Highly absorbable bioactive polymers, methods for their preparation and uses thereof

Abstract

The present invention relates to a superabsorbent polymer obtained by crosslinking at least one polycarboxylic acid. The present invention also relates to a process for producing said superabsorbent polymer, and to its uses and applications, in particular in the agronomic field for diffusing water or active substances.

Description

Technical Field

[0001] This invention relates to highly absorbent polymers obtained by crosslinking at least one polycarboxylic acid. The invention also relates to methods for producing said highly absorbent polymers, and their uses and applications, particularly in the field of agronomy, for the controlled diffusion of water and active substances.

[0002] Therefore, this invention belongs to the field of polymer chemistry and its applications. Background Technology

[0003] The highest-performing hyperabsorbent polymers currently available on the market are produced through chemical cross-linking of monomers that may be environmentally toxic, as these monomers are derived from petrochemicals and are non-biodegradable. These products may release molecules into the environment without systematic monitoring.

[0004] These highly absorbent products primarily originate from chemical reactions that are chemically inconsistent with current goals related to green chemistry, environmental protection, crop health, and animal and human health. Currently known highly absorbent materials, such as cellulose, starch, and chitosan, have the advantages of meeting these green chemistry goals and being biodegradable. However, to date, their technical performance, particularly their liquid absorption capacity, has been lower than that of synthetically produced highly absorbent products (Chang et al., “Superabsorbent polymers used for agricultural water retention,” PolymerTesting 94, 107021, 2021).

[0005] Therefore, there is a need to develop new high-performance, highly absorbable products made from natural and biodegradable components. Summary of the Invention

[0006] The inventors have developed a cross-linked superabsorbent polymer that can serve as an alternative to chemically produced superabsorbent products. This polymer is generated from natural polymers and cross-linked using a method involving divalent and trivalent cations. The inventors have unexpectedly demonstrated that, in the presence of low concentrations of divalent and trivalent cations, the cross-linking reaction of polycarboxylic acids produces a cross-linked polymer with an ultrapure water absorption capacity of 500 to 800 grams of water per gram of polymer, comparable to currently available superabsorbent products produced through chemical synthesis.

[0007] Therefore, the highly absorbent crosslinked polymer according to the present invention constitutes a biodegradable matrix capable of storing and gradually releasing large amounts of water. These properties make it highly valuable for a wide range of applications, particularly in agronomy, health, and optional hygiene. During crosslinking, the presence of a chelating agent further enhances the performance of the biodegradable matrix under standard saline conditions. The highly absorbent crosslinked polymer, the subject of this invention, has positive effects on plant hydration and growth.

[0008] Therefore, the inventors have developed a method for producing the crosslinked polymer.

[0009] The inventors have also developed a method for producing crosslinked polycationic polymers, wherein the crosslinked cationic polymers are capable of forming particles within a biodegradable matrix of a crosslinked polymer according to the invention. When the biodegradable crosslinked polymer matrix contains cationic polymer particles encapsulated with an active agent, treatment of plants with such a matrix rich in the active ingredient results in the sustained release of the active ingredient. Finally, the efficacy of the active ingredient on the treated plants is demonstrated by observations of overexpression of plant compounds involved in their environmental stress responses.

[0010] Furthermore, as highly absorbable products biodegrade, they stimulate the microbiome and plants, thereby particularly activating the systemically acquired resistance (SAR) mechanism.

[0011] Finally, when the natural polymer is a polycarboxylate, the cross-linked polycarboxylate can chelate mineral species that are traditionally unavailable to plants, thereby mobilizing them and improving the bioavailability of these types of minerals to the treated plants.

[0012] The present invention also relates to an ionotropic gelation method for preparing highly absorbable polymer matrices, particularly so-called "bioactive" highly absorbable polymer matrices containing bioactive molecules. The bioactive highly absorbable polymer matrices constitute a combination of biopolymers and cationic oligomers belonging to the chitosan or protein family, and contain, for example, phenolic compounds and plant hormones.

[0013] Therefore, the crosslinked polymer according to the invention offers numerous advantages: its use results in a significant increase in water storage capacity available for plant growth under water stress conditions, time-controlled release of biostimulants and fertilizers encapsulated within the crosslinked polymer during plant growth, and specific stimulation of the plant and its rhizosphere. Furthermore, the crosslinked polymer according to the invention is capable of capturing inorganic nutrients essential for plant growth from the soil or plant microenvironment. Finally, the biodegradation of the polysaccharide polymer produces oligosaccharides that act as elicitors for plants.

[0014] Because of the low concentration of crosslinking agent used in the production method, resulting in a low degree of crosslinking, the highly absorbent crosslinked polymer of the present invention also has the advantage of being obtainable, in particular, in gel form rather than in particle or microbead form. The gel can then be dried and subsequently crushed or ground to produce a dispersible highly absorbent powder. Therefore, the present invention has specific applications in dispersing highly absorbent powder in plant growth beds or as an additive to potting soil in which plants are grown. The dispersible nature of this powder allows for better distribution of the crosslinked polymer according to the present invention, and thus greater efficacy. Detailed Implementation

[0015] The first subject of the present invention is a crosslinked polymer, which is obtained by crosslinking at least one polycarboxylic acid in a solution of a solvent in the presence of a crosslinking agent selected from divalent and trivalent cations, wherein the polymer is characterized in that, during crosslinking, the initial concentration of the crosslinking agent in the solvent is 1 to 30 mM, and the crosslinked polycarboxylic acid polymer is capable of absorbing at least 200 g of water per gram of crosslinked polymer.

[0016] The term "polycarboxylic acid" refers to a molecule containing at least two carboxyl groups, wherein a carboxyl group is a functional group consisting of a carbon atom bonded to an oxygen atom by a double bond and to a hydroxyl group by a single bond. The at least two carboxyl groups are bonded to functional group R, where R is an organic group containing 2, 3, 4, or n carbon atoms. Carboxylic acids particularly include dicarboxylic acids, tricarboxylic acids, tetracarboxylic acids, and polymeric structures consisting of combinations of monomers bearing one or more carboxyl groups.

[0017] The term "polycarboxylic acid" refers to a polycarboxylic acid and its conjugate base, wherein the conjugate base is a polycarboxylate ion, particularly in the form of an alkali metal polycarboxylate, more specifically sodium polycarboxylate, potassium polycarboxylate, calcium polycarboxylate, and magnesium polycarboxylate.

[0018] According to one specific embodiment, in the crosslinked polymer according to the invention, the molecular weight of the polycarboxylic acid is from 1,000 Da to 1,000,000 Da.

[0019] The crosslinked polycarboxylic acid polymers according to the invention are characterized by their ability to retain and absorb liquids, particularly water; this ability is expressed in grams of liquid per gram of crosslinked polymer.

[0020] More specifically, the crosslinked polycarboxylic acid polymers according to the invention are characterized by their "swell index" (or swelling index, SI), expressed in grams (g) of water (or a reference salt solution in which the crosslinked polymer is placed) per gram (g) of the crosslinked polymer. The swelling index of the crosslinked polymers according to the invention is specifically measured using the method described by Dai et al. ("Multifunctional self-assembling hydrogel from guar gum," Chemical Engineering Journal, 330, pp. 1044–1051, 2017). The swelling index can be measured using ultrapure water or a reference salt solution (e.g., 0.9% sodium chloride solution).

