Controlled release encapsulated agrochemicals by biodegradation

Abstract

The present invention relates to a method for preparing a biodegradable microcapsule comprising an agrochemical and a method for preparing a biodegradable microcapsule composition. The present invention also relates to biodegradable microcapsules and to biodegradable microcapsule compositions themselves.

Description

Field of the Invention

[0001] The present invention relates to a method for preparing biodegradable microcapsules containing agricultural chemicals and a method for preparing a biodegradable microcapsule composition. The present invention also relates to the biodegradable microcapsules and the biodegradable microcapsule composition itself.

[0002] background

[0003] Agrochemicals, such as fertilizers and pesticides (e.g., herbicides, insecticides, fungicides), are key to ensuring food production to feed a growing world population. Many agrochemicals are applied to crops by spraying water-based slurries or solutions of the active substances. This enables accurate metering and large-scale spreading of the agrochemicals.

[0004] Agrochemicals are typically encapsulated and applied to crops as a dilute slurry of an insoluble encapsulant suspended in water. This encapsulation protects farmers from the effects of often harmful agrochemicals. It also provides stability in the presence of other chemicals, allowing otherwise incompatible active substances to be delivered in a single formulation. Furthermore, it protects active substances from unwanted degradation due to environmental factors such as UV exposure, evaporation, or washing away from plants and soil by rain.

[0005] However, current encapsulation releases its contents by rupturing through mechanical action (such as friction or pressure), which means that the active substance may remain unavailable for a period of time and then suddenly released all at once. This can expose plants to high local concentrations of these active substances, which can cause damage to the plant.

[0006] Furthermore, the materials currently used to encapsulate such agrochemicals are synthetic and non-biodegradable, causing them to persist in the soil long after the active substances are delivered. They can also be washed into waterways, further contaminating them and entering the food chain.

[0007] Therefore, there is a need for biodegradable encapsulation materials to deliver agricultural chemicals. Commonly used biodegradable encapsulation materials, such as starch, are soluble in water and are therefore not suitable for spraying onto crops as water-based slurries. Since the active substance is released prematurely when the powdered encapsulation is added to water, they do not provide any encapsulation benefits.

[0008] Some degradable polymers, such as polylactic acid and polyvinyl alcohol, can be used as encapsulation shells and are ultimately degradable, but this occurs very slowly, over months and years, which is too slow to effectively release agricultural chemicals. Their release is carried out by mechanical force or via slow diffusion through a porous shell. Biodegradable PLA-based microcapsules are used to construct controlled release delivery systems for pesticides, such as Colloids and Surfaces B: Biointerfaces, Vol. 144, August 1, 2016, pp. 38-45. JP 2006067956 discloses the preparation of biodegradable PLA microcapsules encapsulating useful microorganisms for agricultural use.

[0009] Some biodegradable encapsulants can be made from animal-derived materials such as gelatin or silk, however the use of animal-derived proteins is highly undesirable (Liu, M., Millard, P.-E., Urch, H., Zeyons, O., Findley, D., Konradi, R., Marelli, B., Microencapsulation of High-Content Actives Using Biodegradable Silk Materials, Small 2022, 18, 2201487).

[0010] Therefore, there is a need for a plant-based agrochemical delivery system that rapidly degrades into harmless components in the environment, protects volatile active substances from premature release, releases its cargo in a controlled manner over several days to avoid crop damage and prolong the shelf life of the active substance, protects the active substance from degradation during storage and protects farmers from the effects of the active substance, while being insoluble, stable in water and robust enough to be sprayed. In addition, plant-based biodegradable encapsulants are more compatible with natural active substances (such as essential oils or microorganisms). SUMMARY OF THE INVENTION

[0012] Viewed from a first aspect, the present invention provides a method for preparing biodegradable microcapsules containing an agrochemical, the method comprising:

[0013] (a) forming a suspension of particles comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents, wherein a first co-solvent is an organic acid and a second co-solvent is water, and wherein the solids content of the plant-based protein in the suspension is 5% by weight or greater;

[0014] (b) reducing the size of the protein particles to 20 microns or less by volume as determined by laser diffraction. 50 ;

[0015] (c) dispersing an agricultural chemical in the plant-based protein suspension to form a composition; and

[0016] (d1) spray drying the composition to form microcapsules, or

[0017] (d2) dispersing the composition in an immiscible oil to form microcapsules and removing at least a portion of the oil from the microcapsules.

[0018] Viewed from another aspect the invention provides biodegradable microcapsules comprising an agrochemical obtainable or obtainable by a process as hereinbefore described.

[0019] Viewed from another aspect the invention provides biodegradable microcapsules comprising an agrochemical and one or more plant-based proteins encapsulating the agrochemical, wherein the plant-based protein has a solubility of less than 20%, preferably less than 10%, when measured at a protein concentration of 5% w / w in water at 20°C and pH 7.

[0020] Viewed from another aspect, the present invention provides a method for preparing a biodegradable microcapsule composition, the method comprising: preparing biodegradable microcapsules containing an agricultural chemical according to the method as described above; and suspending the biodegradable microcapsules containing the agricultural chemical in an external aqueous phase.

[0021] Viewed from another aspect, the present invention provides a biodegradable microcapsule composition obtainable or obtainable by a method as hereinbefore described.

[0022] Viewed from another aspect, the present invention provides a biodegradable microcapsule composition comprising the biodegradable microcapsules as hereinbefore described and an external phase. Detailed Description of the Invention

[0024] The present invention relates to a method for preparing biodegradable microcapsules containing agricultural chemicals, the method comprising:

[0025] (a) forming a suspension of particles comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents, wherein a first co-solvent is an organic acid and a second co-solvent is water, and wherein the solids content of the plant-based protein in the suspension is 5% by weight or greater;

[0026] (b) reducing the size of the protein particles to 20 microns or less by volume as determined by laser diffraction. 50 ;

[0027] (c) dispersing an agricultural chemical in the plant-based protein suspension to form a composition; and

[0028] (d1) spray drying the composition to form microcapsules, or

[0029] (d2) dispersing the composition in an immiscible oil to form microcapsules and removing at least a portion of the oil from the microcapsules.

[0030] Any suitable plant-based protein may be used in the present invention. In a preferred method of the present invention, one or more plant-based proteins each have a non-polar amino acid content of less than 50%. The proportion of non-polar amino acids can be determined by analytical methods such as ISO 13903:2005, which hydrolyzes peptides into their constituent amino acids by acidic or alkaline hydrolysis. Residues tested in such analyses include: tryptophan, methionine, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine, valine, and the sum of cystine + cysteine. The percentage of non-polar amino acids is calculated as the sum of the relative abundance of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline.

[0031] In a further preferred method of the present invention, the protein is selected from pea protein, potato protein, soy protein, rapeseed protein, lentil protein, chickpea protein, faba bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, more preferably pea protein and / or potato protein.

[0032] Suitable plant-based proteins also include:

[0033] Brassica: Includes Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongata: elongated mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St Hilarion cabbage, Brassica juncea: Indian mustard, brown and leaf mustard, Sarepta mustard, Brassica napus: napus: rapeseed, canola, rutabaga, Brassica narinosa: broadbeaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, kai-lan, Brussels sprouts, kohlrabi, Brassica perviridis: tender greens, mustard spinach, Brassica rapa (synonym: B. campestris): Chinese cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii): Asian mustard;

[0034] Solanaceae: includes tomatoes, potatoes, eggplants, bell peppers, and hot peppers;

[0035] Cereals: including corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and fonio;

[0036] Pseudocereals: Includes amaranth (love-lies-bleeding, red amaranth, prince-of-Wales-feather), breadnut, buckwheat, chia, cockscomb (also known as quail grass or soko), pitseed goosefoot, and quinoa. quinoa and wattleseed (also known as acacia seeds);

[0037] Leguminosae: Includes Acacia alata (Winged Wattle), Acacia decipiens, Acacia saligna (known by various common names, including coojong, golden wreath wattle, orange wattle, and blue-leafed wattle), Arachis hypogaea (Peanut), Astragalus galegiformis, Cytisus laburnum (common names: laburnum, golden chain, or golden rain), Cytisus supinus, Dolichios lablab (common names include hyacinth bean, lablab-bean bonavist bean / pea), dolichos bean, seim bean, lablab bean), Egyptian kidney bean, Indian bean, bataw, and Australian pea), Ervum lens (Lentil), Genista inctorial (common names include dye's whin, waxen woad, and waxenwood), Glycine max (Soybean), Lathyrus clymenum (peavine or vetchling), Lathyrus odoratus (peavine or vetchling), Lathyrus staivus (peavine or vetchling), Lathyrus Silvetris (peavine or vetchling), Lotus tetragonolobus (asparagus-pea or winged pea), Lupinusalbus (Lupin), Lupinus angustifolius (Lupin), Lupinus luteus (Lupin), Lupinus polyphyllus (Lupin), Medicago sativa (Alfalfa), Phaseolus aureus (Mung Bean), Phaseolus scoccineus (Runner Bean), Phaseolus nanus (Green Bean / French Bean), Phaseolus vulgaris (Green Bean / French Bean), Pisum sativum (Pea), Trifolium hybridum (Clover), Trifolium pretense (Red clover), Vicia faba (Broad bean), Vicia sativa (Vetch), Vigna unguiculate (cowpea);

[0038] Non-legumes: Includes: Acanshosicyos horrida, Aesculushyppocastanum (Conker tree / Horsechestnut), Anacardium occidentale (Cashew tree), Balanites aegyptica, Bertholletia excels (Brazil nut), Beta vulgaris (Sugar beet), Brassica napus (Rapeseed), Brassica juncea (Brown mustard), Brassica nigra (Black mustard), Brassica hirta (Eurasian mustard), Cannabis sativa (Marijuana), Citrullus vulgaris (Watermelon) vulgaris (watermelon species), Citrus aurantiaca (Citrus), Cucurbita maxima (squash), Fagopyrum esculentum (knotweed), Gossypium barbadense (extra-longstaple cotton), Heianthus annuus (sunflower), Nicotiana sp.)(Tobacco plant), Prunus avium (cherry), Prunus cerasus (sour cherry), Prunus domestica (plum), Prunus amygdalus (almond), Ricinus communis (caster bean / caster oil plant), Sesamumindicum (sesame), Sinapis alba (white mustard), Terlfalrea pedata (oyster nut).

[0039] In step (a), the first cosolvent increases the solubility of the one or more plant-based proteins. The first cosolvent can be considered a solubility cosolvent. One or more solubility cosolvents can be present, and the one or more solubility cosolvents can completely or partially dissolve the one or more plant-based proteins.

[0040] According to the present invention, the first co-solvent is an organic acid. An organic acid is an organic compound with acidic properties.

[0041] Preferably, the organic acid is acetic acid, formic acid, gluconic acid, propionic acid, an α-hydroxy acid, and / or a β-hydroxy acid. Preferred α-hydroxy acids include glycolic acid, lactic acid, malic acid, citric acid, and / or tartaric acid, preferably lactic acid. Preferred β-hydroxy acids may include β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxyβ-methylbutyric acid, 2-hydroxybenzoic acid, and carnitine. In particularly preferred methods of the present invention, the organic acid is acetic acid and / or lactic acid.

[0042] The use of organic acids can solubilize plant proteins and also allow for mild hydrolysis of the proteins. For example, without wishing to be bound by theory, the solubility of plant-based proteins in organic acids may be attributed to: i) protonation of the proteins and ii) the presence of an anionic solvation layer that helps reduce hydrophobic interactions. Once initially dissolved in an organic acid, the protonation of plant-based proteins can help stabilize them in their non-solvent (e.g., water).

