STORAGE COMPOSITION FOR AGRICULTURE COMPRISING AT LEAST ONE STABILIZING AGENT

Description

1 STORAGE COMPOSITION FOR AGRICULTURE COMPRISING AT LEAST ONE STABILIZING AGENT The present invention relates to a storage composition for agriculture comprising at least one stabilizing agent and a biological material intended to improve soil fertility or to combat crop pests, biotic and abiotic stress, to a process for forming said storage composition and the use of said stabilizing agent to preserve the viability of a biological material intended to improve soil fertility or to combat crop pests, biotic and abiotic stress. In the field of organic or industrial agriculture, biological materials such as (micro)organisms and nematodes can contribute respectively to improving soil health, protecting crops against pests and pathogens, stimulating plant growth and reducing dependence on chemical inputs,taking into account a reduction in long-term costs thanks to more productive and resilient soils. One of the challenges is to make agriculture more ecological and resilient in order to preserve the environment and the climate. In recent years, advances in the field of agriculture have been marked by the development of biological solutions, responding to the need to move from chemical and synthetic solutions to more sustainable alternatives. Indeed, faced with growing concerns about the impact of chemical products on the environment, biodiversity and human health, players in the agricultural market are turning to biological materials in order to reduce the quantities of chemical inputs. 2025 / 5080 BE2025 / 5080 2 These biological materials can be applied in various forms, solid, oily or aqueous, either in soil or as foliar sprays. Before application to the field, either in soil or as a foliar spray, the biological material must be formulated to maintain its viability and effectiveness. These biological materials,Like bacteria and fungi belonging to the family of (micro)organisms within the scope of the invention, or nematodes, are often transformed into specific products to facilitate their storage and use in the field.10 The preservation of these products depends on several environmental factors. Some (micro)organisms must be stored at an ideal temperature, often between 4°C and 10°C, to prevent their degradation. Humidity must also be controlled, as dry forms can be reactivated prematurely if exposed to a humid atmosphere. In addition, they must be protected from direct light, particularly UV rays, which can degrade them rapidly. Products containing (micro)organisms have a limited shelf life, generally indicated by the manufacturer, beyond which their effectiveness may decrease.20 For example, (micro)organisms are generally preserved in different forms: dry, powdered, or oily. Dry forms, obtained by freeze-drying or in powder form,These forms allow for easy storage and good preservation, provided they are kept away from moisture. They require rehydration before application, which can be inconvenient for the user. Oil-based forms are ready to use but often require stabilizers, emulsifiers, or nutrients to ensure the viability of the biological materials and guarantee a supply chain and application conditions that are realistic compared to market standards. (Micro)organisms can also be encapsulated in granules or microcapsules, allowing for their gradual release into the soil or onto plants. Before application, some (micro)organisms require activation. Products in dry form must be rehydrated, often in a nutrient-enriched solution to stimulate their activity. Furthermore, some products are inoculated onto substrates, such as seeds or organic matter. During the preparation of mixtures,It is crucial to verify the compatibility of (micro)organisms with other agricultural inputs (fertilizers or pesticides) in order not to reduce their effectiveness, but also with any other active ingredients, such as amino acids. The preservation of (micro)organisms must also be able to withstand transport and the associated logistics. For sensitive products, a cold chain is often required, with controlled temperatures to avoid thermal variations that could affect the viability of (micro)organisms or nematodes. It should be noted that, depending on the biological material, formulation and transport may require specific compounds to ensure a stable storage composition over time, with adequate viability, while guaranteeing effectiveness during its application in agriculture, particularly in biostimulation or biocontrol. A storage composition for agriculture that can be easily stored and transported to field application constitutes a real need on the market. Thus,Current solutions are too expensive, restrictive, complex and ineffective. There is therefore a real need to provide a storage composition which includes biological material intended to improve soil fertility or to combat crop pests, biotic stress, and which can be easily preserved and stored for a sufficient period at an affordable cost, while guaranteeing effectiveness when applied in agriculture, particularly in soil or foliar application. To solve this problem, the present invention provides a storage composition for agriculture comprising at least one stabilizing agent, in zwitterionic form, which comprises at least one quaternary ammonium compound and a beneficial biological material intended to improve soil fertility or to control crop pests and promote plant growth by direct and / or indirect action against biotic or abiotic stress, characterized in that said biological material is selected from: -(micro)organisms,or 20 - nematodes to strengthen integrated biological control, and in that said storage composition is aqueous when said biological material is selected by (micro)organisms, and in that it is pasty when said biological material is selected by nematodes. 