[0021] The crosslinked polycarboxylic acid polymer according to the present invention is characterized in that its ability to absorb pure water is greater than or equal to 200, greater than or equal to 300, greater than or equal to 400, greater than or equal to 450, greater than or equal to 500, greater than or equal to 550, greater than or equal to 600, greater than or equal to 650, greater than or equal to 700, greater than or equal to 750, or greater than or equal to 800, expressed in grams of pure water per gram of crosslinked polymer.

[0022] The crosslinked polycarboxylic acid polymer according to the present invention is characterized in that its ability to absorb 0.9% sodium chloride solution is greater than or equal to 20, greater than or equal to 30, greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, or greater than or equal to 80, expressed in grams of 0.9% sodium chloride solution per gram of crosslinked polymer.

[0023] The crosslinked polycarboxylic acid polymers according to the present invention can be further characterized by the following criteria:

[0024] - Its gelation rate,

[0025] - Its viscosity,

[0026] - Its density, expressed in g / cm³ 3 express

[0027] - Its shear rate, in s -1 express,

[0028] - Its dimensional stability when exposed to changing environmental conditions,

[0029] - Its biodegradability, in particular, is expressed as the percentage of mass loss when applied to soil and the assimilation of released monomers by microorganisms (microbial biostimulation).

[0030] - Its mechanical strength properties,

[0031] - Its crosslinking density,

[0032] - The diameter of the polymer fiber.

[0033] These properties can be measured using methods well known to those skilled in the art.

[0034] The term "crosslinking agent" refers to a chemical reagent capable of forming chemical bonds between macromolecular chains, thereby creating a three-dimensional network. In a specific embodiment of the polymer according to the invention, the crosslinking agent, as an activator, comprises or is composed of divalent or trivalent metals. More specifically, the crosslinking agent comprises calcium ions, particularly calcium chloride.

[0035] In one specific embodiment, the crosslinked polymer of the present invention is obtained by crosslinking at least one polycarboxylic acid in a solution of a solvent in the presence of a crosslinking agent selected from divalent and trivalent cations, characterized in that, during crosslinking, the initial concentration of the crosslinking agent in the solvent is 1 to 30 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10 mM, 2 to 7.5 mM, 2 to 6 mM, 3 to 6 mM, 3.5 to 6 mM, and preferably 5 mM.

[0036] Specifically, during crosslinking, the concentration of the polycarboxylic acid in the solvent is 0.1% to 6% by mass / solvent volume (m / v). Specifically, the concentration of the polycarboxylic acid is 0.1% to 4% m / v, particularly 0.1% to 3% m / v, significantly 0.2% to 4% m / v, and more significantly 0.2% to 3% m / v, specifically 0.2% to 2% m / v, particularly 0.2% to 1% m / v, significantly 0.2% to 1% m / v, and more significantly 0.2% to 0.8% m / v, specifically 0.3% to 0.7% m / v, for example 0.5% m / v.

[0037] Among polycarboxylic acids, straight-chain polycarboxylic acids and branched polycarboxylic acids can be distinguished. They can also be further differentiated into natural polycarboxylic acids and synthetic or modified polycarboxylic acids. Finally, polysaccharides constitute an important category of polycarboxylic acids.

[0038] Among polysaccharides, alginic acid (CAS No. 9005-32-7) and alginate / ester derivatives extracted from brown algae or bacteria are well known. Alginates are linear polymers composed of two types of uronic acids: D-mannuronic acid (mannurate) and L-guluronic acid (gulurate), the ratio and distribution of which vary depending on the type of alginate. The number of monomers constituting alginic acid ranges from 100 to 3000. Alkali metal alginates, including sodium alginate, potassium alginate, calcium alginate, and magnesium alginate, are well known.

[0039] In one specific aspect, the crosslinked polymer according to the invention is obtained by crosslinking at least one polycarboxylic acid in solution, said polycarboxylic acid being bio-based alginic acid, more specifically alginic acid extracted from brown algae.

[0040] According to one embodiment, the present invention relates to a polymer obtained by crosslinking alginic acid, said alginic acid comprising 30 to 70% mannulic acid and 30 to 70% guluronic acid by weight of alginic acid.

[0041] More specifically, the crosslinked polymer according to the invention comprises alginate, characterized by the ratio of mannuronic acid monomers to guluronic acid monomers, also known as the "M / G ratio". This ratio can be from 0.1 to 2.

[0042] More specifically, the crosslinked polymer according to the invention comprises alginic acid, wherein the alginic acid is characterized by an "M / G ratio" of 0.2 to 1.8, more specifically 0.4 to 1.8, particularly 0.6 to 1.8, particularly 0.7 to 1.6, particularly 1.2 to 1.6, particularly 1.3 to 1.5, for example 1.4. According to a specific embodiment, the crosslinked polymer according to the invention comprises alginic acid, wherein the alginic acid is characterized by an "M / G ratio" of 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9 or 2.

[0043] More specifically, the crosslinked polymer according to the invention comprises alginate, wherein the alginate is characterized by a molecular weight of 50 to 400 kDa, preferably 100 to 350 kDa.

[0044] According to a first embodiment, the crosslinked polymer according to the invention is obtained by crosslinking alginic acid, which is characterized by an M / G ratio of 0.7 and a molecular weight of 300 kDa ± 10%, referred to herein as "Al M / G 0.7".

[0045] According to a second embodiment, the crosslinked polymer according to the invention is obtained by crosslinking alginic acid, which is characterized by an M / G ratio of 1.4 and a molecular weight of 230 kDa ± 10%, referred to herein as "Al M / G 1.4".

[0046] On the other hand, the crosslinked polymer according to the invention is characterized in that a chelating agent is added to the solvent during crosslinking. Suitable chelating agents include:

[0047] -n-carboxylic acids, where "n" equals 1, 2, 3, or 4, include citric acid and ascorbic acid.

[0048] - Phenolic compounds, including catechol, catechin, ellagitannins, and gallotannins.

[0049] - Salicylate / ester

[0050] - EDTA and EGTA.

[0051] The chelating agent is added at an appropriate concentration such that when the crosslinked polymer of the present invention, obtained by crosslinking in the presence of the chelating agent, is placed in a brine environment, the crosslinked polymer exhibits improved water retention capacity. The inventors have demonstrated that, when citric acid is used as a chelating agent or counterion, the optimal concentration of citric acid in the solvent during the crosslinking step is 0.1 to 5 mM, 0.5 to 2 mM, and preferably 1 mM.

[0052] The present invention also relates to crosslinked polymers, optionally characterized in that a chelating agent is added to the solvent during the crosslinking step, and characterized in that the crosslinked polymer comprises small particles or nanoparticles, the small particles or nanoparticles comprising or consisting of a crosslinked cationic polymer. The crosslinked polymer according to the invention comprises 0.01 to 10% by polymer volume small particles, the small particles comprising or consisting of a crosslinked cationic polymer.

[0053] More specifically, the crosslinked polymer according to this invention comprises 0.1 to 5%, 0.1 to 2%, 0.1 to 1%, 0.1 to 0.5% or 0.18% small particles, said small particles comprising or composed of crosslinked cationic polymers.

[0054] More specifically, the crosslinked cationic polymer is obtained by crosslinking a substance selected from: proteins, chitosan (CAS No. 9012-76-4), urea compounds, monoamine compounds, polyamine compounds, inorganic polymers, and combinations thereof. More specifically, the crosslinked cationic polymer is obtained by crosslinking chitosan.

