Biodegradable microcapsule and preparation method thereof

Abstract

The invention relates to a method for preparing microcapsules and a method for preparing microcapsule compositions. The invention also relates to microcapsules and microcapsule compositions themselves. The invention also relates to the use of the microcapsules and to methods involving the microcapsules, including the preparation of the formulation. The invention also relates to the formulation product itself.

Description

Field of the Invention

[0001] The present invention relates to a method for preparing biodegradable microcapsules and a method for preparing a biodegradable microcapsule composition. The present invention also relates to the biodegradable microcapsules and the biodegradable microcapsule composition itself. The present invention also relates to the use of the biodegradable microcapsules and to a method for preparing the biodegradable microcapsules, including preparing a formulated product. The present invention also relates to the formulated product itself.

[0002] background

[0003] The encapsulation technology related to embedding active ingredients in the external matrix can be used to protect the active ingredients from the impact of its external environment (e.g., exposure to chemicals, air, light, etc.), and protect the external environment from the impact of the active ingredient itself, such as when it is dangerous to handle. For example, when the active ingredient is added to a product formulation (e.g., beverage formulations, fabric conditioning formulations, etc.), this protects the active ingredient, meaning that the active ingredient in the product formulation has an acceptable shelf life and / or will not be activated prematurely. This also means that the external phase of the product formulation will not be negatively affected by the active ingredient. Then, the active ingredient is released when needed (e.g., when the capsule is broken or the capsule is enzymatically degraded).

[0004] Many available encapsulation technologies often rely on the use of synthetic polymers to form a protective shell around the active ingredient. However, synthetic polymers may not be suitable as carriers in drug and food applications or may have limited permitted exposure levels in cosmetic applications. In addition, synthetic polymer shell materials can lead to the formation of microplastics, which are harmful to the environment.

[0005] Spray drying technology allows large-scale preparation of microcapsules, and spray drying of vegetable proteins has been attempted in an attempt to achieve the production of biodegradable microcapsules. However, in order to obtain robust microcapsules with sufficiently small sizes by spray drying technology, it is necessary to spray dried materials with low viscosity (e.g., materials need to be shear-thinned to sufficiently low viscosity to successfully spray dry) at high shear rates, and also with sufficiently high protein solid content. The conventional method for preparing plant protein-based materials suitable for spray drying involves hydrolyzing proteins to decompose them into relatively low molecular weight fragments (e.g., by treating with acid or alkali or enzymes). However, the resulting spray-dried microcapsules are structurally unstable in water or under acidic or alkaline conditions, under elevated temperatures and / or under high shear forces, which means that they are not suitable for being incorporated into product formulations by conventional manufacturing methods, especially in liquid product formulations. In addition, since many oils are unstable under alkaline conditions, the process involved may have a negative impact on the encapsulated material.

[0006] Therefore, there is a need for a method of preparing microcapsules containing active ingredients that are small enough to be incorporated into liquid product formulations, where the microcapsules are biodegradable and stable (i.e., the microcapsules maintain their structural integrity under acidic or alkaline conditions so that the active ingredient is protected during manufacturing and over the shelf life of the product). SUMMARY OF THE INVENTION

[0008] From a first aspect, the present invention provides a method for preparing biodegradable microcapsules, the method comprising:

[0009] (a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the plant-based protein and a second co-solvent decreases the solubility of the plant-based protein;

[0010] (b) inducing the plant-based protein in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel;

[0011] (c) subjecting the plant-based protein hydrogel to a shearing treatment to form a plant-based protein hydrogel slurry;

[0012] (d) dispersing an active ingredient in the plant-based hydrogel slurry to form a composition; and

[0013] (e) drying the composition to form microcapsules.

[0014] Viewed from another aspect, the present invention provides a biodegradable microcapsule obtained or obtainable by the method as hereinbefore described.

[0015] From another aspect, the present invention provides a method for preparing a biodegradable microcapsule composition, the method comprising:

[0016] (a) preparing biodegradable microcapsules according to the method described above; and

[0017] (b) suspending biodegradable microcapsules in the external phase.

[0018] Viewed from another aspect, the present invention provides a biodegradable microcapsule composition obtained or obtainable by the method as hereinbefore described.