[0043] In step (a), the second co-solvent reduces the solubility of the one or more plant-based proteins compared to the first co-solvent. The second co-solvent may be considered a desolubilising co-solvent. One or more desolubilising co-solvents may be present.

[0044] According to the present invention, the second co-solvent is water.

[0045] In preferred methods of the invention, the solvent system comprises a cosolvent ratio of the first cosolvent to the second cosolvent of about 5%-95% v / v, about 10%-90% v / v, about 20%-80% v / v, about 20%-60% v / v, about 25%-55% v / v, about 30%-50% v / v, about 20%, about 30%, about 40%, about 50% or about 60% v / v, most preferably about 30-50% v / v.

[0046] In step (b), the size of the protein particles is reduced to a d value of 20 microns or less by volume as determined by laser diffraction. 50 .

[0047] In a preferred method of the present invention, in step (b), the protein solution is heated to a first temperature above the sol-gel transition temperature of the one or more plant-based protein solutions and then lowered to a second temperature below the sol-gel transition temperature of the one or more plant-based protein solutions to form a hydrogel.

[0048] As used herein, the term "sol-gel transition temperature" refers to the temperature at which the plant-based protein transitions from a liquid state to a hydrogel state. Thus, at temperatures above the sol-gel transition temperature, the plant-based protein will be in a liquid state, while at temperatures below the sol-gel transition temperature, the plant-based protein will be in a hydrogel state.

[0049] In a preferred method of the invention, in step (b) the protein suspension is subjected to a shear treatment comprising a shearing step involving further reduction of the size of the protein particles.

[0050] According to the present invention, the shearing step may comprise a "lower shearing step" and / or a "higher shearing step".

[0051] As used herein, the term "lower shear step" may refer to a process step in which a low level of mechanical energy (preferably by a cutting action) is applied to the material to cause it to primarily break up into large discrete particles. "Lower shear" generally does not include mechanical energy applied to the material by high speed impact (e.g., at speeds greater than 2 ms). -1 The invention also includes any grinding step that comminutes the material (e.g., by differential impact or by shearing). It also generally does not include grinding processes based on cavitation. In a particular embodiment, during the lower shear step, the suspension is broken up to obtain particles such that at least 80% by weight of the suspended particles have a maximum dimension of 1 mm to 100 mm as determined by optical microscopy.

[0052] As used herein, the term "higher shear step" may refer to a process step in which energy is applied to reduce a suspension to small particles, such as to form, for example, a colloidal dispersion. In certain embodiments, during the higher shear step, the suspension is broken up to obtain particles having a particle size d of less than 20 microns, preferably 0.1 to 15 microns, more preferably 0.2 to 10 microns, and most preferably 0.5 to 5 microns, by volume distribution as determined by dynamic laser diffraction. 50 Laser diffraction may be performed according to the method defined herein.

[0053] In preferred methods of the invention, the high shear step may comprise sonication or ultrasonication (e.g. using equipment such as a Bandelin HD4200 or Hielscher UIP1000hdT), high shear mechanical agitation (e.g. using equipment such as a Silverson rotor-stator high shear mixer), high pressure homogenisation or cavitation.

[0054] For the avoidance of doubt, a higher shear step subjects the suspension to a higher level of shear than a lower shear step. Where a process involves both a lower shear step and a higher shear step, the higher shear step must occur after the lower shear step (i.e. they are discrete steps occurring in this particular order).

[0055] A preferred method of the present invention further comprises the step of changing the pH of the plant-based protein suspension such that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

[0056] In a preferred method of the present invention, after said step of changing the pH of the emulsion, the pH of the emulsion is at least 1 pH unit below the isoelectric point of the plant-based protein.

[0057] Preferably, the step of changing the pH of the plant-based protein suspension is performed after step (b) or after step (c).

[0058] During pH adjustment of a plant-based protein suspension, the suspension may pass through the isoelectric point of the protein. Due to the lack of charge repulsion at the isoelectric point, the suspended protein particles in the plant-based protein suspension can rapidly coagulate. To avoid this, pH-adjusting substances can be used to rapidly change the pH, minimizing the time the suspension spends at the isoelectric point.

[0059] The isoelectric point of a plant-based protein is defined as the pH at which the average charge of the solution after ionization has a value of zero. The isoelectric point of a specific plant-based protein can be determined using the method described in Helmick et al., Food Biophysics (2021) 16: 474-483. A preferred isoelectric point method is the experimental method using a zeta potential analyzer as described therein, because the exact value of a given plant protein will vary slightly depending on the plant growth conditions and strain. Alternatively, the calculation method described therein can be used.

[0060] Therefore, in a preferred method of the present invention, the step of changing the pH of the plant-based protein suspension involves adding a pH adjusting substance to the plant-based protein suspension. Preferably, the pH adjusting substance is a solution comprising monovalent metal ions, divalent metal ions or ammonium ions, preferably an alkaline aqueous solution comprising monovalent metal ions, divalent metal ions or ammonium ions. More preferably, the pH adjusting substance is an aqueous hydroxide solution, preferably sodium hydroxide, potassium hydroxide or ammonium hydroxide.

[0061] Preferably, the composition formed in step (c) has a protein solids content in the range of 1 wt% to 25 wt%, more preferably 2 wt% to 20 wt%, even more preferably 3 wt% to 15 wt%, most preferably 4 wt% to 12 wt%, based on the total weight of the composition.

[0062] In step (c), the agrochemical is dispersed in the plant-based protein suspension to form a composition.

[0063] Agrochemicals suitable for use according to the present invention may be solid or liquid at room temperature.

[0064] Agrochemicals are substances used as pesticides for controlling pests and diseases or as plant growth promoters in agriculture, forestry, horticulture and gardening.

[0065] The term pesticide or plant protection agent refers to any substance or mixture of substances that is intended to prevent, eliminate, drive or alleviate any harmful organism. Pesticides can be chemical substances or biological agents, referred to as biological pesticides (biopesticides) (such as viruses or bacteria). They are used to resist harmful organisms, including insects, plant pathogens, weeds, mollusks, birds, mammals, fish, nematodes (round worms) and microorganisms that compete with humans for food, destroy property, spread diseases or cause nuisances. Pesticides include biocides, which are substances that can kill different forms of living organisms. In addition, chemical substances can be obtained by synthetic chemical methods, or alternatively extracted from natural sources, such as plant essential oils or single compounds extracted from essential oils.

[0066] Plant growth promoters include plant growth regulators (PGRs), including biological or synthetic or chemical or bioregulators, micronutrients and macronutrients.

[0067] In a preferred method of the invention, the agricultural chemical is selected from pesticides, including fungicides, herbicides, insecticides, algaecides, molluscicides, acaricides and rodenticides, and antimicrobials, including microbicides, antibiotics, antibacterials, antivirals, antifungals, antiprotozoals and antiparasitics, or a combination thereof.

[0068] Fungicides chemically control fungi. They are compounds used to prevent the spread of fungi in gardens and crops. Fungicides are also used to combat fungal infections. Fungicides can be either contact or systemic. Contact fungicides kill the fungus upon contact with its surface. Systemic fungicides must be absorbed by the fungus before they die.

[0069] Examples of suitable fungicides according to the present invention include the following classes: (3-ethoxypropyl)mercuric bromide, 2-methoxyethylmercuric chloride, 2-phenylphenol, 8-hydroxyquinoline sulfate, 8-phenylmercuric oxyquinoline, 1,2,3-benzothiadiazole-7-carboxylic acid (acibenzolar), acylamino acid fungicides, acypetacs, aldimorph, aliphatic nitrogen-containing fungicides, allyl alcohol, amide fungicides, 1-aminopropyl phosphate (ampropylfos), anilazine, anilide fungicides, antibiotic fungicides, aromatic fungicides, aureofungin, azaconazole, azithiram, azoxystrobin, barium polysulfide, refined benzene benalaxyl-M, benodanil, benomyl, benquinox, bentaluron, benthiavalicarb, benzalkonium chloride, benzamacril, benzamide fungicides, benzamorf, benzoanilide fungicides, benzimidazole fungicides, benzimidazole precursor fungicides, benzimidazolylcarbamate fungicides, benzohydroxamic acid, benzothiazole fungicides, bethoxazin, binapacryl, biphenyl, bitertanol, bithionol, blasticidin-S, Bordeaux mixture mixture), boscalid, bridged diphenyl fungicides, bromuconazole, bupirimate, Burgundy mixture, buthiobate, butylamine, calcium polysulphide, captafol, captan, carbamate fungicides, carbamorph, phenylcarbamate fungicides, carbendazim, carboxin, carpropamid, carvone, Cheshunt mixturemixture), chinomethionat, chlobenthiazone, chloraniformethan, chloranil, chlorfenazole, chlorodinitronaphthalene, chloroneb, chloropicrin, chlorothalonil, tetrachloroquine chlorquinox, chlozolinate, ciclopirox, climbazole, clotrimazole, azole fungicides, azole fungicides (imidazoles), azole fungicides (triazoles), copper (II) acetate, basic copper (II) carbonate, copper fungicides, copper hydroxide, copper naphthenate, copper oleate, copper oxychloride, copper (II) sulfate, basic copper sulfate, copper zinc chromate, cresol, cufraneb, cuprobam, cuprous oxide, cyazofamid, cyclafuramide d), cyclic dithiocarbamate fungicides, cycloheximide, cyflufenamid, cymoxanil, cypendazole, cyproconazole, cyprodinil, dazomet, DBCP, debacarb, decafentin, dehydroacetic acid, dicarboximide fungicides, dichlofluanid, dichlone, dichlorophen, dichlorophenyl dicarboxamide Imine fungicides, dichlozoline, diclobutrazol, diclocymet, diclomezine, dicloran, diethofencarb, diethyl pyrocarbonate, difenoconazole, diflumetorim, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, dinitrophenol fungicides, dinobuton, dinocap, dinocton, dinopenton, dinosulphon, dinoterbon, diphenylamine, dipyrithione, disulphiram, ditalimfos, dithianon, dithiocarbamate fungicides, DNOC, dodemorph, dodicin, dodine, donatodine, drazoxolon,Edifenphos, epoxiconazole, etaconazole, etem, ethaboxam, ethirimol, ethoxyquin, 2,3-dihydroxypropylmercuric ethylmercury, ethylmercuric acetate, ethylmercuric bromide, ethylmercuric chloride, ethylmercuric phosphate, etridiazole, Famoxadone, fenamidone, fenaminosulph, fenapanil, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenitropan, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fentin, ferbam, ferimzone, fluazinam, fludioxonil, flumetover, fluopicolide, fluoximide, fluotrimazole, fluoxastrobin, fluquinconazole, fluorine Flusilazole, flusulphamide, flutolanil, flutriafol, folpet, formaldehyde, fosetyl, fuberidazole, furaxyl, furametpyr, furamide fungicides, furanilide fungicides, furcarbanil, furconazole, furconazole-cis, furfural, furmecyclox, furophanate, glyodin, griseofulvin, guazatine, halacrinate, hexachlorobenzene, hexachlorobutadiene, hexachlorophene, hexaconazole, hexylthiofos, hydrargaphen, Hymexazol, imazalil, imibenconazole, imidazole fungicides, iminoctadine, inorganic fungicides, inorganic mercury fungicides, iodomethane, ipconazole, iprobenfos, iprodione, iprovalicarb, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl, lime sulfur sulphur), mancopper, mancozeb, maneb, mebenil, mecarbinzid, mepanipyrim, mepronil, mercuric chloride, mercuric oxide, mercurous chloride, mercury fungicides, metalaxyl, metalaxyl-M, metam, metazoxolon, metconazole, methasulphocarb, methfuroxam, methyl bromide, methyl isothiocyanate, methylmercury benzoate, methylmercury cyanoguanidine dicyandiamide), methylmercury pentachlorophenoxide, metiram, metominostrobin, metrafenone, metsulphovax, milneb, morpholine fungicides, myclobutanil, myclozolin, N-(ethylmercury)-toluenesulfonanilide, nabam, natamycin, nitrostyrene, nitrothal-isopropyl, nuarimol, OCH, octhilinone, ofurace, organomercury fungicides, organophosphorus fungicides, organotin fungicides, orysastrobin, Oxadixyl, oxathiin fungicides, Azole fungicides, oxine copper, Imidazole (oxpoconazole), oxycarboxin, pefurazoate, penconazole, pencycuron, pentachlorophenol, penthiopyrad, phenylmercuric urea, phenylmercuric acetate, phenylmercuric chloride, phenylmercuric derivatives of catechol, phenylmercuric nitrate, phenylmercuric salicylate, benzenesulfonamide fungicides, phosdiphen, phthalide, phthalimide fungicides, picoxystrobin, piperalin, polycarboxamide bamate), polymeric dithiocarbamate fungicides, polyoxin, polyoxorim, polysulfide fungicides, potassium azide, potassium polysulfide, potassium thiocyanate, probenazole, prochloraz, procymidone, propamocarb, propiconazole, propineb, proquinazid, prothiocarb, prothioconazole, pyracarb olid), pyraclostrobin, pyrazole fungicide, pyrazophos, pyridine fungicide, pyridinitril, pyrifenox, pyrimethanil, pyrimidine fungicide, pyroquilon, pyroxychlor, pyroxyfiir, pyrrole fungicide, quinacetol, quinazamid, quinconazole, quinoline fungicide, quinone fungicide, quinoxaline fungicide, quinoxyfen fen), quintozene, rabenzazole, salicylanilide, silthiofam, simeconazole, sodium azide, sodium o-phenylphenol, sodium pentachlorophenol, sodium polysulfide, spiroxamine, streptomycin, strobilurin fungicides, sulfonanilide fungicides, sulfur, sultropen, TCMTB, tebuconazole, tecloftalam, tecnazene, tecoram,Tetraconazole, thiabendazole, thiadifluor, thiazole fungicides, thicyofen, thifluzamide, thiocarbamate fungicides, thiochlorfenphim, thiomersal, thiophanate, thiophanate-methyl, thiophene fungicides, thioquinox, thiram, tiadinil, tioxymid, tivedo, tolclofos-methyl, tolnaftate, tolylfluanid, toluenemercuric acetate, triadimefon, triadimefon enol), triamiphos, triarimol, triazbutil, triazine fungicides, triazole fungicides, triazoxide, tributyltin oxide, trichlamide, tricyclazole, trifloxystrobin, triflumizole, triforine, triticonazole, unclassified fungicides, undecylenic acid, uniconazole, urea fungicides, validamycin, valinamide fungicides, vinclozolin, zarilamid, zinc cyclohexane, zineb, ziram, zoxamide, and mixtures thereof.