[0055] In one specific embodiment, the crosslinked cationic polymer is obtained by crosslinking in the presence of a crosslinking agent selected from phosphates, citric acid, tannic acid, phenolic compounds, and clay materials.

[0056] More specifically, in the cationic polymer present in the polymer according to the invention, the phosphate is selected from: phosphate derivative solutions, n-polyphosphate / polyphosphate solutions, and phosphate-rich slurries.

[0057] In one specific embodiment, the inorganic polymer in the cationic polymer present in the polymer according to the invention is selected from: clay materials, bentonite, lithium saponite, weathered layers, apatite and hydroxyapatite derivatives, and diatomaceous earth.

[0058] In one specific aspect, the crosslinked polymer according to the invention comprises crosslinked cationic polymer nanoparticles, said nanoparticles containing an active agent. In particular, the active compound may be encapsulated within the nanoparticles.

[0059] The active agent contained in the crosslinked cationic polymer particles may be particularly selected from:

[0060] - Phenolic compounds,

[0061] - Oligosaccharides, especially chitosan,

[0062] - Plant hormones,

[0063] - Amino acids, non-protein amino acids, including the "glycine-like" amino acid family and GABA.

[0064] - Nutrients,

[0065] - Micronutrients,

[0066] - Osmoprotectants, including mannitol, sorbitol, trehalose, and glycine betaine, and

[0067] - Its combination.

[0068] In one specific embodiment, "bioactive crosslinked polymer" refers to a crosslinked polymer comprising a crosslinked cationic polymer according to the present invention, wherein the crosslinked cationic polymer contains a bioactive agent; "Al M / G 0.7 bioactive" refers to an alginate polymer comprising a crosslinked cationic polymer with an M / G ratio of 0.7 according to the present invention, wherein the crosslinked cationic polymer contains a bioactive agent.

[0069] The second subject of the present invention is a method for preparing the polymer according to the invention, comprising at least the following steps:

[0070] a) Add a crosslinking agent selected from divalent and trivalent cations to a polycarboxylic acid solution to obtain a final concentration of the crosslinking agent in the polycarboxylic acid solution of 1 to 30 mM.

[0071] b) Perform a cross-linking reaction to obtain a gel.

[0072] c) Dry the gel obtained in b).

[0073] According to one embodiment of the invention, step b) does not include the step of forming droplets, for example by emulsification or dropwise dispensing.

[0074] In one specific implementation, physical crosslinking is selected from: sonication treatment, heat treatment, and microwave treatment.

[0075] In one embodiment, the method further includes the steps of crosslinking the cationic polymer and combining the crosslinked cationic polymer with the crosslinked polymer of the present invention.

[0076] According to one specific embodiment, the method according to the invention includes the following steps: adding a crosslinking agent selected from divalent and trivalent cations to an alginate solution to obtain a final concentration of the crosslinking agent in the alginate solution of 1 to 30 mM.

[0077] According to yet another preferred embodiment, the method according to the invention includes the step of adding a crosslinking agent selected from divalent and trivalent cations to a 1% carboxylic acid solution at room temperature, wherein the concentration of the crosslinking agent is 3 to 10 mM and the volume ratio of polymer to crosslinking agent is 3:1.

[0078] According to one specific aspect of the method of the invention, the step of drying the gel obtained in b) is carried out at room temperature, or at a temperature of 40 to 80°C (preferably 60°C), or by freeze drying.

[0079] "Freeze-drying" refers to a drying step that results in a product moisture content of 0.1% to 5%. For example, the formed gel can be spread into a 1 cm thick layer and placed in an oven at 60°C for 24 hours. Alternatively, the formed gel can be spread into a 1 cm thick layer and placed in an oven at 40°C for 48 hours. This method ensures uniform drying. Once the polymer is dry, it is ground and stored in a moisture-free environment.

[0080] More specifically, the method for preparing the polymer according to the invention comprises the following steps:

[0081] a) Add calcium chloride to a solution of alginate or its sodium salt with a molecular weight of 50 to 400 kDa to obtain a final concentration of calcium chloride of 1 to 30 mM, preferably 5 mM; this addition can be particularly carried out as follows: alginate is added in a solution of 20 g / L... -1 The concentration was stirred in water and diluted with water to three-quarters of the final volume; calcium chloride was diluted in water to 20 mM, with the volume corresponding to one-quarter of the final volume, in order to obtain a concentration of 5 mM in the final volume.

[0082] b) A cross-linking reaction is carried out by stirring for 10 minutes to obtain a uniform gel.

[0083] c) Dry the gel obtained in b).

[0084] Specifically, step c) can be followed by step d), which involves grinding the gel to produce a powder. This aspect of the invention enables the production of a dispersible powder.

[0085] The present invention also relates to a method for preparing a crosslinked polymer, which further includes the step of preparing a cationic polymer intended to be incorporated into the crosslinked polymer of the present invention.

[0086] The method for preparing cationic polymers includes the following steps:

[0087] - The cationic biopolymer is dispersed in a suitable solvent; simultaneously, a crosslinking agent solution is prepared.

[0088] - After the cationic polymer is fully dispersed, adjust the pH of the polymer solution.

[0089] - Subsequently, the crosslinking agent solution was added while stirring at a medium speed (300 rpm).

[0090] - Once the cross-linking agent has been added, centrifuge the solution.

[0091] - Rinse and resuspend the precipitate for future use.

[0092] More specifically, the method includes the following steps:

[0093] - Disperse five grams of cationic biopolymer in one liter of 1% acetic acid solution; simultaneously, prepare a 0.25% phosphate solution.

[0094] - After the cationic polymer is fully dispersed, adjust the pH of the polymer solution to pH 4.5.

[0095] - Subsequently, while stirring at a moderate speed (300 rpm), a phosphate solution was added at a ratio of 5:3 (cationic polymer to crosslinking agent).

[0096] - Once the cross-linking agent has been added, centrifuge the solution at 10,000 g for 15 minutes at 4°C.

[0097] - Rinse the precipitate twice with ultrapure water and resuspend it for future use.

[0098] Finally, the method according to the invention may further include the step of encapsulating the active substance molecules in the cationic polymer, according to the following steps:

[0099] - Disperse the active substance in a 3:1 ratio (cationic polymer: active substance) until a homogeneous solution is obtained.

[0100] - The dispersion is then crosslinked with a crosslinking agent in the same manner as in the preparation of the polymer support.

[0101] A third subject of the invention is a product, which, based on its weight, comprises 0.01 to 99 wt% of a crosslinked polymer according to the invention or a crosslinked polymer obtained by the method according to the invention. More specifically, based on the weight of the product, the product according to the invention comprises 0.01 to 99 wt%, 0.1 to 99 wt%, 1 to 99 wt%, 5 to 99 wt%, 10 to 99 wt%, 15 to 95 wt%, 20 to 90 wt%, or 30 to 80 wt% of a crosslinked polymer according to the invention, or a crosslinked polymer obtained by the method according to the invention. In addition to the crosslinked polymer, the product may contain excipients or additives.

[0102] In one specific embodiment, the product according to the present invention is selected from:

[0103] - Cell culture support

[0104] - Suitable for use as a water supply or nutrient supply agent in agriculture.

[0105] - Plant biostimulants

[0106] - Absorbent products for medical and / or hygiene purposes, specifically selected from: bandages, active detoxifying hydrogel patches,

[0107] - Reagents used in hydrogel and bio-ink technologies in 3D printing.