[0019] Viewed from another aspect, the present invention provides a biodegradable microcapsule comprising an active ingredient and a plant-based protein carrier comprising a plant-based protein, wherein the plant-based protein carrier encapsulates the active ingredient and wherein the plant-based protein has a solubility of less than 50% when measured at a protein concentration of 5% w / w in an aqueous solution at 25° C. and pH 7.

[0020] Viewed from another aspect, the invention provides a composition comprising the biodegradable microcapsules as hereinbefore described and an external phase.

[0021] Viewed from another aspect, the present invention provides a formulation comprising the biodegradable microcapsules as hereinbefore described.

[0022] Viewed from another aspect, the present invention provides a method for manufacturing a formulated product, the method comprising:

[0023] (a) preparing biodegradable microcapsules according to the method described above; and

[0024] (b) mixing the biodegradable microcapsules with a product formulation.

[0025] Viewed from another aspect, the invention provides the use of the biodegradable microcapsules hereinbefore described in a formulated product.

[0026] definition

[0027] The term "microcapsule" as used herein refers to microparticles in any form. For example, the term encompasses core-shell microcapsules (i.e., microcapsules having a central core containing an active ingredient, wherein the core is surrounded by a plant-based protein hydrogel in the form of a shell). It will be appreciated by those skilled in the art that core-shell microcapsules may have a multi-core morphology (i.e., wherein the core phase is in the form of multiple droplets) or a mono-core morphology (wherein the core phase is in the form of a single droplet). The term also encompasses matrix microcapsules (i.e., microcapsules comprising a plant-based protein hydrogel matrix, wherein the active ingredient is dispersed throughout the matrix). The microcapsules may have a d of 500 nm to 2 mm as determined by laser diffraction. 50 .

[0028] As used herein, the term "lower shear step" may refer to a processing step in which a low level of mechanical energy is applied to a material (preferably by a cutting action) to cause it to primarily break or fragment into large discrete pieces. "Lower shear" generally does not include any treatment step in which a material is subjected to high-speed impact (e.g., at speeds greater than 2 ms). -1The method further comprises a grinding step of crushing or breaking the material by impacting with a differential speed of 0.1% to 0.2% (or 0.1% to 0.2%). It also generally does not include a grinding process based on cavitation. In a specific embodiment, during the lower shear step, the hydrogel is broken to provide fragments such that at least 80% by weight of the hydrogel fragments have a maximum dimension of 1 mm to 100 mm as determined by optical microscopy.

[0029] As used herein, the term "higher shear step" may refer to a treatment step in which energy is applied to reduce the hydrogel into small fragments (e.g., to form a colloidal dispersion). In a particular embodiment, during the higher shear step, the hydrogel is fragmented to provide a hydrogel having a d of 0.5 to 50 microns as determined by laser diffraction. 50 Laser diffraction may be performed as defined herein.

[0030] For the avoidance of doubt, a higher shear step subjects the hydrogel 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).

[0031] 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.

[0032] As used herein, the term "fragrance" (which can be used interchangeably with the term "spice") refers to a component in a formula that can impart or change the smell of a product, such as a fabric conditioner, a hair conditioner, or a substrate such as a fabric or hair. Fragrances are generally used to impart a generally pleasant smell or odor profile to a product to provide a pleasant experience (e.g., a high-end perfume), or to provide sensory cues about the benefits and functions of a product (e.g., the calming effect of a lavender sleep aid, the clean concept of a laundry product), or to mask unpleasant odors (e.g., in an insect repellent product). "Fragrance" may include one or more components, which may be a single chemical entity (referred to herein as a "fragrance material" (which can be used interchangeably with the term "spice material")), or a mixture of different "fragrance materials". Fragrance materials may be produced by a synthetic process, or may be extracted from nature, especially from plants, to obtain naturally occurring plant essential oils and plant extracts, such as orange oil. The fragrance material produced by a synthetic process may be a newly-emerged chemical substance or a fragrance material identical to nature. The fragrance substances of synthetic and natural origin can then be mixed into fragrances by a skilled perfumer (also referred to as a "nose") for use in consumer products. Fragrance substances can be obtained from professional fragrance suppliers (called fragrance houses) as separate chemicals, natural mixtures, or proprietary special mixtures that do not disclose their complete composition. The individual fragrance substances constituting known natural mixtures can be found by reference to magazines commonly used by those skilled in the art (e.g., "Perfume and Flavorist" or "Journal of Essential Oil Research"), or in reference texts such as S. Arctander's books (Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA; and recently reprinted by Allured Publishing Corporation Illinois (1994)) and "Perfume and Flavour Materials of Natural Origin", S. Arctander, Ed., Elizabeth, NJ, listed in 1960. It should be understood that for the purposes of the present invention, "fragrance substances" include pro-fragrances, such as acetal pro-fragrances, ketal pro-fragrances, ester pro-fragrances, hydrolyzable inorganic-organic pro-fragrances, and combinations thereof.The aroma substances can be released from the aroma precursors in various ways, for example by hydrolysis, or by a shift in equilibrium, or by a pH change, or by enzymatic release, or by UV irradiation.