[0070] Herbicides are pesticides used to kill unwanted plants (also known as weeds). Selective herbicides kill specific targets while leaving the desired crop relatively unharmed. Some of these work by interfering with the growth of weeds and are generally based on plant hormones. Herbicides used to clean up wasteland are non-selective and kill all plant material they come into contact with. Herbicides are widely used in agriculture and landscape turf management. They are used in total vegetation control (TVC) programs for highway and railway maintenance. Smaller amounts are used in the management of forestry, pasture systems, and areas designated as wildlife habitats.

[0071] Suitable herbicides may be selected from the group consisting of aryloxycarboxylic acids, such as MCPA; aryloxyphenoxypropionates, such as clodinafop; cyclohexanedione oximes, such as sethoxydim; hydroxybenzonitriles, such as bromoxynil; sulfonylureas, such as nicosulphuron; triazolopyrimidines, such as penoxsulam; triketones, such as mesotrione; triazine herbicides, such as metribuzin, hexaxinone or atrazine. razine); sulfonylurea herbicides such as chlorsulfuron; uracils such as lenacil, bromacil or terbacil; urea herbicides such as linuron, diuron, siduron or neburon; acetanilide herbicides such as alachlor or metolachlor; thiocarbamate herbicides such as benthiocarb and triallate; oxadiazolone herbicides, such as Oxadiazon Oxazolidinone herbicides, phenoxyacetic acid; diphenyl ether herbicides, such as fluazifop, acifluorfen, bifenox or oxyfluorfen; dinitroaniline herbicides, such as trifluralin; organic phosphonate / salt herbicides, such as glufosinate salts and esters and glyphosate salts and esters; and / or dihalogenated benzonitrile herbicides, such as bromoxynil or ioxynil; benzoic acid herbicides; dipyridylium herbicides, such as paraquat; and other herbicides, such as isoflurane, Particularly preferred herbicides may be selected from 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine, cyproconazole, dicamba as a benzoic acid, glyphosate, glufosinate, imazapic as an imidazolinone, isopropylamine as a chloroacetamide, picloram, clopyralid and triclopyr as a pyridinecarboxylic acid or synthetic auxins, their corresponding water-soluble salts and esters, and mixtures thereof.

[0072] Insecticides are pesticides used against all developmental forms of insects and include ovicides and larvicides used against the eggs and larvae of insects. Insecticides are used in agriculture, medicine, industry and households.

[0073] Suitable insecticides may include those selected from the group consisting of chlorinated insecticides such as, for example, Camphechlor, DDT, hexachlorocyclohexane, gamma-hexachlorocyclohexane, methoxychlor, pentachlorophenol, TDE, Aldrin, Chlordane, Chlordecone, Dieldrin, Endosulphan, Endrin, Heptachlor, Mirex, and mixtures thereof; organophosphorus compounds such as, for example, Acephate; ate), Azinphos-methyl, Bensulide, Chlorethoxyfos, Chlorpyrifos, Chlorpyriphos-methyl, Diazinon, Dichlorvos (DDVP), Dicrotophos, Dimethoate, Disulphoton, Ethoprop, Fenamiphos, Fenitr othion), Fenthion, Fosthiazate, Malathion, Methamidophos, Methidathion, Methyl-parathion, Mevinphos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Phorate, Phosalone, Phosmet t), Phostebupirim, Pirimiphos-methyl, Profenofos, Terbufos, Tetrachlorvinphos, Tribufos, Trichlorfon and mixtures thereof; Carbamates such as, for example, Aldicarb, Carbofuran, Carbaryl, Methomyl, 2-(1-methylpropyl)phenylmethylcarbamate and mixtures thereof;Pyrethroids, such as, for example, Allethrin, Bifenthrin, Deltamethrin, Permethrin, Resmethrin, Sumithrin, Tetramethrin, Tralomethrin, Transfluthrin and mixtures thereof; Plant toxin-derived compounds, such as, for example, Derris, rotenone, Pyrethrum, Neem (Azadirachtin), nicotine, caffeine and mixtures thereof; Neonicotinoids, such as imidacloprid; Abamectins, for example emamactin; diazines, such as indoxacarb; and / or o-formamidobenzamides, such as chlorantraniliprole (rynaxypyr).

[0074] Acaricides are pesticides that kill mites. Antibiotic acaricides, carbamate acaricides, formamidine acaricides, mite growth regulators, organochlorines, permethrins, and organophosphate acaricides all fall into this category. Molluscicides are pesticides used to control mollusks, such as moths, slugs, and snails. These substances include metaldehyde, methiocarb, and aluminum sulfate. Nematicides are a class of chemical pesticides used to kill parasitic nematodes (helminths).

[0075] Agrochemicals also include plant growth regulators (PGRs). PGRs are synthetic or biological compounds used to regulate plant growth, such as increasing branching, inhibiting shoot growth, increasing flowering, removing excess fruit, or altering fruit ripening. They can be divided into five categories: auxin-related compounds, gibberellins and gibberellin biosynthesis inhibitors, cytokinins, abscisic acid, and compounds that affect ethylene status.

[0076] Agrochemicals also include nutrients. A composition can contain at least one nutrient. Nutrients are chemical elements and compounds that are desirable or necessary to promote or improve plant growth. Nutrients are often described as either macronutrients or micronutrients.

[0077] Micronutrients generally refer to trace metals or trace elements and are generally applied in lower doses. Suitable micronutrients include trace elements selected from zinc, boron, chlorine, copper, iron, molybdenum and manganese.

[0078] The micronutrients may be in soluble form or included as insoluble solids and may be in the form of salts or chelates. Preferably, the micronutrients are in the form of carbonates or oxides. Preferably, the micronutrients may be selected from zinc, calcium, molybdenum, manganese or magnesium. Particularly preferred micronutrients for use in the present invention may be selected from zinc oxide, manganese carbonate, manganese oxide or calcium carbonate.

[0079] Macronutrients generally refer to those containing nitrogen, phosphorus and potassium, and include fertilizers such as ammonium sulfate, as well as water conditioners. Suitable macronutrients include fertilizers and other nitrogen, phosphorus or sulfur containing compounds, and water conditioners.

[0080] Suitable fertilizers include inorganic fertilizers that provide nutrients such as nitrogen, phosphorus, potassium or sulfur. Examples of such fertilizers include:

[0081] For nitrogen as a nutrient: nitrates and / or ammonium salts, such as ammonium nitrate, including in combination with urea, for example calcium ammonium nitrate, ammonium nitrate sulfate, ammonium phosphates, in particular monoammonium phosphate, diammonium hydrogen phosphate and polyammonium phosphate, ammonium sulfate, and less commonly calcium nitrate, sodium nitrate, potassium nitrate and ammonium chloride;

[0082] For phosphorus as a nutrient: acidic forms of phosphorus, such as phosphoric acid, pyrophosphoric acid or polyphosphoric acid, but more usually in salt form, such as ammonium phosphate, in particular ammonium dihydrogen phosphate, diammonium hydrogen phosphate and ammonium polyphosphate, potassium phosphate, in particular potassium dihydrogen phosphate and potassium polyphosphate;

[0083] For sulfur as a nutrient: ammonium sulfate and potassium sulfate, for example mixed with magnesium sulfate.

[0084] The microcapsules may contain at least one micronutrient and / or at least one macronutrient.

[0085] The use of some of these synthetically derived agrochemicals and the presence of their residues in food and water are raising health and safety concerns. Furthermore, the use of chemical pesticides can impact the environment and biodiversity. The continued and sometimes inadequate use of chemical pesticides is also contributing to the development of pathogen resistance, leading to potential food safety issues.

[0086] In response to the growing demand to reduce the use of agrochemicals and develop alternative methods to protect crops from pathogens and pests, research and development in the field of biopesticides has grown exponentially over the past 20 years.