[0108] - Soil and wastewater treatment agents (removal of heavy metals and recalcitrant molecules, including fertilizers, pesticides, pharmaceuticals, and hydrocarbons).

[0109] - Encapsulation of microorganisms for use in agriculture, healthcare, and water and soil remediation.

[0110] A fourth subject of this invention is the use of the crosslinked polymer according to the invention for water supply and / or biostimulation of plant growth. A biostimulant is defined as any component, substance, or microorganism capable of reducing fertilizer use to promote plant health without causing any subsequent harmful effects. It can be applied in various ways: directly to plants or soil, where it will provide effective nutrition, improve tolerance to abiotic stresses, and promote optimal plant growth.

[0111] The product according to the invention can be specifically used for:

[0112] - Provides additional support to the soil to increase its water-holding capacity and improve crop survival rates under water stress.

[0113] - Use targeted molecular biostimulation to stimulate plants and enhance their defense systems to combat abiotic stresses.

[0114] - Generate prebiotic nutrients in the rhizosphere to enhance plant-microbe interactions.

[0115] The present invention also relates to the use of cross-linked polymers obtained by the method according to the invention for the biostimulation of water supply and / or plant growth.

[0116] The present invention also relates to the use of products for the purpose of providing water and / or acting as biostimulants for plant growth, said products comprising a crosslinked polymer according to the invention or comprising a crosslinked polymer obtained by the method according to the invention.

[0117] The present invention also relates to the use of products comprising the crosslinked polymer according to the invention for the repair, adsorption, and chelation of metals. Highly absorbent hydrogels can adsorb metals by following the method according to the invention and producing thin films in membrane form (monolayer or multilayer). Attached Figure Description

[0118] In Example 1 Figure 1 The swelling index (in grams of water per gram of polymer) of crosslinked polymers prepared in the presence of different amounts of crosslinking agent is shown. The conditions shown from left to right are: I) containing 0.05 M Ca 2+ Al M / G 0.7, II) containing 0.1 M Ca 2+ Al M / G 0.7, III) containing 0.5 M Ca 2+ Al M / G 0.7 (IV) contains 0.05 M Ca 2+ Al M / G 1.4, V) containing 0.1 M Ca 2+ Al M / G 1.4, VI) containing 0.5 M Ca 2+Al M / G 1.4. White and black bars represent drying times of 0.5 and 24 hours, respectively.

[0119] In Example 1 Figure 2 Histograms showing the mass (in grams) of the swollen gel prepared from Al M / G 1.4 as a function of calcium chloride concentration (in mM) during crosslinking, and curves showing the gelation rate (in percentage) as a function of calcium chloride concentration (in mM).

[0120] In Example 1 Figure 3 The figure shows a thick line curve representing the mass (in grams) of the swollen gel prepared from Al M / G 1.4 as a function of calcium concentration (left y-axis), and a thin line curve representing the mass (in grams) of the dry gel prepared from Al M / G 1.4 as a function of calcium concentration (right y-axis).

[0121] In Example 1 Figure 4 The viscosity of Al M / G 1.4 as a function of time is shown. The first graph shows the viscosity of the Al M / G 1.4 solution in water over a 50-minute time period. The second plateau corresponds to the cross-linked polymer formed from Al M / G 1.4 upon the addition of 5 mM calcium chloride.

[0122] In Example 1 Figure 5 The swelling index (in grams of water per gram of polymer) of alginate gels or their sodium salts, cross-linked in the presence of 5 mM calcium and then dried at room temperature for 48 hours or freeze-dried for 48 hours, is shown. Measurement times were 0.5 hours or 21 hours. The test conditions were: a) Al M / G 0.7 dried at room temperature, b) Al M / G 1.4 dried at room temperature, c) Al M / G 0.7 freeze-dried, and d) Al M / G 1.4 freeze-dried.

[0123] In Example 1 Figure 6 The swelling index (in grams of water per gram of polymer) of alginate gels or their sodium salts crosslinked in the presence of 10 mM calcium and then dried at room temperature for 48 hours or freeze-dried for 48 hours is shown. Measurements were taken over 30 minutes or 21 hours. The test conditions were: a) Al M / G 0.7 dried at room temperature, b) Al M / G 1.4 dried at room temperature, c) Al M / G 0.7 freeze-dried, and d) Al M / G 1.4 freeze-dried.

[0124] In Example 1 Figure 7The water absorption kinetics, expressed as grams of water per gram of polymer, are shown as a function of time in minutes for crosslinked polymers containing Al M / G 1.4 alginate (white squares) and crosslinked polymers containing alginate with an M / G ratio of 0.7 (diamonds).

[0125] In Example 2 Figure 8 The swelling index (in grams of water per gram of polymer) of the crosslinked polymer (curve with black circles) prepared from Al M / G 1.4 and the commercially available highly absorbent polymer (potassium polyacrylate or PAK) (curve with crosses) is shown as a function of time (in minutes) under saline conditions (0.9% NaCl solution).

[0126] In Example 2 Figure 9 This is a histogram showing the swelling index (in grams of water per gram of polymer) of seven different polymers under saline conditions (0.9% NaCl solution). The white and black histograms represent the conditions after 30 minutes and 1440 minutes of swelling, respectively, and are shown from left to right as follows: 1) Potassium polyacrylate (PAK); 2) Al M / G1.4 prepared without counterions (CI); 3) Al M / G1.4 prepared in the presence of 1 mM citric acid; 4) Al M / G1.4 prepared in the presence of 2.5 mM citric acid; 5) Al M / G1.4 prepared in the presence of 5 mM citric acid; 6) Al M / G1.4 prepared in the presence of 7.5 mM citric acid; 7) Al M / G1.4 prepared in the presence of 10 mM citric acid.

[0127] Figure 10 The water absorption kinetics of Al M / G 1.4 bioactive crosslinked polymer (black square) containing an anti-counterionic agent (1 mM citric acid) and commercially available high-absorbency polymer PAK (white diamond) are shown as a function of time in minutes, expressed as grams of water per gram of polymer, under saline conditions (0.9% NaCl solution).

[0128] Example 3 Figure 11 The absorption and desorption kinetics of the cross-linked polymer containing Al M / G 1.4 bioactivity (black circle) and the commercially available highly absorbent polymer PAK (cross) are shown as a function of time in minutes, expressed as grams of water per gram of polymer.

[0129] Example 4 Figure 12This is a histogram showing the swelling index (in grams of water per gram of polymer) in ultrapure water as a function of the proportion of calcium alginate (CaAl) in Al M / G 1.4 alginate, from left to right representing 30%, 40%, 50%, or 70% calcium alginate. For each percentage of calcium alginate, the values ​​120, 990, 1200, and 2460 represent swelling time in minutes.

[0130] Example 5 Figure 13 The lengths of roots treated by root application are shown (from left to right): untreated roots, roots treated with a polymer prepared from alginate with an M / G ratio of 0.7, and roots treated with a polymer prepared from alginate with an M / G ratio of 1.4; different letters indicate significant differences according to Tukey's test (p = 0.01).

[0131] Example 5 Figure 14 The lengths of seedlings treated with the following root application methods are shown (from left to right): untreated roots; roots treated with a polymer prepared from Al M / G 1.4; and roots treated with a polymer prepared from Al M / G 0.7; different letters indicate significant differences according to the Tukey test (p = 0.01).