[0033] Fragrances can be described in terms of their odor intensity, detection threshold, odor saturation, and their characteristics. In fragrance encapsulation, it is preferred to use fragrances with low odor detection thresholds and high intensity to maximize the noticeability of even small levels of fragrance encapsulated and released.

[0034] In order to impart an odor, a fragrance substance must be volatile, even if only to a small degree, because the molecules must travel through the air into the nose, where they attach to specific nerve receptors and trigger signals within the olfactory system. Fragrance substances can be classified according to their volatility. Preferably, fragrance substances are liquid at 20°C and atmospheric pressure, but occasionally they can be solid and can be mixed with other liquid fragrance substances or solvents. Typically, the fragrance industry refers to the volatility and substantiality of substances by roughly classifying them into the following three categories: substances with the lowest volatility and highest substantiality are classified as base notes, substances with medium volatility and substantiality are classified as middle notes, and substances with the highest volatility and lowest substantiality are classified as top notes. This is based on the odor perception of the substance and is quite subjective. One way to objectively classify the volatility of fragrance substances is to classify them according to their vapor pressure.

[0035] As used herein, the term "vapor pressure" means the partial pressure of a given chemical in the air at a defined temperature (e.g., 25°C) and standard atmospheric pressure (760 mmHg). It defines the affinity of a chemical species for the gas phase rather than the liquid or solid state. The higher the vapor pressure, the higher the proportion of the substance in the closed head space when equilibrium is reached. It is also related to the evaporation rate of the aroma substance, which is defined in an open environment when the substance leaves the system. Vapor pressure can be easily determined according to the reference program ACD / Percepta Desktop Software Version 14.0 (built on August 26, 2021) (Advanced Chemistry Development, Inc (ACD / Labs), Toronto, Canada, www.acdlabs.com).

[0036] A physical parameter related to the encapsulation of fragrance substances is their hydrophobicity, which can be defined in terms of their partition coefficient P. The term "partition coefficient" as used herein refers to the ratio of the equilibrium concentrations of a substance in n-octanol and water, and is a measure of the difference in solubility of the substance between these two solvents. The term "logP" as used herein refers to the logarithm to the base 10 of the partition coefficient P. logP can be easily determined according to the reference program ACD / Percepta Desktop Software Version 14.0 (built on August 26, 2021) (Advanced Chemistry Development, Inc (ACD / Labs), Toronto, Canada, www.acdlabs.com). The logP value is predicted from the SMILE string of the fragrance molecule. Three different types of logP values ​​can be selected from the software. logP Classic is based on an algorithm that takes into account a database of experimental logP values ​​while using the principle of separated carbon. logP GALAS is based on an algorithm that takes into account a database of compound training sets and adjusts the values ​​using data from structurally similar compounds. The consensus logP is a model based on the first two algorithms and can be expressed as: consensus logP = a × logP Classic + b × logP GALAS, where a and b are the coefficients of the model. The latter value (consensus logP) is the logP value indicated in this article.