2025 / 5080 BE2025 / 5080 5 The storage composition according to the invention can be in aqueous or pasty form depending on the biological material considered. Preferably, a person skilled in the art should understand that the pasty form means a dynamic viscosity at a given temperature that is higher than the dynamic viscosity at that given temperature of the aqueous form. The storage composition according to the invention is effective thanks to the presence of said stabilizing agent, which comprises at least one quaternary ammonium compound. Indeed, this allows the biological material to be protected from its surrounding environment. Thus, the viability of the biological material is preserved in a stable manner over time. In other words,The effectiveness of the biological material can be guaranteed at the time of its application in agriculture, particularly in soil or foliar applications. The storage composition according to the invention allows, preferably, storage for at least 24 months with maintenance of viability, or even a loss of viability of at most, a logarithmic scale of residual CFU of the biological material. It has been observed that the stabilizing agent, in combination with (micro)organisms, makes it possible to provide an aqueous storage composition that is easy to preserve and transport until the time of its application. This is also the case when the biological material is selected from fungi. When the biological material is selected from nematodes,It has been observed that the final storage composition 25 is in the form of a pasty storage composition. This also allows it to benefit from the preservation and transport properties indicated above. The storage composition according to the invention can be supplied at an affordable cost, which makes it a major advantage in the context of the stabilization of biological materials as defined in section 5 of the invention. During application, the user can benefit from a ready-to-use storage composition without necessarily adding any processing steps or additional ingredients. Furthermore, the storage composition according to the invention can 10 be stored at ambient temperature, which is particularly advantageous in terms of storage and transport. Furthermore, the storage composition can be maintained at a wide pH range, which offers flexibility to the user. Preferably, the storage composition according to the invention has a pH between 4 and 13, preferably between 5 and 11, and more preferably between 6 and 10. Preferably,The biological material is in a state of biological dormancy, corresponding to a state of temporary inactivity. This preserves the viability of the biological material and its surrounding environment. Surprisingly, a slight or even no logarithmic decrease was observed after one month of storage, which is particularly advantageous for the user. Advantageously, the (micro)organisms are in the form of spores and the nematodes in the form of resistant cysts. 2025 / 5080 BE2025 / 5080 7 According to an advantageous method, the said storage composition has a storage temperature between -5° and 30°C, preferably between 0° and 25°C, more preferably between 5° and 25°C, for a period of at least 1 month, preferably at least 2 months, more preferably at least 3 months, more preferably at least 5-6 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months with maintenance of the viability of the biological material during storage,possibly during an agricultural or horticultural application. It has been observed that the storage temperature as defined 10 makes it possible to reduce logistics and transport costs. Indeed, the storage composition according to the invention can be obtained at ambient temperature and also maintained and transported at ambient temperature, which constitutes a definite advantage compared to state-of-the-art storage compositions. 15 Preferably, said pasty storage composition has a higher viscosity than the aqueous storage composition. This allows nematodes to be considered as biological material and to be stored and transported while preserving their viability before application in agriculture or horticulture.20 Preferably, said (micro)organisms, such as bacteria, are chosen from the group comprising the genera Azospirillum spp., Bacillus spp., Lactobacillus spp., Clostridium spp., Streptomycess spp., Pseudonoma spp., Azotobacters spp., Cellvibrios spp., Rhizobium spp., Ensifers spp., Allorhizobium spp., Enterobacters spp., and mixtures thereof.25 Preferably,said (micro) organisms, such as bacteria, are chosen from the group comprising the species Bacillus 2025 / 5080 BE2025 / 5080 8 subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacilluslicheniformis, Bacillusthuringiensis,Bacilluscereus,Bacillusmegaterium,Bacilluspumilus, Bacillusanthracis,Clostridiumbotulinum,Clostridiumdifficile,Clostridium perfringens,Clostridiumtetani,Clostridiumacetobutylicum,Clostridium thermocellum,Lactobacillusacidophilus,Lactobacillusplantarum,5 Lactobacillusrhamnosus,Lactobacilluscasei,Lactobacillusdelbrueckii, Lactobacillus reuteri, Lactobacillus salivarius, and mixtures thereof. The selection of these (micro)organisms in combination with the stabilizing