[0132] Example 5 Figure 15 The root lengths (from left to right) are shown by the following root application treatments: untreated roots (control); roots treated with the bioactive component (cationic polymer: 10 mg ChNP); roots treated with the bioactive crosslinked polymer (Al M / G 1.4 bioactive) containing Al M / G 1.4 rich in counterions and bioactive molecules according to the present invention; different letters indicate significant differences according to the Tukey test (p = 0.01).

[0133] Figure 16 The stomatal conductance as a function of volumetric water content (“VWC”) in winter wheat plants (1 month old) during 16 days of water stress is shown, as well as the treatments they received, which consisted of: no treatment (black curve), a cross-linked polymer prepared with Al M / G 1.4 according to the invention; a cross-linked polymer prepared with Al M / G 1.4 containing bioactive molecules according to the invention (Al M / G 1.4 bioactive); and a commercial chemical compound (PAK).

[0134] Figure 17The relative water content (from left to right) of winter wheat plants (grown for 1 month) under the following treatments are shown: no treatment; cross-linked polymer prepared with Al M / G 1.4 according to the invention; cross-linked polymer prepared with Al M / G 1.4 containing bioactive molecules according to the invention (Al M / G 1.4 bioactive); commercially available highly absorbable polymer (PAK) after 14 days of severe water stress due to water restriction; different letters indicate significant differences according to Tukey's test (p = 0.01).

[0135] Figure 18 The relative water content of winter wheat plants after one month of growth is shown (from left to right): no treatment; cross-linked polymer prepared with Al M / G 1.4 according to the invention; cross-linked polymer prepared with Al M / G 1.4 containing bioactive molecules according to the invention (Al M / G 1.4 bioactive); commercially available highly absorbable polymer (PAK) after 16 days of severe water stress due to water restriction; different letters indicate significant differences according to Tukey's test (p=0.01).

[0136] Figure 19 This is a histogram showing the water potential (MPa) in the leaves of winter wheat plants after 3 months of growth at noon; in a kinetic study involving high-intensity water stress due to water restriction for up to 11 days, the plant treatments were as follows: no treatment; commercially available highly absorbable polymer (PAK); cross-linked polymer prepared according to the invention using Al M / G 1.4; cross-linked polymer prepared according to the invention using Al M / G 1.4 (Bioactive); different letters indicate significant effects according to the Tukey test (p = 0.01).

[0137] Figure 20 This is a histogram showing the relative water content (% water) in the leaves of winter wheat plants after 3 months of growth; in a kinetic study involving high-intensity water stress caused by water restriction for up to 11 days, the plant treatments were as follows: no treatment; commercially available highly absorbable polymer (PAK); cross-linked polymer prepared according to the invention using Al M / G 1.4; cross-linked polymer prepared according to the invention using Al M / G 1.4 containing bioactive molecules (Al M / G 1.4 bioactive); different letters indicate significant differences according to the Tukey test (p = 0.01).

[0138] Figure 21This is a histogram showing the percentage of electrolyte leakage in the leaves of winter wheat plants after 3 months of growth; in a kinetic study involving high-intensity water stress due to water restriction for up to 11 days, the plant treatments were as follows: no treatment; commercially available highly absorbable polymer (PAK); cross-linked polymer prepared according to the invention using Al M / G 1.4; cross-linked polymer prepared according to the invention using Al M / G 0.7 containing bioactive molecules (Al M / G 0.7 bioactive); different letters indicate significant differences according to the Tukey test (p = 0.01).

[0139] Figure 22 The transformation of various alginates from solution to gel is shown, along with the quantification of swollen gels and the dry gel content as a function of calcium concentration. Gels formed from Al M / G 0.7 bis (A) alginate are represented by solid lines with hollow rhombuses (swollen gel) and solid lines with solid rhombuses (dry gel). Gels based on Al M / G 1.4 bis (B) alginate are represented by dashed lines with hollow circles (swollen gel) and dashed lines with solid circles (dry gel).

[0140] Figure 23 The gelation percentage and swollen gel content of two alginate types with different mannuronic acid and guluronic acid ratios are shown: Al M / G 0.7 bis (A) (62% G and 38% M) and Al M / G 1.4 bis (B) (57% G and 43% M), based on the quantification of swollen gel and the gelation percentage as a function of calcium concentration. Gels formed from Sigma alginate are shown by dark solid lines (% gelation rate) and black bars (swollen gel). Gels from S900-NS are shown by light solid lines (% gelation rate) and gray bars (swollen gel).

[0141] Figure 24 The quantification of swollen and dry gel content of AL M / G 1.4 alginate as a function of calcium concentration is shown. Changes in swollen gel mass are shown by a dark solid line, and changes in dry gel mass are shown by a light line. Points represent mean ± SE.

[0142] Figure 25The expression of genes selected as biomarkers of immune response in wheat treated with Al M / G 1.4 bis (A), Al M / G 0.7 bis (B), Al M / G 1.4 (C), and XPC (D) alginate (XPC: highly cross-linked calcium alginate) for 7 days (7-day-old seedlings, 7 days after treatment) is shown. PR-1, PR-2, and PR-3 are genes encoding type 1, type 2, and type 3 pathogen response proteins (pathogenesis-related proteins). PAL and CAT are genes encoding phenylalanine aminolysase and catalase, respectively, which are involved in the metabolism of phenylpropanoids and the catabolism of hydrogen peroxide.

[0143] Example

[0144] Example 1: Preparation and characterization of highly absorbent crosslinked polymers using the method according to the present invention, and comparison with crosslinked polymers not within the scope of the present invention.

[0145] 1.1 Materials and Methods

[0146] The following products are used:

[0147] - Chitosan oligomers (water-soluble fungal-derived oligochitosan, 1.28 kDa, ChibioBiotech Co.),

[0148] - Chitosan polymers (acid-soluble fungal chitosan polymer, 150 kDa and 98% DDA; water-soluble fungal-derived chitosan polymer, 32.4 kDa and 98% DDA, Chibio Biotech Co.),

[0149] - Sodium alginate with an M / G ratio of 1.4 (expressed as Al M / G 1.4) and a molecular weight (Mw) of 300 kDa (Algaia, Lannillis, France).

[0150] - Sodium alginate with an M / G ratio of 1.4 (expressed as Al M / G 1.4 bis), and a molecular weight of 161.7 kDa (Algaia, Lannillis, France).

[0151] - Sodium alginate with an M / G ratio of 0.7 (expressed as Al M / G 0.7) and a molecular weight Mw of 230 kDa (Algaia, Lannillis, France).

[0152] - Sodium alginate with an M / G ratio of 0.7 (expressed as Al M / G 0.7 bis) and a molecular weight Mw of 222 kDa (SigmaAldrich, lot number W201502).

[0153] - Calcium alginate with an M / G ratio of 1.6 (denoted as Al M / G 1.6) (Algaia, Lannillis, France).

[0154] - Calcium chloride (Sigma-Aldrich, CAS 10035-04-8),

[0155] - Calcium citrate (Sigma-Aldrich, CAS 5785-44-4),

[0156] - Sodium citrate dihydrate (Sigma-Aldrich, CAS 6132-04-3),

[0157] - Potassium citrate (Sigma-Aldrich, CAS 6100-05-6),

[0158] - Sodium monophosphate, sodium pyrophosphate, sodium tripolyphosphate and polyphosphates (Sigma-Aldrich, CAS 7558-80-7; CAS 7722-88-5; CAS 7758-29-4),

[0159] - Diatomaceous earth (Sigma-Aldrich, CAS 68855-54-9),

[0160] - Commercially available highly absorbent polymer PAK (Alquera, CAS 25608-12-2).