[0037] Another aspect related to the encapsulation of aroma substances is their Hansen Solubility Parameters (HSP). The term HSP refers to a solubility parameter method proposed by Charles Hansen, which was originally used to predict the solubility of polymers in a given solvent, as described in Charles Hansen's The Three Dimensional Solubility Parameter and Solvent Diffusion Coefficient, Danish Technical Press (Copenhagen, 1967). This method was later reapplied to many other molecules. Aromatic substances (or flavor substances or solvents) and their interactions with the environment are defined by three forces: atomic dispersion forces, molecular permanent dipole forces, and molecular hydrogen bonding forces. Substances with similar HSP parameters are more likely to be miscible. These forces can be quantified by 3 values: δ D , the Hansen dispersion value associated with van der Waals interactions (intermolecular forces); δ Ρ , the Hansen polarity value associated with the dipole moment (charge); and δ Η , Hansen hydrogen bond ("h bond") value. Solubility parameter δ (MPa1 / 2 ) is defined as δ 2 =δ D 2 +δ P 2 +δ H 2 =E / V, where E is the cohesive energy of the solvent and V is the molar volume. The HSP value for a given substance can be obtained from the HSPiP (Hansen Solubility Parameters in Practice) software available at www.hansen-solubility.com in two main different ways. These values ​​can be obtained by retrieving from a master data set containing more than 20,000 substances by name or CAS number; or by entering the SMILE string of a given molecule in the DIY part of the software and predicting using the Y-MB (Yamamoto-Molecular Breaking) method. In addition, determining the HSP sphere relative to a given fragrance substance is a good way to predict solubility preferences in a mixture of fragrance substances. The radius Ro of the HSP sphere is defined as Ro=Ra / RED, where Ra is the HSP distance between two molecules (1 and 2), expressed as: Ra 2 =4(δ D1 -δ D2 ) 2 +(δ P1 -δ P2 ) 2 +(δ H1 -δ H2 ) 2 , and RED is the relative energy difference. The RED value can also be extracted from or predicted by the HSPiP software, and a good solvent for a given substance should exhibit a RED value less than or equal to 1, while a solvent exhibiting a RED value greater than 1 should be considered a poor solvent for the given substance.

[0038] The aroma substance may be selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds and Schiff bases.

[0039] Preferred aldehyde aroma substances include, but are not limited to, α-amylcinnamaldehyde, anisealdehyde, decanal, lauryl aldehyde, methyl nonyl acetaldehyde, methyl octyl acetaldehyde, nonanal, benzaldehyde, nerol, geranal, 1,1-diethoxy-3,7-dimethyloct-2,6-diene, 4-isopropylbenzaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, α-methyl-p-isopropyldihydrocinnamaldehyde, 3-(3-isopropylphenyl)butyraldehyde, α-hexylcinnamaldehyde, 7-hydroxy-3,7-dimethyloct-1-aldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, octanal, phenylacetaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, hexanal, 3,7-dimethyloctanal, 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-butanal aldehyde, nonanal, octanal, 2-nonenal undecenal, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexenyl-1)-2-butenal, 2,6-dimethyloctanal, 3-(p-isopropylphenyl)propanal, 3-phenyl-4-pentenal citronellal, o- / p-ethyl-α,α,9-decenal, dimethyldihydrocinnamaldehyde, p-isobutyl-α-methylhydrocinnamaldehyde, cis-4-decen-1-al, 2,5-dimethyl-2-vinyl-4-hexenal, trans-2-methyl-2-butenal, 3-methylnonanal, α-sweet orange aldehyde, 3-phenylbutyraldehyde, 2,2-dimethyl-3-phenylpropanal, m-tert-butyl-α-methyldihydrocinnamaldehyde, geranyloxyacetaldehyde, trans-4-decen-1-al, methoxycitronellal, and mixtures thereof.