agent of the present invention leads to a particularly stable storage composition over time, the viability of which can be maintained during storage and transport, and allows for effective application, particularly in the following fields: biocontrol, biostimulant, horticulture, integrated pest management, conventional agriculture,agriculturebiologique,etc. Avantageusement,lesdits(micro)organismes,telsqueles15 champignons,sontchoisisdanslegroupecomprenantlesgenres Trichodermaspp.,Glomusspp.,Beauveriaspp.,Saccharomycesspp., Penicilliumspp.,Metarhiziumspp.,Kluyveromycesspp.,Torulasporaspp., Zygosaccharmoycesspp.,etleursmélanges. Avantageusement,lesdits(micro)organismes,telsqueles20 champignons,sontchoisisdanslegroupecomprenantlesespèces Trichodermaharzianum,Trichodermaviride,Glomusintraradices,Glomus etunicatum,Beauveriabassiana,Aspergillusniger,Trichoderma asperellum,Metarhiziumanisopliae,Lecanicilliumlecanii,Paecilomyces lilacinus,Pochoniachlamydosporia,Trichodermakoningii,Penicillium25 bilaiae,Chaetomiumglobosum,and their mixtures. 2025 / 5080 BE2025 / 5080 9 The selection of these fungal families in combination with the stabilizing agent of the present invention leads to a particularly stable storage composition over time, the viability of which can be maintained during storage and transport and allow for effective application, particularly in the following 5 areas: biocontrol, biostimulant, horticulture, integrated pest management, conventional agriculture, organic agriculture, seed treatments, etc. Advantageously, said nematodes are chosen from the group comprising the genera Steiner nemas spp., Heterorhabditis spp., and Aphelenchoides spp. and their mixtures. Advantageously, said nematodes are chosen from the group comprising the species Steinernemacarpocapsae, Steinernemafeltiae, Steinernemaglaseri, Steinernemariobrave, Heterorhabditisbacteriophora, Heterorhabditisgidis, Heterorhabditis indica, Heterorhabditismarelatus, Aphelenchusavenae,and their mixtures. Nematodes enable the biological control of crop pests through their entomopathogenic action, thus contributing to reducing or eliminating the use of chemical pesticides20 or any other chemical inputs. Furthermore, the selection of nematodes in combination with the stabilizing agent of the present invention leads to a particularly stable, pasty storage composition over time, the viability of which can be maintained during storage and transport, and allows for effective application.25 Preferably, said at least one quaternary ammonium comprises the radicals R1, R2, R3, and R4, which are alkyl groups, and where R4 is linked to a polar union selected from the group comprising carboxylate (COO-), hydrogen sulfite (HSO3-), and phosphate (PO42- or PO33-). -), boronate (BO₂⁻ or B(OH)₃⁻) and their combinations. It has been observed that quaternary ammonium compounds are zwitterionic molecules, which allows for a composition of aqueous or pasty storage.which offers advantages in terms of storage and transport compared to oily or solid storage compositions. Advantageously, said stabilizing agent according to the invention is chosen from the group comprising betaine, betaine glycine, N,N-Dimethylglycine, betaine phosphate, betaine boronate, betaine HCl, betaine hydrogen sulfite, and mixtures thereof. It has been observed that these betaine-based compounds stabilize biological matter by maintaining it in a dormant state while preserving the viability of the biological matter. Indeed, the betaine-based compounds as defined in the present invention regulate the osmotic pressure of the environment and also prevent the premature termination of bacterial spores. Furthermore, betaine allows the protection of plant cells, of plants,against osmotic stress and dehydration and contributes to maintaining water balance in tissues. This biostimulant effect will result in stimulated plant growth as well as an effect on crop quality. These effects are due to better management of water stress (drought, heatwave, desiccation of fruit after harvest), salt stress and resistance to temperature changes (spring frost). Advantageously, betaine is of plant origin and comes from the valorization of eco-products from the processing of sugar beets, such as sugar beet molasses. This allows us to remain on a bio-based biological solution that respects the environment and the ecosystem and is in line with the perspective of sustainable agriculture. Preferably, the said stabilizing agent has a concentration between 5 and 50% by weight, preferably between 10 and 40%, more preferably between 15 and 35%.relative to the total weight of the storage composition. 10 It has been observed that a concentration of the stabilizing agent between 5 and 50% by weight, preferably between 10 and 40%, more preferably between 15 and 35% relative to the total weight of the storage composition, allows, according to the invention, adequate solubility of the stabilizing agent. 15 Advantageously, said (micro)organisms are present at a concentration between 10⁶ and 10¹² CFU / mL. It has been observed that this range allows for the best biostimulating efficacy without saturating the biological activity or compromising the viability of the (micro)organisms. 