[0087] Biopesticides include microorganisms selected from bacteria, cyanobacteria, microalgae, fungi, viruses, nematodes, protozoa and yeasts, which can be in any combination and which are capable of killing undesirable organisms. These microorganisms may be in a dormant state or in an inactivated form or exist as spores. The microcapsules of the present invention may include Bacillus thuringiensis, Bacillus thuringiensis var. kurstaki (Bt), B. thuringien, Bacillus thuringiensis var. tenebrionid, Bacillus thuringiensis var. aizawai, Bacillus thuringiensis japonensis, Bacillus popilliae, Bacillus lentimorbus, Bacillus sphaericus, Bacillus pumilus, Bacillus subtilis, Bacillus firmus, Burkholderia cepacia, and the like. cepacia), Bacillus amyloliquefaciens, Bacillus licheniformis, Erwinia amylovora, Pasteuria penetrans, Pasteuria ausage, Psedomonas spp., Streptomyces griseoviridis, and Xanthomonas campestris pv. Poannua, as well as those microorganisms as given in Kumar, J., Ramlal, A., Mallick, D., and Mishra, V. An Overview of Some Biopesticides and Their Importance in Plant Protection for Commercial Acceptance. Plants 2021, 10, 1185.

[0088] Among natural alternatives to chemical pesticides, products based on plant extracts and / or essential oils (EOs) have attracted increasing attention due to their very low human toxicity, high volatility, and rapid degradation as generally recognized as safe (GRAS) compounds.

[0089] The microcapsules of the present invention, wherein the agrochemical is a biopesticide, EO or EO component, are suitable for use in agrochemical formulations that can be used in plant production that can be certified as organic by organizations such as the USDA (United States Department of Agriculture) or Ecocert in Europe.

[0090] Essential oils have a strong odor and are produced by aromatic plants as secondary metabolites. They are typically obtained from several plant parts by steam distillation. They consist of a mixture of volatile compounds (20 to 100), although in most cases they are characterized by two or three main compounds that represent the majority of the EO (20%-70%). For example, the EO of lemon (Citrus limon) primarily consists of limonene and β-pinene. EO can contain molecules with a wide range of chemical functionalities, such as terpenes and terpenoids (e.g., limonene, linalool); as well as aromatic and aliphatic molecules (e.g., cinnamaldehyde, safrole). These components are characterized by low molecular weight and, therefore, high volatility and rapid evaporation. They are also often very sensitive to oxygen and light. This makes them difficult to apply directly to crops and fields as agrochemicals, and therefore encapsulation that gradually releases the EO or its components is advantageous.

[0091] Essential oils have long been known for their antimicrobial and medicinal properties. The latter has particularly fueled the development of aromatherapy, where they are used as bactericides (e.g., tea tree and cinnamon EOs), fungicides (Lavandulaspica EOs), or virucides (Cinnamomum camphora).

[0092] Over the past 20 years, the antibacterial and antifungal properties of essential oils have been evaluated against a variety of plant pathogens to determine their potential as alternative plant protection products (see, for example, Baser, KHC; Buchbauer, G.'s Handbook of Essential Oils, 3rd ed.; CRC Press: Boca Raton, FL, USA, 2020, Chapter 24, which provides a review of the use of EOs in agriculture).

[0093] The complex composition of essential oils is of interest because they can be used as multi-site chemicals, reducing the risk of resistance. Alternatively, antibacterial and antifungal properties can be achieved by extracting the main components from EO and using a single natural active substance or a simple mixture of several natural components. For example, the main component of EO from Thymus vulgaris is thymol, while the main component of EO from Rosmarinus officinalis is cineole. These single components can be extracted and used as agricultural active substances.

[0094] In a preferred method of the present invention, the agrochemical is an essential oil of natural origin and is selected from the following list of natural plant sources: European fir (Abies alba), balsam fir (Abies balsamea), Siberian fir (Abiessibirica), garlic (Allium sativun), amyris (Amyris balsamifera), dill (Anethumgraveolens), Amazon rosewood (Aniba rosaeodora var. Amazonica), celery (Apiumgraveolens var. Dulce), Luzon olive (Canarium luzonicum), caraway (Carum carvi), North African cedar (Cedrus atlantica), cedar (Cedrus deodara), camphor tree, cinnamon (Cinnamomum cassia), Ceylon cinnamon (Cinnamomum zeylanicum), camphor (Cinnamosma fragrans), lemon (Citrus aurantifolia), lime (Citrus aurantium), bergamot (Citrus bergamia), lemon (Citrus lime), grapefruit (Citrus paradisi), mandarin (Citrus reticulata), sweet orange (Citrus sinensis), copaiba (Copaifera officinalis), cilantro (Coriandrum sativum), Corydothymus capitatus, cumin (Cuminum cymincum), columnar cypress (Cupressus sempervirens var. Stricta), lemongrass (Cymbopogon citratus), flexuosus (Cymbopogon flexuosus), giant citronella (Cymbopogon giganteus), martini var. Motia (Russa citronella), citronella (Cymbopogon nardus), maple citronella (Cymbopogon winterianus), lemon eucalyptus (Eucalyptus citriodora), broadleaf eucalyptus (Eucalyptus citriodora). dives), Eucalyptus globulus, Eucalyptus radiata, Eucalyptus smithii, Eugeniacaryophyllus), fennel (Foeniculum vulgare), Fujian cypress (Fokienia hodginsii), fragrant white bead (Gaultheria fragrantissima), star anise (Illicum verum), North American juniper (Juniperus virgiana), laurel (Laurus nobilis), lavender (Lavendula angustifolia), Lavendula x burnatii clone grosso, lemon scale (Leptospermum petersonii), mountain pepper (Litsea citrata), Melaleuca alternifolia, Melaleuca cajputii, Melaleuca quinquenervia, wild mint (Mentha arvensis), lip mint (Mentha pulegium), lemon mint (Mentha x citrata), peppermint (Mentha x piperita), Monarda fistulosa), nutmeg (Myristica fragrans), myrtle (Myrtus communis), myrtle, basil (Ocimum basilicum), holy basil (Ocimum sanctum), origanum compactum, winter oregano (Origanum heracleoticum), sweet oregano (Origanum majorana), Bourbon geranium (Pelargonium x asperum), allspice (Pimenta racemosa), anise (Pimpinella anisum), maritime pine (Pinus pinaster), maritime pine turpentine (Pinus pinaster térébenthine), Scots pine (Pinus sylvestris), black pepper (Piper nigrum), rosemary, Salvia lanvandulifolia, sage (Salvia officinalis), summer savory (Satureja hortensis), winter savory (Satureja montana), benzoin (Styrax benzoe), North American cedar (Thuyaoccidentalis), frankincense thyme (Thymus mastichina), borneol thyme (Thymussatureioides), silver thyme, Trachyspermum amni, vanilla (Vanilla fragrans Auct), vetiver (Vetiveriazizanoides), ginger (Zingiber officinale).

[0095] In addition, other essential oils with known effects as agrochemicals can be found in Chang Y, Harmon PF, Treadwell DD, Carrillo D, Sarkhosh A and Brecht JK (2022) Biocontrol Potential of Essential Oils in Organic Horticulture Systems: From Farm to Fork. Front. Nutr. 8: 805138. Table 1 provides examples of EOs that act on plant pathogenic fungi / oomycetes, Table 2 provides examples of EOs that act on plant pathogenic bacteria, and Table 4 provides examples of EOs that exhibit herbicidal properties.

[0096] In a preferred method of the present invention, the main components of the essential oil can be extracted from the EO and then encapsulated. These components may include borneol, camphor, carvacrol, β-caryophyllene, camphene, cinnamaldehyde, eucalyptol, α-curcumene, β-cymene, diallyl disulfide and diallyl trisulfide, eucalyptol, eugenol, eugenyl acetate, geranial, α-humulene, limonene, myrcene, neral, α-pinene, γ-pinene, γ-terpinene, terpinolene, α-thujone, thymol and vanillin.

[0097] In a preferred method of the present invention, the agrochemical is dispersed in a carrier phase. Preferably, the carrier phase is a solvent, fat, wax or microbial growth medium.

[0098] In a preferred process of the present invention, the carrier phase is a solvent.

[0099] Preferably, the solvent is one with low volatility (eg, having a vapor pressure of less than 0.1 Torr at 25°C, preferably less than 0.01 Torr at 25°C, preferably less than 0.001 Torr at 25°C).

[0100] Preferably, the solvent has low or no odor.

[0101] Preferably, the solvent has at least two Hansen solubility parameters selected from the group consisting of atomic dispersion (δD) less than 20, dipole moment (δP) less than 8, and hydrogen bonding (δH) less than 11. More preferably, the solvent has at least two Hansen solubility parameters selected from the group consisting of atomic dispersion (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0102] Preferably, the solvent has a viscosity greater than 1.07 g / cm 3 A solvent having this property can advantageously prevent emulsion separation of the microcapsules (e.g. in the final product formulation).

[0103] Preferably, the solvent contains only low levels of materials having alcohol functional groups (e.g., primary alcohol functional groups). In preferred methods of the invention, the solvent contains less than 40% wt, more preferably less than 20% wt, of alcohol-containing materials based on the total weight of the solvent. In particularly preferred methods of the invention, the solvent does not contain alcohol-containing materials.

[0104] In a preferred method of the present invention, the carrier phase is a solvent selected from the group consisting of carboxylic acid esters, fatty acid esters, phthalates, triols, diols, rosin resins, isoparaffins, terpenes and vegetable oils or combinations thereof.

[0105] Preferably, the solvent is selected from 840, 812N, 829, 829ECO, Coco 810, 810N, 128. 808, T-C7, 8810, PPG 810, OE, DO and 818, Limonene, benzyl benzoate, diethyl phthalate, isopropyl myristate, triethyl citrate, dipropylene glycol and propylene glycol, triacetin, glycerol, 1,3-propylene glycol or a combination thereof, preferably 812N.

[0106] Preferably, the solvent is a vegetable oil selected from the group consisting of coconut oil, corn oil, canola oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil, sesame oil, soybean oil and sunflower oil. Other examples of vegetable oils are given in CTFA Cosmetic Ingredient Handbook, JM Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Vegetable oils are oils from plant sources. Optionally, the solvent is derived from a vegetable oil.

[0107] In a preferred process of the invention, the carrier phase is a fat or wax having a melting point of less than 60°C, preferably less than 45°C, preferably less than 25°C.

[0108] Preferably, the wax is selected from 100. 142 and 154 or a combination thereof, preferably 100.

[0109] In a preferred method of the present invention, the carrier phase is a microbial growth medium, such as lysogeny broth (LB), minimal synthetic defined (SD) medium or M9 minimal medium.

[0110] The preferred method of the present invention may further comprise a post-treatment step of the microcapsules. Preferably, the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step or a coating formation step.

[0111] In a preferred method of the present invention, the post-treatment step includes a non-covalent cross-linking step. Preferably, the non-covalent cross-linking step includes treating the microcapsules with a non-covalent cross-linking agent selected from sodium tripolyphosphate (NaTPP), sodium hexametaphosphate, and phenolic compounds (e.g., tannic acid, caffeic acid, etc.).

[0112] In a preferred method of the present invention, the post-treatment step comprises a covalent cross-linking step. Preferably, the covalent cross-linking step comprises treating the microcapsules with a covalent cross-linking agent selected from the group consisting of genipin, epoxy compounds, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and polyamide-based cross-linking resins (e.g., ) or a combination thereof.

[0113] In a preferred method of the present invention, the post-treatment step comprises a coating formation step.

[0114] In a preferred method of the present invention, the coating forming step comprises subjecting the microcapsules to a complex coacervation step using a polysaccharide. Preferably, the polysaccharide is selected from xanthan gum, gellan gum and chitosan or a combination thereof.

[0115] In a preferred method of the present invention, the coating forming step comprises treating the microcapsules with a mineral aqueous solution to form a mineral coating. Preferably, the mineral aqueous solution comprises iron salt, calcium salt, phosphate, carbonate, titanium salt or zinc salt or a combination thereof.