[0040] Preferred ester fragrance substances include, but are not limited to, allyl cyclohexylpropionate, allyl heptanoate, allyl isopentyl acetate (allyl amyl glycolate), allyl hexanoate, amyl acetate (n-amyl acetate), amyl propionate, benzyl acetate, benzyl propionate, benzyl salicylate, cis-3-hexenyl acetate, citronellyl acetate, citronellyl propionate, cyclohexyl salicylate, dihydroisojasmonate, dimethylbenzyl methanol acetate, ethyl acetate, ethyl acetoacetate, ethyl butyrate, ethyl 2-methylbutyrate, ethyl 2-methylvalerate, fenchyl acetate (1,3,3-trimethyl-2-norbornyl acetate), tricyclodecenyl acetate, tricyclodecenyl propionate, geranyl acetate, cis-3-hexenyl isobutyrate, hexyl acetate, cis-3-hexenyl salicylate, n-hexyl salicylate, isobornyl acetate, linalyl acetate, p-tert-butylcyclohexyl acetate, (-)-L-menthyl acetate, o-tert-butylcyclohexyl acetate, methyl benzoate, methyl dihydroisojasmonate, α-methylbenzyl acetate, methyl salicylate, 2-phenylethyl acetate, prenyl acetate, cedryl acetate, cyclabute isobutyrate, phenylethyl phenylacetate, pinyl formate, citronellyl anthranilate, tricyclo[5.2.1.0-2,6] decane-2-carboxylic acid ethyl ester, n-hexyl ethyl acetoacetate, 2-tert-butyl-4-methylcyclohexyl acetate, formic acid, 3,5,5-trimethylhexyl ester, phenylethyl crotonate, cyclogeranyl acetate, geranyl crotonate, ethyl geranate, geranyl isobutyrate, 3,7-dimethyl-ethyl 2-nonynoic acid-2,6-octadienoic acid methyl ester, citronellyl valerate, 2-hexenylcyclopentanone, cyclohexyl anthranilate, L-citronellyl tiglic acid, butyl tiglic acid, amyl tiglic acid, geranyl octanoate, 9-decenyl acetate, 2-isopropyl-5-methylhexyl-1-butyrate, n-pentyl benzoate, 2-methylbutyl benzoate (and its mixture with amyl benzoate), dimethyl benzyl carbinyl propionate, dimethyl benzyl acetate carbinyl acetate), trans-2-hexenyl salicylate, dimethyl benzyl carbinyl isobutyrate, 3,7-dimethyloctyl formate, rose formate, rose isovalerate, rose acetate, rose butyrate, rose propionate, cyclohexyl ethyl acetate, neryl butyrate, tetrahydrogeranyl butyrate, myrcenyl acetate, 2,5-dimethyl-2-vinylhex-4-enoic acid methyl ester, 2,4-dimethylcyclohexane-1-methyl acetate, ocimenyl acetate acetate), linalyl isobutyrate, 6-methyl-5-heptenyl-1 acetate, 4-methyl-2-pentyl acetate, 2-methylbutyric acid n-pentyl ester, propyl acetate, isopropenyl acetate, isopropyl acetate, 1-methylcyclohex-3-ene-carboxylic acid methyl ester, propyl tiglate, propyl / isobutylcyclopent-3-enyl-1-acetate (α vinyl), butyl 2-furoate, ethyl 2-pentenoate, (E)-3-pentenoate methyl ester, 3-methoxy-3-methylbutyl acetate, n-pentyl crotonate, n-pentyl isobutyrate, propyl formate, furfuryl butyrate, methyl angelate, new Methyl valerate, isopentenyl hexanoate, furfuryl propionate, diethyl malate, isopropyl 2-methylbutyrate, dimethyl malonate, bornyl formate, styryl acetate, 1-(2-furyl)-1-propanone, l-citronellyl acetate, 3,7-dimethyl-1,6-nonadien-3-yl acetate, neryl crotonate, dihydromyrcenyl acetate, tetrahydromyrcenyl acetate, lavandulyl acetate, 4-cyclooctenyl isobutyrate, cyclopentyl isobutyrate, 3-methyl-3-butenyl acetate, allyl acetate, geranyl formate, cis-3-hexenyl hexanoate and mixtures thereof.

[0041] Preferred alcohol flavoring substances include, but are not limited to, benzyl alcohol, β-γ-hexenol (2-hexen-1-ol), cedarol, citronellol, cinnamyl alcohol, p-cresol, cuminol, dihydromyrcenol, 3,7-dimethyl-1-octanol, dimethylbenzyl alcohol, eucalyptol, eugenol, fenchol, geraniol, hydratopic alcohol, chrysanthemum alcohol, chrysanthemum oleoresin ... alcohol), isononanol (3,5,5-trimethyl-1-hexanol), linalool, methyl chavicol (artemisinin), methyl eugenol (eugenol methyl ether), nerol, 2-octanol, patchouli alcohol, phenylhexanol (3-methyl-5-phenyl-1-pentanol), phenylethyl alcohol, α-terpineol, tetrahydrolinalool, tetrahydromyrcenol, 4-methyl-3-decen-5-ol, l-3,7-dimethyloctan-1-ol, 2-(furfuryl-2)-heptanol, 6,8-dimethyl-2-nonanol, ethyl norbornyl cyclohexanol, β-methylcyclohexaneethanol, 3,7-dimethyl-(2),6-octenyl (diene)-1-ol, trans-2-undecen-1-ol, 2-ethyl-2-isopentenyl-3-hexenol, isobutyl benzyl carbinol, dimethyl benzyl carbinol carbinol), ocimene alcohol, 3,7-dimethyl-1,6-nonadien-3-ol (cis and trans), tetrahydromyrcenol, α-terpineol, 9-decenol-1,2-(2-hexenyl)-cyclopentanol, 2,6-dimethyl-2-heptanol, 3-methyl-1-octen-3-ol, 2,6-dimethyl-5-hepten-2-ol, 3,7,9-trimethyl-1,6-decadien-3-ol, 3,7-dimethyl-6-nonen- 1-ol, 3,7-dimethyl-1-octyne-3-ol, 2,6-dimethyl-1,5,7-octatrienol-3, dihydromyrcenol, 2,6-trimethyl-5,9-undecadienol, 2,5-dimethyl-2-propylhex-4-enol-1, (Z)-3-hexenol, o-, m-, p-methylphenylethanol, 2-methyl-5-phenyl-1-pentanol, 3-methylphenylethanol, para-methyl dimethyl benzyl carbinol, methyl benzylcarbinol, p-methylphenylethanol, 3,7-dimethyl-2-octen-1-ol, 2-methyl-6-methylene-7-octen-4-ol, and mixtures thereof.