20 Advantageously, said nematodes are present at a concentration between 1 million and 1 billion IJs / kg. It has been observed that this range allows for the best efficacy biostimulant without saturating the biological activity or compromising the viability of the nematodes.25 The unit IJs means the infectious juveniles (IJs) which are a stage in the life cycle of the nematodes,for example those belonging to the Steinernematidae and Heterorhabditidae families. Nematodes are parasites of insects and are commonly used as biological control agents to manage pest insect populations in agricultural and horticultural environments. Preferably, the pasty storage composition according to the invention comprises moisturizing, texturizing, unloading, nutritive, antimicrobial, antifungal, stabilizing agents or milk proteins selected from a group comprising glycerol, polyethylene glycol, sodium alginate, modified starch, cellulose and modified cellulose, polysaccharides, clay, perlite, vermiculite, yeast extracts, sucrose, glucose, casein and mixtures thereof. Preferably, the storage composition according to the invention comprises a preservative selected from the group comprising a mixture containing cinnamal, surfactin, pine oil, 15-1,2-octanediol, glycerol triacetate, a mixture based on 1,2-alkanediol; 3-phenyl-1-propanol,and their mixtures. A preservative containing cinnamaldehyde, surfactin, pine oil, 1,2-octanediol, a mixture based on 1,2-alkanediol; 3-phenyl-1-propanol, isothiazolinone, Sovinol®20 P850, Sovinol®P850 / N, Sovinol®P740 / O, Sovinol®P540 / N, Sovinol®P540 and mixtures thereof. The invention also relates to a method for forming a storage composition according to the invention for preserving the viability of a beneficial biological material intended to improve soil fertility or to combat crop pests and promote plant growth by direct and / or indirect action against biotic stress. abiotic, comprising the following steps: - Selection of a biological material from among: (micro)organisms, or nematodes arranged to reinforce integrated biological control - Dissolving said (micro)organisms with the stabilizing agent supplied in liquid form to provide an aqueous storage composition,or a mixture of said nematodes with the stabilizing agent to provide a pasty storage composition, - Storage of said storage composition. According to an advantageous method, said storage step is carried out at a temperature between -5° and 30°C, preferably between 0° and 25°C, more preferably between 5° and 25°C and maintained, for a period of at least 1 month, preferably at least 2 months, more preferably at least 3 months, more preferably at least 6 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months with maintenance of viability during storage and, possibly during agricultural or horticultural application. All the aforementioned characteristics of the composition according to the invention are also applicable to the process as described in the invention. The invention also relates to the use of a stabilizing agent and biological material in an agricultural storage composition.said stabilizing agent being in the form of 2025 / 5080 BE2025 / 5080 14 zwitterionic and comprising at least one quaternary ammonium, said beneficial biological material being intended to improve soil fertility or to control crop pests and promoting plant growth by direct and / or indirect action against biotic or abiotic stress, characterized in that said biological material is selected from: -(micro)organisms, or -nematodes to strengthen integrated biological control, and in that said storage composition is aqueous when said biological material is selected from (micro)organisms, and in that it is pasty when said biological material is selected from nematodes. Preferably, the use of the storage composition according to the invention makes it possible to preserve the viability of the biological material 15 until application in agriculture or horticulture, preferably in foliar or soil form. More preferably,Use of the storage composition according to the invention to preserve biostimulant or biocontrol biological materials during their storage and transport. Advantageously, the use of the storage composition according to the invention is carried out in the following fields: biocontrol, biostimulant, horticulture, integrated pest management, conventional agriculture, organic agriculture. 2025 / 5080 BE2025 / 5080 15 Other embodiments of the use of the storage composition according to the invention are indicated in the appended claims. In the context of the present invention, the expression "(micro)organisms" means all microscopic organisms belonging to the taxonomic kingdom of bacteria and / or fungi. For the purposes of the present invention,The term "pasty storage composition" means a composition exhibiting a dynamic viscosity at a given temperature that is greater than that of the aqueous composition of the invention at that given temperature. The expression "stabilizing agent" refers to a substance that maintains the integrity of (micro)organisms and nematodes by preserving their viability, biological activity, and dormant or vegetative state. The expression "preservative" refers to a substance that delays and / or prevents the deterioration of (micro)organisms and nematodes due to the proliferation and development of contaminants or chemical reactions. The expression "biological matter in a state of biological dormancy, corresponding to a state of temporary inactivity" means that the biological matter is in porulating, vegetative, or cyst-resistant form. Thus, when biological matter is not active, we can speak of biological dormancy. This term describes a state where the metabolism of biological matter is considerably reduced or suspended.allowing it to survive in unfavorable conditions until the environment becomes