[0161] 1.2 Preparation of cross-linked polymers

[0162] The anionic polymer used is:

[0163] - Sodium alginate containing mannuronic acid to guluronic acid in a ratio of 0.7, or in other words, an "M / G ratio of 0.7", with a molecular weight of approximately 230 kDa, is expressed as "Al M / G 0.7".

[0164] - Sodium alginate with a mannulic acid to guluronic acid ratio of 1.4, in other words, an M / G ratio of 1.4, and a molecular weight of approximately 300 kDa, is expressed as "Al M / G 1.4".

[0165] The solvent is pure water. The crosslinking agent is calcium chloride, which is present in the solvent at a concentration of 0 to 500 mM during the crosslinking reaction.

[0166] Comparative crosslinking polymers not covered by this invention were prepared under the reaction conditions shown in Table 1 below:

[0167] Table 1

[0168]

[0169] The highly absorbent crosslinked polymer according to the present invention was also prepared under the reaction conditions shown in Table 2 below:

[0170] Table 2

[0171]

[0172] The cross-linked polymer is prepared as follows:

[0173] - Using an Ultraturrax running at maximum speed, mix a polymer solution containing 0.5% m / v polymer with a crosslinking agent solution at a ratio of 1:3 for 10 minutes.

[0174] - Stir magnetically for 30 minutes at room temperature.

[0175] Rinse once more with ultrapure water.

[0176] - Dry at 40°C for 24 to 48 hours.

[0177] - Homogenize by grinding at maximum speed for 3 minutes.

[0178] The swelling index was then determined using the method described by Dai et al. (2017) and Zhang et al. (“Protocol efficiently measuring theswelling rate of hydrogels”, MethodsX 7, 100779, 2020).

[0179] 1.3 Characterization of shear rate-viscosity relationship and gelation rate

[0180] Rheological analysis was performed using an AR-2000 rheometer (TA Instruments, UK) equipped with a cone-plate (52 µm gap) and a Peltier heating system for precise control. Rheological data were collected and analyzed using TA Instruments' RheologyAdvantage software (V5.7.0). The analysis was conducted by applying pulses from 0.001 to 1000 s⁻¹. -1 The flow was measured by measuring the shear rate.

[0181] The gelation time of alginate was determined using a peak-holding step, by applying a 1-second pressure during stepwise measurements. -1The shear rate was measured. Measurements were performed on an uncrosslinked polymer and then continued for 60 seconds during the crosslinking process.

[0182] 1.4 Characterization of the swelling index of crosslinked polymers

[0183] The swelling index (SI) of the crosslinked polymer was determined in water according to the protocol described by Dai et al. (2017), and expressed as grams of water absorbed per gram of polymer. The swelling index was then measured in water and in a 0.9% NaCl aqueous solution.

[0184] The swelling index of dried hydrogels was determined using the tea bag method with ultrapure water and a saline solution (0.9% NaCl). 50 to 200 mg of dried polymer powder was placed in a tea bag and sealed. The tea bag was then placed in the water / saline solution, and the time was recorded. The swelling index was then calculated based on the mass difference.

[0185] 1.5 Swelling Index and Drying Method

[0186] After preparing the crosslinked polymer, it was dried in a ventilated oven at 40°C or freeze-dried to achieve a dry matter content of 90 to 95%. After drying, the swelling index was measured using the tea bag method.

[0187] 1.6 Behavior of counter ions under ion stress

[0188] The method is identical to that disclosed in Example 1.2, using a calcium concentration of 5 mM, with a slight modification in the dispersion step: a counterion is added at a concentration of 0 to 20%, in this case being a citrate ion. The method described in Example 1.2 is then continued until a highly absorbent hydrogel powder is obtained. The swelling index of the polymer is then evaluated using the tea bag method at an ionic strength of 0.9%; this method provides in-depth understanding of the ionic activity for various applications (e.g., salinity of different agricultural soils).

[0189] 1.7 Results

[0190] Crosslinking agent concentration and swelling index

[0191] Figure 1 The swelling index of the crosslinked polymer as a function of the crosslinking agent concentration is shown. Crosslinked polymers that do not conform to the present invention, i.e., those prepared with high concentrations of calcium ions (0.05 to 0.5 M), result in structurally solidified gels, but which remain rigid and lack flexibility. Therefore, high calcium concentrations are not suitable for allowing proper, gradual swelling.

[0192] Crosslinking agent concentration and gelation rate

[0193] Figure 2Includes a histogram depicting the mass of the swollen gel, expressed in grams, as a function of the calcium chloride concentration, expressed in mM, during crosslinking; the curves superimposed on the histogram show the gelation rate, expressed as a percentage, for each crosslinked polymer according to the invention as a function of the calcium chloride concentration.

[0194] Figure 3 On one hand, it shows the mass of the swollen gel as a function of calcium concentration for each crosslinked polymer according to the invention (thin line curve and left y-axis), and on the other hand, it shows the mass of the dry gel as a function of calcium concentration (thick line curve, right y-axis).

[0195] These results indicate that a calcium concentration of 5 mM is optimal for producing stable, continuous gels with sufficient swelling capacity.

[0196] Figure 4 The gelation of the Al M / G 1.4 solution (first plateau) in the presence of 5 mM calcium ions (second plateau) is shown in the "two-step peak hold" experiment, confirming the gelation after the addition of 5 mM calcium salt, as evidenced by the increase in viscosity at low shear rates (0.5 Hz at 25 °C).

[0197] Drying method and absorption capacity

[0198] Figure 5 The swelling index of sodium alginate gels crosslinked in the presence of 5 mM calcium and dried or freeze-dried at room temperature is shown in grams of water per gram of polymer, obtained by swelling for 30 minutes (0.5 hours) or 21 hours. Results obtained with sodium alginate Al M / G 1.4 and Al M / G 0.7 were similar under various drying conditions. These results indicate that low calcium concentrations allow for perfect stability of partially crosslinked continuous gels, corresponding to low crosslinking densities, compared to rigid gels.

[0199] Figure 6 The swelling index of sodium alginate gel crosslinked in the presence of 10 mM calcium and dried or freeze-dried at room temperature is shown in grams of water per gram of polymer, obtained by swelling for 30 minutes (0.5 hours) or 21 hours.

[0200] Therefore, the gel according to the invention offers the following advantages: it is easy to produce, requires a small amount of input, and its swelling capacity is not reduced by airflow drying compared to freeze drying.

[0201] Absorption kinetics in water

[0202] The absorption kinetics of polyanionic matrices derived from crosslinking two types of alginate (Al M / G 1.4 and Al M / G 0.7) in the presence of 5 mM calcium were compared. Figure 7The absorption kinetics of the two polymers prepared as described in Section 1.2 above are shown. The water absorption capacities of the two types of crosslinked polymers according to the invention are comparable and exceed 500 g water / g polymer.

[0203] Example 2: Preparation of cross-linked polyanionic matrix in the presence of counterions; swelling index under high ionic strength.

[0204] In a 0.9% saline solution (23.8 mS / cm) 2 In the presence of (ISO 17190-5:2001), the crosslinked polyanionic matrix prepared from Al M / G 1.4 alginate in the presence of a crosslinking agent (5 mM calcium) and in the absence of counterions tends to collapse (white cubes), unlike the matrix (black rhombuses) prepared from the commercial superabsorbent PAK. Figure 8 To address this issue, counterions are added during the preparation of the crosslinked polymer. The counterion used is citric acid, added at concentrations of 1 mM, 2.5 mM, 5 mM, 7.5 mM, or 10 mM.