[0042] Preferred ketone aroma substances include, but are not limited to, oxahedralidone, benzyl acetone, benzophenone, L-carvone, cis-jasmone, 4-(2,6,6-trimethyl-3-cyclohexen-1-yl)-but-3-en-4-one, ethyl amyl ketone, α-ionone, ionone β, ethyl ketone, octahydro-2,3,8,8-tetramethyl-2-acetanaphthalene, α-irone, 1-(5,5-dimethyl-1-cyclo-hexen-1-yl)-4-penten-1-one, 3-nonanone, ethyl hexyl ketone, menthone, 4-methylacetophenone, γ-methyl ionone, methyl amyl ketone, methyl heptenone (6-methyl-5-hepten-2-one), methyl heptenone, methyl hexyl ketone, δ- Muskenone, 2-octanone, 2-pentyl-3-methyl-2-cyclopenten-1-one, 2-heptylcyclopentanone, α-methylionone, 3-methyl-2-(trans-2-pentenyl)-cyclopentenone, octenylcyclopentanone, n-pentylcyclopentenone, 6-hydroxy-3,7-dimethyloctanoic acid lactone, 2-hydroxy-2-cyclohexen-1-one, 3-methyl-4-phenyl-3-butene-2-one, 2-pentyl-2,5,5-trimethylcyclopentanone, 2-cyclopentylcyclopentanol-1,5-methylhexane-2-one, γ-dodecalactone, δ-dodecalactone, γ-nonalactone, δ-nonalactone, γ-octyl lactone, δ-undecalactone, γ-undecalactone, alpha damascena damascone), beta damascone, gamma damascone, delta damascone, and mixtures thereof.

[0043] Preferred ether fragrance substances include, but are not limited to, diphenyl ether, p-tolyl methyl ether, 4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydro-cyclopenta(G)-2-benzopyran, β-naphthyl methyl ether, methyl isobutenyl tetrahydropyran, 5-acetyl-1,1,2,3,3,6-hexamethylindane (phantolide), 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene (tonalid), 2-phenethyl-3-methylbut-2-enyl ether, ethylgeranyl ether, phenethyl isopropyl ether, and mixtures thereof.

[0044] Preferred olefin flavoring substances include, but are not limited to, alloocimene, camphene, β-caryophyllene, cadinene, diphenylmethane, d-limonene, limonene, β-myrcene, p-cymene, 2-α-pinene, β-pinene, α-terpinene, γ-terpinene, terpinolene, 7-methyl-3-methylene-1,6-octadiene, and mixtures thereof.

[0045] Preferred nitrile aroma substances include, but are not limited to, 3,7-dimethyl-6-octenonitrile, 3,7-dimethyl-2(3),6-nonadienenitrile, (2E,6Z)-2,6-nonadienenitrile, n-dodecanenitrile, and mixtures thereof.

[0046] Preferred Schiff base aroma materials include, but are not limited to, citronellal nitrile, nonanal / methyl anthranilate, N-octylene-methyl anthranilate, hydroxycitronellal / methyl anthranilate, cyclamen aldehyde / methyl anthranilate, methoxyphenylpropionaldehyde / methyl anthranilate, ethyl p-aminobenzoate / hydroxycitronellal, citral / methyl anthranilate, 2,4-dimethylcyclohex-3-enecarboxaldehyde methyl anthranilate, hydroxycitronellal indole, and mixtures thereof.