[0116] As will be appreciated by the skilled artisan, multiple post-processing steps may be performed. For example, in a preferred method of the invention, the microcapsules are subjected to a non-covalent cross-linking step (e.g., using NaTPP), followed by a coating step (e.g., using chitosan), and optionally a further non-covalent cross-linking step (e.g., using NaTPP).

[0117] The present invention also provides biodegradable microcapsules comprising an agrochemical, which are obtained or obtainable by the method as described above.

[0118] The present invention also provides a method for preparing a biodegradable microcapsule composition, the method comprising:

[0119] - preparing biodegradable microcapsules containing agricultural chemicals according to the method as described above; and

[0120] - Biodegradable microcapsules containing agrochemicals are suspended in the external aqueous phase.

[0121] Preferably, the external phase is an external aqueous phase, preferably hard water with a total hardness of at least 40 mg / L CaCO3, or an acidic buffer solution with a pH of 4.0 to 5.5.

[0122] Preferred methods of the present invention further comprise adding one or more suspending agents to the external phase.

[0123] Preferably, the one or more suspending agents are selected from gum arabic, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil type 1, magnesium aluminum silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, polyvinyl pyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers (such as cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylate and cross-linked polyvinyl pyrrolidone), and swelling clays (such as bentonite and laponite).

[0124] The biodegradable microcapsule composition may also contain a preservative and / or antimicrobial agent, such as an organic acid or its ester or salt, such as ascorbic acid or its ester or salt, for example ascorbyl palmitate, sorbic acid or its ester or salt, for example potassium sorbate, benzoic acid or its ester or salt, for example methyl and propyl benzoic acid and 4-hydroxybenzoate, propionic acid or its ester or salt, for example sodium propionate, phenol or its ester or salt, for example sodium 2-phenylphenol; 1,2-benzisothiazolin-3-one; or formaldehyde itself or as paraformaldehyde; or an inorganic material, such as sulfite and its salts, typically present in an amount of 0.01 wt.% to 1 wt.% of the formulation.

[0125] Biodegradable microcapsule compositions can include dyes or pigments combined with agricultural chemicals. Examples of suitable dyes include: anthraquinone, triphenylmethane, phthalocyanine and derivatives thereof, and diazonium salts. Pigment dispersions can contain pigments such as Pigment Red 112 (CAS 6535-46-2), Pigment Red 2 (CAS 6041-94-7), Pigment Red 48:2 (CAS 7023-61-2), Pigment Blue 15:3 (CAS 147-14-8), Pigment Green 36 (CAS 14302-13-7), Pigment Green 7 (CAS 1328-53-6), Pigment Yellow 74 (CAS 6358-31-2), Pigment Orange 5 (CAS 3468-63-1), Pigment Violet 23 (CAS 6358-30-1), Pigment Black 7 (CAS 97793-37-8), and Pigment White 6 (CAS 98084-96-9).

[0126] Examples of suitable effect pigments include pearlescent pigments of varying particle sizes. Effect pigments with a particle size of 15 μm or less, or 60 μm or less, are typically used. The particle size of the effect pigment is typically no greater than 200 μm, preferably no greater than 100 μm. Typically, the particle size of the effect pigment is 1 μm or greater. Another effect pigment may be aluminum.

[0127] The present invention also provides a biodegradable microcapsule composition, which is obtained or obtainable by the method as described above.

[0128] The present invention also provides biodegradable microcapsules comprising an agrochemical and one or more plant-based proteins encapsulating the agrochemical, wherein the plant-based protein has a solubility of less than 20%, preferably less than 10%, when measured at a protein concentration of 5% w / w in water at 20° C. and pH 7. Low solubility is highly desirable in order to maintain the structural integrity of the microcapsules when they are added to water to prepare an aqueous composition for use, or if it rains immediately after the microcapsules are applied to a field or crop.

[0129] Protein solubility is determined using the following protocol: a known amount of microcapsules is added to an aqueous solution, which is then centrifuged to separate the soluble and insoluble portions. After centrifugation, the liquid supernatant (i.e., the soluble portion) is removed without obtaining any solids (i.e., the insoluble portion) deposited at the bottom. The resulting supernatant is analyzed for nitrogen content. The protein content in the supernatant is then calculated using a coefficient of 6.25 based on nitrogen content. Protein solubility is defined as the amount of protein in the supernatant divided by the amount of protein in the entire aqueous solution.

[0130] In the preferred biodegradable microcapsules of the present invention, after incubation at 20°C for 10 days in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, at least 25%, more preferably at least 40%, even more preferably at least 50%, preferably at least 60% of the initially encapsulated agrochemical remains present inside the microcapsules as determined by GC.

[0131] In the preferred biodegradable microcapsules of the present invention, the one or more plant-based proteins encapsulating the agrochemicals are selected from soy protein, pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, faba bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, preferably pea protein and / or potato protein.

[0132] In the preferred biodegradable microcapsules of the present invention, the one or more plant-based proteins encapsulating the agrochemicals have been pretreated with an organic acid. Preferably, the organic acid is acetic acid, formic acid, propionic acid, α-hydroxy acid and / or β-hydroxy acid. Particularly preferably, the organic acid is acetic acid and / or lactic acid.

[0133] In the preferred biodegradable microcapsules of the present invention, the one or more plant-based proteins encapsulating the agrochemicals have a protein secondary structure having at least 40% intermolecular β-sheets, at least 50% intermolecular β-sheets, at least 60% intermolecular β-sheets, at least 70% intermolecular β-sheets, at least 80% intermolecular β-sheets, or at least 90% intermolecular β-sheets, wherein the intermolecular β-sheet content percentage is measured by FTIR.

[0134] In preferred biodegradable microcapsules of the present invention, the one or more plant-based proteins encapsulating the agrochemicals are selected from pea proteins and soy proteins, and the agrochemicals are selected from essential oils and components of essential oils.

[0135] In the preferred biodegradable microcapsules of the present invention, the agricultural chemical is dispersed in a carrier phase.

[0136] In preferred biodegradable microcapsules of the present invention, the microcapsules have a d value of less than or equal to 500 μm, less than or equal to 250 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm as determined by laser diffraction. 90 diameter.

[0137] In the preferred biodegradable microcapsules of the present invention, the plant-based protein encapsulating the agrochemical has been non-covalently modified by a non-covalent cross-linking agent, or the plant-based protein has been covalently modified by a covalent cross-linking agent, or the plant-based protein has a coating deposited thereon.

[0138] The present invention also provides a biodegradable microcapsule composition comprising the biodegradable microcapsule as described above and an external phase.

[0139] Preferably, the external phase is an external aqueous phase, preferably hard water with a total hardness of at least 40 mg / L CaCO3, or an acidic buffer solution with a pH of 4.0 to 5.5.

[0140] The biodegradation of plant protein microcapsule shells occurs easily in the presence of enzymes, particularly proteases. Most soil microorganisms, including bacteria and fungi, produce proteases to recycle soil organic matter by cracking cell wall proteins. These enzymes, such as Streptomyces griseus, are easily found in soil (see Vranova, V., Rejsek, K., Formeanek, P. (2013)). Proteolytic activity in soil: A2 Review, Applied Soil Ecology, 70, p. 23-32). These proteases similarly crack the microcapsule protein shells, releasing active substances within days and weeks. When specific microorganisms are added to a buffered slurry of microcapsules and incubated, the in vitro release rate of the active substance can be measured.

[0141] In the preferred biodegradable microcapsules of the present invention, after incubation at 20° C. in the dark for 14 days in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 and in the presence of 5.76% of Streptomyces griseus protease in 10 mM NaOAc + 5 mM CaCl2 in an amount relative to the mass of the microcapsules, at least 20%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 50% of the initially encapsulated agrochemical is released from the microcapsules as determined by GC, while in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 and in the presence of 5.76% of Streptomyces griseus protease in 10 mM NaOAc + 5 mM CaCl2 in an amount relative to the mass of the microcapsules, at least 20%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 50% of the initially encapsulated agrochemical is released from the microcapsules, while in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 and in the presence of 10 mM NaOAc + 5 mM CaCl2 in an amount relative to the mass of the microcapsules, at least 20%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 50% of the initially encapsulated agrochemical is released from the microcapsules. After incubation in CaCl2 at 20°C in the dark for 14 days, less than 50%, more preferably less than 40%, even more preferably less than 30%, most preferably less than 20% of the initially encapsulated agrochemical is released from the microcapsules as determined by GC.

[0142] The biodegradation of the microcapsules can be confirmed in the standard aerobic soil biodegradation test ISO 17556:2019. Empty powdered microcapsules containing only the shell material and no active ingredient are prepared by spray drying. The shells are then incubated with soil as an inoculum at a constant temperature (preferably 20 to 25°C) in the dark or in diffuse light. The water holding capacity, pH and organic matter content of the soil are measured and controlled. The ratio of carbon in the sample to nitrogen in the soil is also controlled. Soil biodegradation is measured as carbon dioxide production and / or oxygen demand in a respirometer. The level of biodegradation is expressed as a percentage by comparing the oxygen consumption with the theoretical oxygen demand or by comparing the amount of carbon dioxide released with the theoretical amount. Biodegradation is measured at regular intervals and the test is continued until a constant level of biodegradation is present or for up to 6 months. A reference material is also tested, and for the test to be valid, its biodegradation needs to exceed 60% during the plateau phase or at the end of the test.

[0143] In the preferred biodegradable microcapsules of the present invention, the biodegradation rate of the plant-based protein based on CO2 evolution after 28 days as measured according to ISO 17556:2019 is 40% to 100%, more preferably 50% to 100%, most preferably 60% to 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1a Shown at ×20 magnification are intact multi-core microcapsules from Example 2a after 7 days in the incubator.

[0146] Figure 1b Shown at ×20 magnification are fragmented microcapsules and protein aggregates from Example 2b after 7 days in the incubator.

[0147] Figure 2 Showing the soil biodegradation profile of microcapsule shells. Example

[0148] Material

[0149] - Pea protein isolate (80 wt% protein, 4 wt% carbohydrates) (ProEarth P16109) was purchased from Cambridge Commodities Ltd, UK. The isoelectric point was determined to be 4.5 using the zeta potential analyzer method described in the reference cited above.

[0150] -Glacial acetic acid was purchased from Fisher Scientific, UK

[0151] - Lactic acid 85% was purchased from Sigma-Aldrich Gillingham, UK

[0152] -Thymol was purchased from Fisher Scientific, UK

[0153] - 812N was purchased from 1010 Leochemicals, Germany

[0154] - 840 was purchased from 1010 Leochemicals, Germany

[0155] - Polyglycerol polyricinoleate (PGPR) was purchased from Danisco, Denmark

[0156] -Maltodextrin was purchased from Sigma-Aldrich Gillingham, UK

[0157] -Polysorbate 80 was purchased from Sigma-Aldrich Gillingham, UK

[0158] Potassium hydroxide was purchased from Sigma-Aldrich Gillingham, UK

[0159] -Ethanol was purchased from Fisher Scientific, UK

[0160] - Streptomyces griseus (type XIV, ≥3.5 units / mg solid, powder) purchased from Sigma-Aldrich Gillingham, UK

[0161] - All components used for phosphate buffered saline (PBS) (pH 7.4) were purchased from Fisher Scientific, UK and prepared in the following ratios: 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4

[0162] -Sodium acetate trihydrate was purchased from Alfar Aesar, UK

[0163] -Calcium chloride dihydrate was purchased from Fisher Scientific, UK

[0164] -Methylparaben was purchased from Alfar Aesar, UK

[0165] method

[0166] Total thymol loading

[0167] Thymol levels in the microcapsules were determined by extracting the thymol and injecting it into a gas chromatography (GC) column. Quantification was achieved using a calibration curve of thymol diluted in ethanol.

