glutamic acid homopolypeptides - in the amount of5%. low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - up to 5% by volume and the rest is water.