conducive to its activity. From 2025 / 5080 BE2025 / 5080 16 onwards, biological material is said to be beneficial given that it is intended to improve soil fertility or to combat crop pests and promote plant growth by direct and / or indirect action against biotic or abiotic stress. The term "viability" used in the context of the present invention should be understood as the viability of (micro)organisms, expressed in logarithmic form, in a sample. It is defined by the following logarithmic relation: Logarithmic reduction = Log10( ) Where: -N0: Initial number of viable (micro)organisms (before preservation). -Nt: Number of viable (micro)organisms remaining after preservation. Advantageously, the storage composition can be obtained by adding, for example, 36.08 g of betaine glycine powder (97% purity w / w) and bringing it to 100 mL using deionized water. Alternatively,A ready-to-use solution containing 35% betaine glycine can be used for dissolving (micro)organisms. In the context of the invention and according to preferred embodiments, two forms of betaine are used: - Betaine powder with a concentration of 97% glycine betaine, which is then diluted in water for 17 tests. The pH of betaine powder diluted to 35%, 25%, and 15% by weight can vary between 6 and 7. - Liquid betaine with 35% glycine betaine is ready to use. The pH of liquid betaine is approximately 10. The examples were carried out with betaine powder, i.e., at a pH of approximately 7. Example 1 - Storage Composition Element Quantity Water 65% by weight Bacillus 10⁸ CFU Betaine 35% by weight Storage Time Number of pores / ml 0 1.55 x 10⁸ 24 hours 2.51 x 10⁸ 7 days 2.61 x 10⁸ 1 month 2.42 x 10⁸ 2 months 3.16 x 10⁸ 3 months 2.84 x 10⁸ 5 months 2.41 x 10⁸ 6 months 2.93 x 10⁸ It was surprising to find that the compounds based on glycine,In particular, glycine betaine at 35% and the number of spores remaining are shown in the table over time after 6 months of incubation. 2025 / 5080 BE2025 / 5080 18 Comparative Example 1 – Acidic pH Spores were dissolved in different acidifying solutions (citric acid or lactic acid) and their stability was observed over time. Samples were taken and serial dilutions of the samples were performed. These dilutions were spread on Petri dishes. The bacterial colonies that grew represent the quantity of bacteria present in the sample, called "CFU" for colony-forming units. Here are the samples tested and the quantity of bacteria counted: 10 Sample 1: Bacillus spores in water Sample 2: Bacillus spores with SOVINOLP740 (preservative) at 0.2% Sample 3: Bacillus spores with SOVINOLP740 (preservative) at 0.2% pH<4 with lactic acid 15 Sample 4: Bacillus spores with SOVINOLP740 (preservative) at 0.2% pH<4 with citric acid Sample 5: Bacillus spores with SOVINOLP740 (preservative) at 0,1% pH < 4 with Citric Acid 20 Compounds % difference compared to T0 H2O 98.21% SOVINOLP7400.2% 99.50% SOVINOLP7400.2% lactic acid 99.95% SOVINOLP7400.2% citric acid 99.55% SOVINOLP7400.1% citric acid 99.09% 2025 / 5080 BE2025 / 5080 19 It follows from this example that Bacillus pores are not stable in water. Furthermore, an acidic pH generated by citric or lactic acid does not allow the pores to be stabilized in aqueous solution. And, the solvent also does not seem to help the stabilization of the pore(s). 5. The articles "a" and "an" are used here to refer to one or more (i.e., at least one) of the grammatical objects of the article and may be replaced by an article that designates a plural, such as "at least two," "at least three," "several," etc. The expressions "in a mode of realization," "according to a mode of realization," and others generally mean that the particular feature, structure, or characteristic following the expression is included in at least one mode of realization of this disclosure.and may be included in more than one embodiment of this disclosure. It is important to note that such expressions do not necessarily refer to the same embodiment. If the text indicates that a component or feature "may" or "could" be included or have a characteristic, it is not necessary that this particular component or feature be included or have the characteristic. 20 If it appears here, the term "comprising" or "containing" and derivatives thereof are not intended to exclude the presence of any additional component, step, or procedure, whether disclosed or not here. To avoid any doubt, the term "comprising" may include any additional element / additive, adjuvant, or compound, unless otherwise indicated. 25 By contrast, the term "essentially consisting of", if it appears here, excludes from the scope of any subsequent quotation any other 2025 / 5080 BE2025 / 5080 20 component, step or procedure, except those not essential to operability, and the term "consisting of", if used, excludes any component,step or procedure not specifically defined or indicated. The terms "or" and "and / or", unless otherwise indicated, refer to the members indicated individually as well as in any combination. For example, the expression A and / or B refers to A alone, B alone, or A and B. Furthermore, the term and any equivalent to "comprising" may be replaced by "consisting of". Also, the terms "obtainable" or "can be obtained" or any similar or derived expressions may be replaced by "directly obtained". It is understood that the present invention is in no way limited to the embodiments described above and that many modifications may be made to them without departing from the scope of the annexed claims. 2025 / 5080 BE2025 / 5080,