[0168] To measure the total level of thymol in the microcapsule sample, the capsules are first broken up by sonication. For example, 10-50 mg of dried microcapsules are added to 3.0 g of deionized (DI) water and 10% KOH and vortexed to mix thoroughly. The solution is then sonicated for 1 minute using a Bandelin Sonopuls HD4200 and a small probe TS104 at 30% amplitude (approximately 1.5 kJ total). During sonication, the temperature is kept below 20°C using ice to avoid any loss of thymol through evaporation. An optical microscope is used to visually inspect whether the capsules are completely broken. If not, the sonication step is repeated. Ethanol is added to the mixture, which is then mixed and centrifuged. The supernatant is collected and retained as the first extract. The residue is mixed with ethanol and centrifuged. The supernatant is collected and added to the first extract. The mixture is then appropriately diluted and injected into a GC for analysis.

[0169] Slurry solid content

[0170] The solid content of the protein slurry is measured by the remaining mass after drying. Approximately 5 g of the slurry is pipetted into a small polypropylene dish and the mass is accurately recorded. The dish is placed in a 40 ° C oven and dried overnight. After removing the dish from the oven, dry mass is measured immediately, and the solid content of the protein slurry is calculated as the percentage of the initial wet mass.

[0171] Slurry particle size

[0172] The particle size of the slurry particles is measured by laser diffraction. For most slurries, this is performed with an Anton Paar laser diffraction particle size analyzer PSA1190. The measurement is performed by diluting the slurry in an aqueous solution adjusted to the same pH with acetic or lactic acid within 1 hour of manufacture. The test material is diluted to the required concentration to ensure the absence of agglomerates and the desired optical density (typically 5-15% obscuration) for measurement. The referenced d 50 This is for volume distribution, as calculated via general analysis using Mie theory.

[0173] For samples with high acid concentrations, the PSA1190 equipment is not compatible. In such cases, alternative laser diffraction equipment, such as the Malvern Pananalytical Mastersizer 3000E, may be used. When such equipment is not available, the particle size in high acid slurries is measured using the Malvern Pananalytical Zetasizer, which uses dynamic light scattering (DLS). This method is comparable to the laser diffraction method. The measurement is performed by diluting the still hot high lactic acid dispersion after sonication at 80°C with 35% w / v lactic acid. The test material is diluted to the required concentration in hot diluent to prevent protein gelation and to have the desired optical density (0.1% to 1% w / v) for measurement. The cited d 50 is for volume distribution, as calculated via general analysis using Mie theory.

[0174] Slurry pH

[0175] The undiluted slurry pH was measured using a calibrated Mettler Toledo FiveEasy F20 according to the equipment supplier's instructions.

[0176] Viscosity of the composition for spray drying

[0177] The viscosity of the spray-dried composition was measured within 1 hour of composition preparation using an Anton Paar MCR 92 rheometer. The rheometer was set up using a cone (1 degree, 50 mm diameter) and plate geometry. The viscosity was measured at a temperature of 20°C and a shear rate of 50 g / s.

[0178] Microcapsule particle size

[0179] The particle size of the final microcapsules is measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. If the microcapsules are available as a powder, they are added to reverse osmosis water and diluted to the desired concentration to have the desired optical density (typically 5-15% obscuration) for measurement. The dispersion should be checked via optical microscopy for any larger microcapsule agglomerates. If these agglomerates are visible, 0.5% by weight of acetic acid can be added to ensure that the primary particles are well dispersed. The referenced d 50 is for volume distribution, as calculated via general analysis using Mie theory.

[0180] Alternatively, particle diameter can be measured using optical microscopy (e.g., using an open Frame microscope equipped with a CellCam 200CR camera, an Aura Pro phase contrast illuminator, and 4x, 10x, and 20x universal flat-field fluorite objective). Microcapsule powder is added to reverse osmosis water or a single-strength buffer as needed. In this type of method, for each corresponding sample, particle diameter is taken from the average size measurement of 50 microcapsules. The grid of the Hirschmann counting chamber (Fuchs Rosenthal) is then used to calibrate the optical microscope. Using the straight line tool in ImageJ 1.53, particle diameter is measured from two center-edges, and the overlay text function is enabled to avoid repeating capsules.

[0181] Light microscopy

[0182] Light microscopy images were obtained using an open frame microscope equipped with a CellCam 200CR camera, an Aura Pro phase contrast illuminator, and 4x, 10x, and 20x universal plan fluorite objectives. Samples for light microscopy were prepared by adding dried microcapsules to reverse osmosis water. A cover glass was then placed on top of the sample and the image was captured.

[0183] Example 1: Preparation of thymol microcapsules

[0184] Preparation of protein hydrogels

[0185] Reverse osmosis (RO) water (1120g) was added to a 2-liter stainless steel container and 216g of pea protein isolate was added. The container was placed in a 92°C water bath and mixed with an overhead stirrer at 1500rpm. After stirring for 3 minutes, glacial acetic acid (480g) was added. The mixture was stirred at 1500rpm for 15 minutes and then at 1200rpm for 30 minutes, ensuring that the temperature of the mixture exceeded 85°C for at least 10 minutes. The mixture was poured into a tray to a depth of approximately 10mm and left to stand overnight at room temperature.

[0186] Then the hydrogel is subjected to shearing as follows. Via the low shear cutting step, the protein hydrogel is cut into approximately 1cm cubes. The cubes are divided into two 75 micron filter bags, and each filter bag is then immersed in a bucket equipped with 16L RO water. This forms a crude protein hydrogel slurry in the filter bag. Soak the hydrogel cubes and stir for 90-150 minutes with an overhead stirrer at 600-800rpm. This step is carried out to reduce the concentration of acetic acid in the hydrogel by diffusing into the continuous aqueous phase. The pH value of the wash water is then measured, and if it is higher than 3.2, then continue to soak for another 30 minutes. If it is lower than 2.9, then drain half of the water and replace with fresh RO water, then continue to soak for another 30 minutes. The filter bag is then hung above the bucket to drain for 5 minutes. The washed gel from the two filter bags was transferred to a 5-liter beaker and homogenized with a Silverson mixer at 5000 rpm for 5 minutes, 6000 rpm for 5 minutes, and 7000 rpm for 5 minutes. The smooth slurry was then transferred to a 1 L Nalgene bottle (800 g each) and exposed to high shear ultrasonic treatment (Hielscher UP500Hdt) while cooling with ice until 250 kJ was applied, oscillating once every 75 kJ. The hydrogel slurry was then passed through a 200 micron sieve before use. The pH was measured to be 3.0, and the particle size d was 0.04477 g by volume distribution. 50 It is 11 microns.

[0187] The solids content was measured to be 9.3 wt%.Dilute acetic acid (3 wt% in DI water) was added to reduce the protein content to 8.0 wt% without significantly affecting the pH or particle size distribution.

[0188] Preparation of spray-dried microcapsules

[0189] 112.12 g of the diluted dispersion was homogenized using a Silverson L5M-A high shear mixer (8000 rpm for 2 minutes) and 17.27 g of the Thymol was diluted at 40% by weight in 812N. The mixture was further homogenized using a Silverson at 8000 RPM for 5 minutes. The expected droplet size was approximately 5 microns.

[0190] The sample was then spray dried using a Buchi B290 spray dryer. The air inlet temperature was 130°C at a Q flow setting of 40. A two-fluid nozzle with a 1.4 mm tip size was used. The aspirator flow rate was set to 100%. The fluid was pumped into the spray dryer using a syringe pump at a speed setting of 13% (4-5 ml / min). The resulting dried microcapsule powder was collected from a collection tank. The total thymol loading of the microcapsules was analyzed according to the method described herein. It was found to be 3.9 wt%.

[0191] Example 1a: Second preparation of thymol microcapsules

[0192] Preparation of spray-dried microcapsules

[0193] 103.25 g of the diluted dispersion prepared in Example 1 was homogenized using a Silverson L5M-A high shear mixer (8000 rpm for 2 minutes) and 16.81 g of Thymol was diluted at 40% by weight in 812N. The mixture was further homogenized using a Silverson at 8000 RPM for 5 minutes. The expected droplet size was approximately 5 microns.

[0194] The sample was then spray dried according to the process in Example 1.

[0195] The resulting dry microcapsule powder was collected from a collection jar. The total thymol loading of the microcapsules was analyzed. It was found to be 4.9 wt%.

[0196] Example 1b: Preparation of comparative maltodextrin thymol microcapsules

[0197] 45 g of maltodextrin was dissolved in 105 g of deionized water and mixed with a magnetic stirring bar until a uniform slurry was formed. In addition to adding 0.14 g of polysorbate 80, 15.23 g of Thymol diluted at 40 wt% in 812N was added to the slurry.The mixture was homogenized using a Silverson L5M-A high shear mixer at 8000 rpm for 5 minutes.

[0198] The sample was then spray dried using a Buchi B290 spray dryer. The air inlet temperature was 130° C. with a Q flow setting of 40. A two-fluid nozzle with a 1.4 mm tip size was used. The aspirator flow rate was set to 100%. The fluid was pumped into the spray dryer using a syringe pump at a speed setting of 13% (4-5 ml / min).

[0199] The resulting dry microcapsule powder was collected from a collection jar. The total thymol loading of the microcapsules was analyzed. It was found to be 3.4 wt%.

[0200] Example 2: Enzyme release test

[0201] Protease from Streptomyces griseus was prepared as a stock solution of 10 mg / ml enzyme in 10 mM sodium acetate + 5 mM calcium chloride solution (pH 7.5).

[0202] According to Table 1, the thymol microcapsule powder of Example 1 was suspended in phosphate-buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben, so that each sample contained 5 mg of thymol. The control sample, Example 2a, had no enzyme added, and only sodium acetate and calcium chloride solutions were added. The test sample, Example 2b, had a protease solution added.

[0203]

[0204] Table 1 - Composition of biodegradation test samples with and without protease

[0205] The samples were mixed on day 0 and placed in an incubator at 37°C in the dark.

[0206] Figure 1a Shown are intact multi-core microcapsules of Example 2a after 7 days in the incubator in the absence of proteases. Figure 1b Shown are the fragmented microcapsules and protein aggregates of Example 2b after 7 days incubation in the presence of proteases.

[0207] On day 7, the samples were centrifuged at 4900 rpm for 37 minutes, and the supernatant was transferred to a 100 ml bottle. Ethanol was added to achieve a tenfold dilution of the supernatant sample. The concentration of thymol in the supernatant was measured by gas chromatography. This was used to calculate the amount of thymol released from the capsules.

[0208] 2.9ml deionized water and 0.1ml 10% KOH solution are added to the sediment material after each sample centrifugation. With mixture with 30% amplitude ultrasonic treatment (Bandelin Sonopuls HD4200, with probe TS104) 2 minutes, vortex and centrifuge at 4900rpm for 5 minutes. 100 μ l supernatant from each sample is diluted ten times with ethanol, then the concentration of thymol is measured by gas chromatography. This is used to calculate the amount of thymol remaining in the supernatant at the end of the experiment, and this amount is considered to be the amount of thymol that was not initially released in the first supernatant. The results are shown in Table 2.