[0205] Behavior under ion stress in the presence of counterions

[0206] The method for preparing the gel in the presence of counterions is similar to that previously disclosed in Example 1.2, with a slight modification in the dispersion step: counterions are added at a concentration of 0.001 to 20%, more specifically 0.01 to 10%, in this case the counterions are citrate ions. The method disclosed in Example 1.2 is then continued until a highly absorbent hydrogel powder is obtained. Subsequently, following the procedure disclosed in Example 1.4 above, the swelling index of the polymer is determined using the tea bag method at an ionic strength of 0.9%.

[0207] The crosslinked polymer according to the present invention is prepared as follows:

[0208] - Using Ultraturrax® running at maximum speed, mix the Al M / G 1.4 alginate solution, 5 mM crosslinking agent solution, and anti-ion solution for 10 minutes.

[0209] - Stir magnetically for 30 minutes.

[0210] Rinse once more with ultrapure water.

[0211] - Dry,

[0212] - Homogenization.

[0213] In a 0.9% saline solution (23.8 mS / cm) 2The swelling index of cross-linked polymers containing counterions was determined in the presence of (ISO 17190-5:2001) for 24 hours.

[0214] The results showed that the presence of citric acid at a concentration of at least 1 mM improved the performance of the matrix in a saline environment and prevented it from collapsing over time. Figure 9 ).

[0215] Therefore, the addition of counterions such as citric acid / sodium citrate pairs increases the liquid retention capacity in saline solutions. These counterions act as chelating agents within the crosslinked polymer matrix.

[0216] Swelling index kinetics under saline conditions

[0217] In the presence of a 0.9% NaCl aqueous solution, and with 5 mM calcium as a crosslinking agent and 1 mM chelating agent such as citric acid, the crosslinked polyanionic polymer does not collapse. Figure 10 (The curve with black squares). This cross-linked polyanionic polymer contains a bioactive substance; it is represented in this specification as Al M / G 1.4 bioactive and prepared as disclosed in Example 4. Its absorption in saline solution is comparable to that of the commercial superabsorbent PAK (rhomboid). Figure 10 ,diamond).

[0218] Absorption and desorption kinetics

[0219] Figure 11 The results show that the cross-linked polymer (black circle) according to the invention has a greater water absorption capacity than the commercially available superabsorbent polymer PAK (cross). The cross-linked polymer according to the invention has the advantages of being bio-based and biodegradable, while PAK is produced through chemical synthesis, and its high absorbency properties are maintained during rehydration and drying. The cross-linked polymer and PAK according to the invention were gradually dried at room temperature for 6 days.

[0220] Example 3: Mass production of highly absorbent matrix

[0221] In order to increase the volume of the highly absorbent matrix that can be produced, the inventors have developed a method in which the crosslinked polymer comprises sodium alginate and calcium alginate.

[0222] Preparation of cross-linked polymers

[0223] The crosslinked polymer was prepared as follows: a sodium alginate paste was prepared by adding 200 mL of water to every 100 g of sodium alginate polymer (500 g / L sodium alginate) at a high shear rate. A calcium alginate suspension was prepared by adding 200 mL of water to every 100 g of calcium alginate polymer (500 g / L calcium alginate). The ratio of sodium alginate (NaAl) to calcium alginate (CaAl) was adjusted based on the mass percentage of calcium alginate added: 30% CaAl-70% NaAl; 40% CaAl-60% NaAl; 50% CaAl-50% NaAl; 70% CaAl-30% NaAl. Figure 12 ).

[0224] Mix the paste and suspension in the desired proportions, with or without adding chelating agents or active substances.

[0225] The resulting gel was spread onto a non-stick tray and dried at 40°C for 48 hours.

[0226] Example 4: Production of cross-linked polymers containing cross-linked cationic polymer nanoparticles

[0227] The cationic polymer is a high molecular weight, highly deacetylated chitosan (>99% deacetylation).

[0228] Five grams of cationic polymer were dispersed in one liter of 1% acetic acid solution. Simultaneously, a 0.25% phosphate solution was prepared. After the cationic polymer was completely dispersed, the pH of the polymer solution was adjusted to 4.5. Subsequently, the phosphate solution was added at a ratio of 5:3 (cationic polymer to crosslinking agent) while stirring at a moderate speed. Once the crosslinking agent addition was complete, the solution was centrifuged at 10,000 g for 15 minutes at 4°C. The precipitate was washed twice with ultrapure water and resuspended for later use.

[0229] Preparation of nanoparticles loaded with active molecules

[0230] The active substance was dispersed at a ratio of 3:1 (cationic polymer: active substance) until a homogeneous solution was obtained. The dispersion was then crosslinked with a crosslinking agent in the same manner as described below for the preparation of a cationic polymer matrix.

[0231] Absorption properties of the highly absorbent polymer according to the present invention

[0232] The crosslinked polymer, which is mixed with cationic polymers and / or diatoms and / or anionic polymers and crosslinked with divalent to trivalent cations according to method 1, has a swelling index of 250 to 800 g water / g polymer.

[0233] In the presence of a 0.9% NaCl aqueous solution, the absorption capacity of the superabsorbent polymer according to the present invention is comparable to that of the commercial superabsorbent polymer PAK (potassium polyacrylate), i.e., 70 g water / g polymer.

[0234] In terms of ionic strength, hydrogels that are mixed with citrate and / or phosphate and / or cationic polymers and / or anionic polymers according to method 1 and crosslinked with divalent or trivalent cations can have a swelling index of 40 to 60 g solution / g polymer.

[0235] Figure 12 The results shown illustrate the properties of various mixtures of sodium alginate and calcium alginate. Approximately 1 kg of gel can be produced. The drying time is shorter than that of polymers containing only sodium alginate. These results demonstrate that the crosslinked polymers according to the present invention can be produced in large quantities.

[0236] Example 5: The role of cross-linked polymers rich in bioactive molecules in plant growth

[0237] Low molecular weight chitosan can be used as a biostimulant, as can sodium alginate and calcium alginate.

[0238] As shown in Example 2, a crosslinked polymer matrix was prepared from Al M / G 0.7 sodium alginate, Al M / G 1.4 sodium alginate, or calcium alginate in the presence of 5 mM CaCl2 and 1 mM citric acid.

[0239] Wheat seedlings were treated with a cross-linked polymer matrix at a concentration of 1 g / L. The lengths of the roots, leaves, and seedlings of the treated wheat were measured and compared with those of the untreated wheat.

[0240] Figure 13 and 14 The results showed that the raw materials (Al M / G 0.7; Al M / G 1.4; and calcium alginate) have the intrinsic ability to promote the growth of seedling roots and leaves.

[0241] A bioactive cross-linked polymer matrix was prepared from sodium alginate with an M / G ratio of 1.4 in the presence of 5 mM calcium ions, 1 mM citric acid, and 10 mg / L of chitosan oligosaccharide nanoparticles.

[0242] Wheat seedlings received root treatment consisting of application of a bioactive cross-linked polymer matrix (Al M / G 1.4 bioactive) containing 10 mg / L chitosan oligosaccharide nanoparticles at a concentration of 3 g / L, while other wheat seedlings received root treatment consisting of application of 10 mg / L chitosan nanoparticles (ChNP 10 mg).

[0243] After growing for 7 days under root treatment, the root length of wheat treated under different conditions was measured and compared. Figure 15 The results presented demonstrate the synergistic effect of the intrinsic effects of the components in the combined formulation according to the present invention.

[0244] A polymer matrix prepared from sodium alginate and containing chitosan promotes root growth and enhances seedling and leaf growth in a wheat model.

[0245] Example 6: The role of cross-linked polymer matrices rich in or low in bioactive molecules under environmental stress conditions.

[0246] As shown in Example 2, a cross-linked polymer matrix was prepared from Al M / G 1.4 sodium alginate, and cross-linked with 5 mM calcium chloride and 1 mM citric acid as counterions. The matrix was dried in a ventilated oven at 40°C for 24 to 48 hours, and then ground at high speed for 3 minutes until particles were obtained.

[0247] Then, a cross-linked bioactive polymer matrix was prepared as shown in Example 4.

[0248] A cross-linked bioactive polymer matrix (containing sodium alginate (Al M / G 1.4), calcium chloride (5 mM CaCl2), chitosan nanoparticles (a high molecular weight polymer chitosan cross-linked with TPP), and citric acid (1 mM)) was applied at a concentration of 3 g / L. The effect of seedling treatment with this cross-linked bioactive polymer matrix on plant growth was compared with the effects of a polymer matrix (as in Example 1), potassium polyacrylate (a positive control of a highly absorbent matrix), and a negative control (no highly absorbent matrix) on seedlings.

[0249] The effect was assessed in two separate growth stages: the first at 1 month of growth (tillering stage) and the second at 3 months of growth (late stem elongation stage to early flowering stage).

[0250] Stomatal conductance and relative water content in wheat leaves after one month of growth

[0251] Figure 16 The results show how stomatal conductance of winter wheat plants treated with the polymer matrix according to the invention varies with soil moisture content, the matrix optionally being rich in bioactive molecules (chitosan). Treatment of plants with Al M / G 1.4 bioactive compounds resulted in partial stomatal closure, which led to maintenance of tissue water content in one-month-old plants.

[0252] Figure 17 and 18The results showed that, under water stress, plants treated with a chitosan-rich substrate, denoted as “Al M / G 1.4 bioactivity”, maintained a 6% higher water content than the control.

[0253] Figure 19 This study shows the changes in water potential in wheat leaves subjected to water stress based on the applied treatment. It can be observed that the water potential changes more rapidly in the negative control. Furthermore, when comparing relative water content (…),… Figure 20 At this stress level, a sharp decrease in relative tissue water content can be observed, while polymer treatment helps maintain turgor pressure even at very low water potentials. The inventors observed that, over an 11-day stress period, the bioactive matrix (Al M / G 1.4 bioactive) maintained tissue water content at approximately 50%, comparable to that of a highly absorbent potassium polyacrylate matrix. Potassium polyacrylate is widely used in agriculture to increase soil absorption and runoff infiltration.

[0254] Figure 21 Changes in electrolyte leakage during stress are shown. The percentage of leakage is a stress marker indicating damage to the cell membrane. The inventors observed that the Al M / G 1.4 bioactive treatment reduced cell damage induced by severe water stress, and compared with the control, a significant 50% reduction in cell damage indicated by electrolyte leakage was observed.

[0255] Example 7: Quality and Stability

[0256] The transition of AlM / G 1.4, AlM / G 1.4 bis, and AlM / G 0.7 bis alginate solutions from a viscous state to a gel state was quantitatively analyzed. Figures 22 to 24 A 5 g / L alginate solution was prepared, and a series of calcium concentrations were tested on different types of alginate. For Al M / G 1.4 alginate, 5 g / L solutions were cross-linked with calcium at concentrations of 1 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM. For Al M / G 1.4 bis and Al M / G 0.7 bis alginate, three calcium concentrations (2.5 mM, 5 mM, and 10 mM) were tested.

[0257] like Figure 24 As shown, the results indicate that the swollen Al M / G 1.4 alginate gel exhibits a transformation range of 3.5 mM to 7 mM to produce a water-retaining gel.

[0258] Figure 22 and 23The gelation rates of Al M / G 1.4 bis and Al M / G 0.7 bis alginate shown in the figure are similar to those observed for Al M / G 1.4 alginate, with a maximum swelling gel concentration of 5 mM. The same behavior was observed at a concentration of 10 mM, at which the gel swelled only slightly but exhibited a high degree of gelation.

[0259] Example 8: Bioactivity

[0260] like Figure 25 As shown, alginates (Al M / G 1.4 and Al M / G 1.4bis) with mannuronic acid content greater than 55% exhibit promising biological activities in plants.

[0261] AlM / G 1.4 bis alginate with 57% mannuronic acid has been shown to affect the activation of the "pathogenesis-related" PR-2 protein. AlM / G 1.4 alginate followed the same trend, while AlM / G 0.7 alginate with a lower mannuronic acid content (40%) did not lead to the same significant induction as alginate with a mannuronic acid content greater than 55%. Furthermore, in the case of calcium-crosslinked alginate (XPC), biological activity was less pronounced.

Claims

1. A crosslinked polymer obtained by crosslinking at least one polycarboxylic acid in a solution of a solvent in the presence of a crosslinking agent selected from divalent and trivalent cations, wherein the polymer is characterized in that, during crosslinking, the initial concentration of the crosslinking agent in the solvent is 1 to 30 mM, and the crosslinked polycarboxylic acid polymer is capable of absorbing at least 200 g of water per gram of crosslinked polymer.

2. The crosslinked polymer according to claim 1, characterized in that, The crosslinking agent is a divalent metal, including calcium, or a trivalent metal.

3. The crosslinked polymer according to claim 1 or 2, characterized in that, The polycarboxylic acid is alginic acid, which comprises 30 to 70% mannulic acid and 30 to 70% guluronic acid by weight of alginic acid.

4. The crosslinked polymer according to any one of the preceding claims, characterized in that, During crosslinking, a chelating agent is added to the solvent, the chelating agent being selected from: n-carboxylic acids, where "n" equals 1, 2, 3, or 4; phenolic compounds; salicylates / esters; EDTA; and EGTA.

5. The crosslinked polymer according to any one of the preceding claims, characterized in that, It contains nanoparticles, which comprise or are composed of cross-linked cationic polymers.

6. The crosslinked polymer according to claim 5, characterized in that, The nanoparticles contain an active agent.

7. The crosslinked polymer according to claim 6, characterized in that, The active agent is selected from: phenolic compounds, oligosaccharides, plant hormones, amino acids, non-protein amino acids, nutrients, micronutrients, osmotic protectants, and combinations thereof.

8. A method for producing a crosslinked polymer as defined in any one of claims 1 to 7, the method being characterized by comprising at least the following steps: a) Add a crosslinking agent selected from divalent and trivalent cations to a polycarboxylic acid solution to obtain a final concentration of 1 to 30 mM in the polycarboxylic acid solution. b) Perform a cross-linking reaction to form a gel. c) Dry the gel obtained in b).

9. A product, based on the weight of the product, comprising 0.01 to 99% by weight of a polymer as defined in any one of claims 1 to 7 or a polymer obtained by the method according to claim 8.

10. Use of the polymer as defined in any one of claims 1 to 7, or the polymer obtained by the method according to claim 8, or the product as defined in claim 9, for the purpose of biostimulating water supply and / or plant growth.

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