[0209] Extractable thymol% in supernatant % extractable thymol in the remaining shell material Example 2a 6％ 94％ Example 2b 49％ 51％

[0210] Table 2 - Percentage of thymol released and retained after 7 days of incubation

[0211] The release study confirmed that in the absence of enzymes, in Example 2a, a large portion of the active ingredient (over 90%) remained encapsulated over 7 days. This slurry is suitable for storage prior to application to the field and crops. In the presence of enzymes, the plant protein shell was broken down and the active substance was released, resulting in a release of over 45% after 7 days.

[0212] According to Table 3, the thymol microcapsule powder of Example 1a was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben, such that each sample contained 4 mg of thymol. A control sample, Example 2c, had no enzyme added, but only sodium acetate and calcium chloride solutions. A test sample, Example 2d, had a protease solution added. Eight replicates were prepared for each of Example 2c and Example 2d so that samples could be analyzed at different incubation time points (including times t = 0, 4 hr, 24 hr, 48 hr, 72 hr, 1 week, 10 days, and two weeks).

[0213]

[0214] Table 3 - Composition of replicated biodegradation test samples with and without protease for time series studies

[0215] The samples were mixed and placed in an incubator at 37°C in the dark on day 0, except for the t=0 sample (which was analyzed immediately). At each time point, the samples were centrifuged at 4900 RPM for 30 minutes, and the supernatant was transferred to a 50 ml Falcon tube. Ethanol was added to achieve a tenfold dilution of the supernatant sample. The concentration of thymol in the supernatant was measured by gas chromatography, which was used to calculate the amount of thymol released from the capsule.

[0216] In the sediment material after each sample centrifugation, 2.9ml DI water and 0.1ml 10% KOH solution are added. With mixture with 30% amplitude ultrasonic treatment (Bandelin Sonopuls HD4200, with probe TS104) 2 minutes, vortex and centrifuge at 4900rpm for 5 minutes. To dilute 100 μ l supernatant liquors of each sample with ethanol ten times, then measure the concentration of thymol by gas chromatography. This is used to calculate the amount of thymol remaining in the sample when the experiment is finished, and this amount is considered to be the amount of thymol that did not initially release in the first supernatant liquor. The results are shown in Table 4 and Table 5.

[0217] Example 2c Extractable thymol% in supernatant % extractable thymol in the remaining shell material t＝0 3 97 t=4hr 6 94 t=24hr 5 95 t=48hr 6 94 t=72hr 5 95 t = 1 week 8 92 t = 10 days 7 93 t = 2 weeks 6 94

[0218] Table 4 - Percentage of thymol released and retained in samples without protease

[0219] Example 2d Extractable thymol% in supernatant % extractable thymol in the remaining shell material t＝0 5 95 t=4hr 3 97 t=24hr 5 95 t=48hr 21 79 t=72hr 27 73 t = 1 week 51 49 t = 10 days 36 64 t = 2 weeks 49 51

[0220] Table 5 - Percentage of thymol released and retained in samples containing protease

[0221] This release study confirmed that, in the absence of enzymes, a large portion of the active ingredient (over 90%) remained encapsulated in Example 2c over a two-week period. This slurry is suitable for storage prior to application to the field and crops. In the presence of enzymes, the plant protein shell was broken down, and the active was released in a time-dependent manner, achieving nearly 50% release within two weeks.

[0222] Example 2e: Comparative Maltodextrin-Thymol Release Test

[0223] According to Table 6, the thymol microcapsule powder of Example 1b was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben so that each sample contained 4 mg of thymol.

[0224]

[0225] Table 6 - Composition of test samples containing maltodextrin-thymol capsules

[0226] The samples were prepared, mixed, and incubated for one hour. The samples were centrifuged at 4900 RPM for 10 minutes, and the supernatant was transferred to a 50 ml Falcon tube. Ethanol was added to achieve a tenfold dilution of the supernatant sample. The concentration of thymol in the supernatant was measured by gas chromatography, which was used to calculate the amount of thymol released from the capsules in buffered water.

[0227] In the sediment material after sample centrifugation, add 2.9ml deionized water and 0.1ml 10% KOH solution. With mixture with 30% amplitude ultrasonic treatment (Bandelin Sonopuls HD4200, with probe TS104) 2 minutes, vortex and centrifuge at 4900rpm for 5 minutes. 100 μ l supernatant is diluted ten times with ethanol, then by gas chromatography measurement thymol concentration. This is used to calculate the amount of thymol that remains in the sample when the experiment is finished, and this amount is considered to be the amount of thymol that does not initially release in the first supernatant. The results are shown in Table 7.

[0228] Example 2e Extractable thymol% in supernatant % extractable thymol in the remaining shell material t = 1 hour 42 58

[0229] Table 7 - Percentage of Thymol Released and Retained in Samples Containing Maltodextrin-Thymol Capsules

[0230] This release study demonstrated that, for the maltodextrin capsules in Example 2e, even in the absence of any enzyme, a significant portion of the active ingredient (over 40%) was released after one hour in aqueous buffer. This type of slurry is unsuitable for storage prior to application to fields and crops. Once prepared as an aqueous composition or in the presence of environmental water, such as rainwater, it prematurely releases the active ingredient, demonstrating a lack of controlled-release properties.

[0231] Example 3: Thymol Soil Release Test

[0232] The thymol microcapsule powder of Example 1a was added to a soil water medium extracted from soil at Cambridge Science Park, UK, and the release of thymol was measured over time. The soil was collected at 52°13'53.5"N 0°08'44.3"E, or 'tape.steps.chief' according to the what3words convention. The soil water was prepared by adding 16.6g of soil to 33.2g of reverse osmosis water and inverting several times to form a slurry. The slurry was centrifuged at 4900RPM for 5 minutes. The supernatant (soil water) was collected and passed through a 10μm filter. According to Table 8, the thymol microcapsule powder of Example 1a was suspended in the filtered soil water so that each sample contained 4mg of thymol.

[0233] Test samples Mass of microcapsule powder (mg) Soil water mass (mg) Example 3a 82 4000

[0234] Table 8 - Composition of test samples containing soil water

[0235] On day 0, samples were mixed and placed in a dark incubator at 37°C. At each time point, samples were centrifuged at 4900 RPM for 30 minutes, and the supernatant was transferred to a 50 ml Falcon tube. Ethanol was added to achieve a tenfold dilution of the supernatant sample. The thymol concentration in the supernatant was measured by gas chromatography and used to calculate the amount of thymol released from the capsules.

[0236] In the sediment material after each sample centrifugation, add 2.9ml DI water and 0.1ml 10% KOH solution. With mixture with 30% amplitude ultrasonic treatment (Bandelin Sonopuls HD4200, with probe TS104) 2 minutes, vortex and centrifuge 5 minutes at 4900rpm. To dilute ten times with ethanol from 100 μ l supernatant of each sample, then measure the concentration of thymol by gas chromatography. This is used to calculate the amount of thymol that is retained in the sample when the experiment is finished, and this amount is considered to the amount of thymol that is not initially released in the first supernatant. The results are shown in Table 9.

[0237] Example 3a Extractable thymol% in supernatant % extractable thymol in the remaining shell material t=24hr 5 95 t=72hr 8 92 t = 1 week 45 55

[0238] Table 9 - Percentage of thymol released and retained in samples containing soil water

[0239] The release study confirmed that the active substance was released in the presence of soil water, resulting in a 45% release after 7 days. Microscopic observation of the soil water after 72 hours showed the presence of bacteria that were naturally present in the sampled soil. These bacteria produce proteases that break down the shell of the plant protein microcapsules.

[0240] Example 4: Preparation of thymol microcapsules via double emulsion process

[0241] Preparation of protein dispersions

[0242] A protein slurry of 10% w / w pea protein isolate and 35% w / w lactic acid was prepared by adding 12 g of pea protein isolate, 58.8 g of reverse osmosis water, and 49.4 g of 85% lactic acid to a 250 ml Nalgene bottle and shaking. The pH was measured to be 1.78. 100 ml of the protein dispersion was sonicated (Bandelin Sonopuls HD4200 with probe TS104) at 80% amplitude input of 100 kJ.

[0243] According to the method described herein, since the acid content of the dispersion is 35% w / w, the particle size distribution is measured by using dynamic light scattering. Within 2 hours of sonication, 2 ml of the slurry is diluted into 18 ml of 35% w / w lactic acid at 80°C, mixed thoroughly, and then diluted 10 times with 35% w / w lactic acid at 80°C to a final solid concentration of 0.1 w / v%. Particle size distribution by volume 50 The value is 29.9 nm (+ / - 6.4 nm based on 3 repetitions). This value is comparable to that obtained by laser diffraction.

[0244] Preparation of double emulsion microcapsules

[0245] The primary emulsion was prepared immediately after sonication of the protein dispersion.

[0246] Preparation of 80% w / w thymol in 812N solution and store in hot water until ready to use. The 812N mixture was added to the hot dispersion with overhead stirring at 600 rpm for 30 seconds to prepare a primary emulsion.

[0247] preparation 840 and 0.5% w / w polyglyceryl ricinoleate (PGPR) and heated to 53°C. The primary emulsion was then manually poured into 300 ml of heated The mixture of 840 and PGPR was stirred at 1000 rpm for 2 minutes. The stirring was then reduced to 700 rpm for 4 minutes, with an ice bath added midway. The stirring was further reduced to 500 rpm for 24 minutes. The microcapsules were allowed to settle at 4°C for 2 hours.

[0248] Excess oil was decanted, and the settled microcapsules were transferred to a 1 L beaker containing 400 ml of 100 mM sodium tripolyphosphate (NaTPP) and 4% w / w polysorbate 80. The microcapsules were stirred at 150 rpm for 15 minutes using a 4-blade Teflon impeller to remove excess oil. The microcapsules were collected on a 200 μm sieve and resuspended in pH 3 reverse osmosis water in a separatory funnel. The microcapsules were again collected on a 75 μm sieve and resuspended in pH 3 reverse osmosis water. The washing process was repeated three more times. Finally, the microcapsules were resuspended in a solution of 0.1 M sodium citrate, 20 mM calcium chloride, and 0.1% w / w sodium benzoate. The total thymol loading of the microcapsules was analyzed according to the methods herein. It was found to be 1.02 wt %. Due to the elongated shape of the microcapsules, the particle size was manually determined by light microscopy and Image J analysis. The average length and width were measured to be 143 μm and 98 μm, respectively.

[0249] Example 5: Enzyme release test

[0250] Protease from Streptomyces griseus was prepared as a stock solution of 10 mg / ml enzyme in 10 mM sodium acetate + 5 mM calcium chloride solution (pH 7.5).

[0251] The microcapsules of Example 4 were poured onto a 38 μm sieve and washed with 450 ml of PBS pH 7.4 containing 0.2% w / v methylparaben for pH adjustment. The microcapsules were dried from below the sieve by capillary action using a paper towel for accurate dry mass measurement. According to Table 10, the microcapsules were suspended in PBS (pH 7.4) containing 0.2% w / v methylparaben so that each sample contained 4 mg of thymol. Samples were prepared in duplicate and measured at two time points (0 day and 14 day).

[0252] The control sample Example 5a had no enzyme added, and only sodium acetate and calcium chloride solutions were added. The test sample Example 5b had protease solution added.

[0253]

[0254] Table 10 - Composition of biodegradation test samples with and without protease

[0255] On day 0, the samples were mixed and placed in a dark incubator at 37°C. On day 0, one sample each, with and without protease, was centrifuged at 4900 rpm for 30 minutes to accelerate microcapsule sedimentation. The supernatant was transferred to a 50 ml Falcon tube. Ethanol was added to achieve a tenfold dilution of the supernatant sample. The thymol concentration in the supernatant was measured by gas chromatography. This was used to calculate the amount of thymol released from the microcapsules on day 0.

[0256] On day 14, one of each sample was analyzed again with and without protease to determine the level of thymol in the supernatant, but without the centrifugation step prior to transfer of the supernatant.

[0257] In the sedimentation material of each sample, add 2.9ml deionized water and 0.1ml 10%KOH solution respectively.With mixture with 30% amplitude ultrasonic treatment (Bandelin Sonopuls HD4200, band probe TS104) 2 minutes, vortex and centrifuge 5 minutes at 4900rpm, with fragmentation microcapsule and discharge remaining thymol.To dilute ten times with 100 μ l supernatant of each sample with ethanol, then pass through the concentration of gas chromatography measurement thymol.This is used to calculate the amount of thymol that is retained in the supernatant when the experiment is finished, and this amount is considered to the amount of thymol that did not discharge in the supernatant at first the 0th day.The results are presented in Table 11 and Table 12.

[0258] Example 5c Extractable thymol% in supernatant % extractable thymol in the remaining shell material t＝0 5 95 t = 14 days 4 96

[0259] Table 11 - Percentage of thymol released and retained after 14 days of incubation in the absence of protease

[0260] Example 5d Extractable thymol% in supernatant % extractable thymol in the remaining shell material t＝0 5％ 95％ t = 14 days 68％ 32％

[0261] Table 12 - Percentage of thymol released and retained after 14 days of incubation with protease

[0262] This release study demonstrated that, in the absence of protease, as in Example 5c, a large portion of the active ingredient (over 95%) remained encapsulated in water after 14 days. This slurry is suitable for storage prior to application to the field and crops. In the presence of protease, as in Example 5d, the plant protein shell ruptured and the active was released, resulting in a release of 68% after 14 days.

[0263] Example 6: ISO biodegradation test

[0264] Empty spray-dried microcapsules were prepared for biodegradation testing of shell materials. Protein hydrogel dispersions were prepared as in Example 1 and spray-dried using a Buchi B290 spray dryer without adding any agrochemicals. The air inlet temperature was 120° C. at a Q flow setting of 50. A two-fluid nozzle with a 1.4 mm tip size was used. The aspirator flow rate was set to 120%. A peristaltic pump was used to pump the fluid into the spray dryer at a speed setting of 30% (9 ml / min). The dried microcapsule powder formed was collected from a collection tank.

[0265] The biodegradation of these spray-dried microcapsules in soil was tested using the standard aerobic soil biodegradation test ISO 17556:2019. Empty powdered microcapsules, containing only the shell material but no agrochemical, were incubated with soil as an inoculum in the dark at 25°C. The soil's water holding capacity, pH, and organic matter content were measured and controlled. The ratio of carbon in the samples to soil nitrogen was also controlled. Soil biodegradation was measured as carbon dioxide production in a respirometer. The level of biodegradation was expressed as a percentage by comparing the amount of carbon dioxide released to the theoretical amount. The test was performed in duplicate.

[0266] Biodegradation was measured at regular intervals and the test was continued for 180 days. The results for the microcapsule shells (2 replicates) can be found in Figure 2 The shell material biodegraded immediately and at a very high rate. After 10 days, the biodegradation rate had reached 53%. After 28 days, the biodegradation rate was 65%. This rate slowed over time, reaching 73.0% + / - 0.6% biodegradation after 180 days. This confirms that the microcapsule shells are highly biodegradable by soil microorganisms.

[0267] A reference material, microcrystalline cellulose, was also tested. For the test to be valid, its biodegradation needed to exceed 60% at the plateau phase, or the end of the test. After 180 days, the absolute biodegradation level of the control was 82.7% + / - 0.2%, making the test valid.

Claims

1. A method for preparing biodegradable microcapsules containing agricultural chemicals, the method comprising: (a) forming a suspension of particles comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents, wherein a first co-solvent is an organic acid and a second co-solvent is water, and wherein the plant-based protein in the suspension has a solids content of 5% by weight or greater; (b) reducing the size of the protein particles to 20 microns or less by volume as determined by laser diffraction. 50 ; (c) dispersing an agricultural chemical in the plant-based protein suspension to form a composition; (d) (d1) spray drying the composition to form microcapsules containing the agricultural chemical, or (d2) dispersing the composition in an immiscible oil to form microcapsules containing the agricultural chemical and removing at least a portion of the oil from the microcapsules; and The method further comprises the step of changing the pH of the plant-based protein suspension so that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

2. The method according to claim 1 , wherein the one or more plant-based proteins each have non-polar amino acids in an amount of less than 50%, preferably wherein the protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, more preferably wherein the protein is pea protein and / or potato protein.

3. The method according to claim 1 or claim 2, wherein the first co-solvent is acetic acid, formic acid, gluconic acid, propionic acid, α-hydroxy acid and / or β-hydroxy acid, preferably lactic acid.

4. The method of any one of claims 1 to 3, wherein in step (b), the protein solution is heated to a first temperature above the sol-gel transition temperature of the one or more plant-based protein solutions and then lowered to a second temperature below the sol-gel transition temperature of the one or more plant-based protein solutions to form a hydrogel.

5. The method according to any one of claims 1 to 4, wherein in step (b), the protein suspension is subjected to a shearing treatment comprising a shearing step involving further reducing the size of the protein particles, preferably wherein the particle size d by volume as determined by dynamic laser diffraction is reduced to 0. 50 Reduced to a range of 0.1 to 15 microns, preferably 0.2 to 10 microns, preferably 0.5 to 5 microns.

6. The method according to any one of claims 1 to 5, wherein the step of changing the pH of the plant-based protein suspension is performed after step (b) or after step (c).

7. The process according to any one of claims 1 to 6, wherein the composition formed in step (c) has a protein solids content in the range of 1 wt% to 25 wt%, preferably 2 wt% to 20 wt%, more preferably 3 wt% to 15 wt%, even more preferably 4 wt% to 12 wt%, based on the total weight of the composition.

8. The method according to any one of claims 1 to 7, wherein the agrochemical is selected from the group consisting of pesticides, including fungicides, herbicides, insecticides, algaecides, molluscicides, acaricides and rodenticides, and antimicrobials, including microbicides, antibiotics, antibacterials, antivirals, antifungals, antiprotozoals and antiparasitics, or a combination thereof.

9. The method according to any one of claims 1 to 8, wherein the agrochemical is dispersed in a carrier phase.

10. The method according to any one of claims 1 to 9, further comprising subjecting the microcapsules containing agricultural chemicals to a post-treatment step, preferably wherein the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step or a coating formation step.

11. The method of claim 10, wherein the non-covalent cross-linking step comprises treating the microcapsules containing the agricultural chemical with a non-covalent cross-linking agent selected from the group consisting of sodium tripolyphosphate, sodium hexametaphosphate, and phenolic compounds.

12. The method of claim 10, wherein the covalent cross-linking step comprises treating the microcapsules containing the agrochemical with a covalent cross-linking agent selected from the group consisting of genipin, epoxy compounds, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and polyamide-based cross-linking resins, or combinations thereof.

13. The method according to claim 10, wherein the coating forming step comprises: (i) subjecting the microcapsules containing the agrochemical to a complex coacervation step using a polysaccharide; and / or (ii) treating the microcapsules containing the agricultural chemicals with an aqueous mineral solution.

14. Biodegradable microcapsules containing an agrochemical, obtained or obtainable by the method according to any one of claims 1 to 13.

15. A method for preparing a biodegradable microcapsule composition, the method comprising: (a) preparing biodegradable microcapsules containing agricultural chemicals according to the method according to any one of claims 1 to 13; as well as (b) suspending the biodegradable microcapsules containing the agricultural chemicals in an external aqueous phase.

16. A biodegradable microcapsule composition obtained or obtainable by the method of claim 15.

17. Biodegradable microcapsules comprising an agrochemical and one or more plant-based proteins encapsulating the agrochemical, wherein the plant-based protein has a solubility of less than 20%, preferably less than 10%, when measured at a protein concentration of 5% w / w in water at 20°C and pH 7.

18. The biodegradable microcapsule according to claim 17, wherein after incubation in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 at 20°C for 10 days, at least 25%, more preferably at least 40%, even more preferably at least 50%, most preferably at least 60% of the initially encapsulated agrochemical remains present inside the microcapsule as determined by GC.

19. The biodegradable microcapsules according to claim 17 or claim 18, wherein at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% of the initially encapsulated agrochemical is released from the microcapsules after incubation at 20°C in the dark for 14 days in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 and in the presence of 5.76% of Streptomyces griseus protease in 10 mM NaOAc + 5 mM CaCl2 relative to the mass of the microcapsules, while at least 20%, preferably at least 30%, more preferably at least 40%, and most preferably at least 50% of the initially encapsulated agrochemical is released from the microcapsules in phosphate buffered saline (PBS) comprising 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 and in the presence of 5.76% of Streptomyces griseus protease in 10 mM NaOAc + 5 mM CaCl2 relative to the mass of the microcapsules. After incubation in CaCl2 at 20°C in the dark for 14 days, less than 50%, preferably less than 40%, more preferably less than 30%, most preferably less than 20% of the initially encapsulated agrochemical is released from the microcapsules as determined by GC.

20. The biodegradable microcapsule according to any one of claims 17 to 19, wherein the percentage of biodegradation of the plant-based protein based on CO2 evolution after 28 days, as measured according to ISO 17556:2019, is from 40% to 100%, more preferably from 50% to 100%, even more preferably from 60% to 100%.

21. The biodegradable microcapsule according to any one of claims 17 to 20, wherein the one or more plant-based proteins encapsulating the agricultural chemical are selected from pea protein, potato protein, soy protein, rapeseed protein, lentil protein, chickpea protein, faba bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, preferably wherein the one or more plant-based proteins encapsulating the agricultural chemical are pea protein and / or potato protein.

22. The biodegradable microcapsule according to any one of claims 17 to 21, wherein the one or more plant-based proteins encapsulating the agricultural chemical have a protein secondary structure having at least 40% intermolecular β-sheets, at least 50% intermolecular β-sheets, at least 60% intermolecular β-sheets, at least 70% intermolecular β-sheets, at least 80% intermolecular β-sheets, or at least 90% intermolecular β-sheets, wherein the intermolecular β-sheet content percentage is measured by FTIR.

23. The biodegradable microcapsule according to any one of claims 17 to 22, wherein the one or more plant-based proteins encapsulating the agricultural chemical are selected from pea protein and soy protein, and wherein the agricultural chemical is selected from essential oils and components of essential oils.

24. The biodegradable microcapsule according to any one of claims 17 to 23, wherein the agricultural chemical is dispersed in a carrier phase.

25. The biodegradable microcapsule according to any one of claims 17 to 24, wherein the microcapsule has a d value of less than or equal to 500 μm, less than or equal to 250 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm as determined by laser diffraction. 90 diameter.

26. The biodegradable microcapsule according to any one of claims 17 to 25, wherein the plant-based protein encapsulating the agricultural chemical has been non-covalently modified by a non-covalent cross-linking agent, or the plant-based protein has been covalently modified by a covalent cross-linking agent, or the plant-based protein has a coating deposited thereon.

27. A biodegradable microcapsule composition comprising the biodegradable microcapsule according to any one of claims 17 to 26 and an external phase.

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