[0047] As used herein, the term "flavoring agent" refers to a component in a formula that can impart or change the taste and smell of a product (such as toothpaste or food). Flavoring agents are generally used to impart a generally pleasant taste and smell, or taste and smell characteristics to a product, to simply provide a pleasant experience (such as in food), or to cover up an unpleasant taste or smell (such as in medicine). Flavoring agents or flavoring substances can be described according to their odor intensity, detection threshold, and quality. "Flavoring agents" can include one or more components, which can be a single chemical entity (referred to herein as "flavoring substances"), or a mixture of different "flavoring substances". Flavoring substances can be produced by synthetic processes, or can be extracted from nature, especially from plants, to produce naturally occurring vegetable and animal oils and secretions, such as vanilla extract. Flavoring substances of synthetic and natural origin can then be mixed into flavoring agents by skilled flavorists for use in consumer products. Flavoring substances can be obtained from professional flavoring suppliers (referred to as flavoring houses) as separate chemicals, natural mixtures, or proprietary special mixtures that do not disclose their complete composition. The individual flavoring substances that make up the known natural mixtures can be found by reference to magazines commonly used by those skilled in the art (e.g., "Perfume and Flavorist" or "Journal of Essential Oil Research"), or in reference texts such as books by S. Arctander (Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA; and recently reprinted by Allured Publishing Corporation Illinois (1994)); "Perfume and Flavour Materials of Natural Origin", S. Arctander, Ed., Elizabeth, NJ, 1960; and "Flavourings", E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998. It should be understood that flavoring agents can be volatile or have volatile components that can be detected by the nose in the same manner as fragrance agents. Thus, flavoring substances can also be classified according to their physical properties (e.g., volatility and hydrophobicity) using the methods described above for fragrance substances. Flavour substances can also be described in terms of their Hansen Solubility Parameters using the methods described above for aroma substances.

[0048] The sources of flavor substances include essential oils, concretes, absolutes, resins, resinous substances, balsams and tinctures. Preferred flavor substances include anise oil, ethyl-2-methylbutyrate, vanillin, cis-3-heptenol, cis-3-hexenol, trans-2-heptenal, butyl valerate, 2,3-diethylpyrazine, methylcyclopentenolone, benzaldehyde, valerian oil, 3,4-dimethoxyphenol, amyl acetate, amyl cinnamate, γ-butyrolactone, trimethylpyrazine, phenylacetic acid, isovaleraldehyde, ethyl maltol, ethyl vanillin, ethyl valerate, ethyl butyrate, cocoa extract, coffee extract, peppermint oil, spearmint oil, clove oil, Anethole, cardamom oil, wintergreen oil, cinnamaldehyde, ethyl-2-methylvalerate, γ-hexenolactone, 2,4-decadienal, 2,4-heptadienal, methylthiazolol (4-methyl-5-b-hydroxyethylthiazole), 2-methylbutanethiol, 4-mercapto-2-butanone, 3-mercapto-2-pentanone, 1-mercapto-2-propane, benzaldehyde, furfural, furfuryl alcohol, 2-mercaptopropionic acid, alkylpyrazine, methylpyrazine, 2-ethyl-3-methylpyrazine, tetramethylpyrazine, polysulfide, dipropyl disulfide, methylbenzyl disulfide, alkylthiophene, 2,3-dimethylthiophene, 5-methylfurfural, acetylfuran, 2,4-decadienal, guaiacol, phenylacetaldehyde, b-decanoic acid, d-limonene, acetoin, amyl acetate, maltol, ethyl butyrate, levulinic acid, piperonal, ethyl acetate, n-octanal, n-valeraldehyde, n-hexanal, diacetyl, monosodium glutamate, monopotassium glutamate, sulfur-containing amino acids (such as cysteine), 2-methylfuran-3-thiol, 2-methyldihydrofuran-3-thiol, 2,5-dimethylfuran- 3-thiol, tetramethylpyrazine, propyl propenyl disulfide, propyl propenyl trisulfide, diallyl disulfide, diallyl trisulfide, diallyl disulfide, diallyl trisulfide, 4-methyl-2-[(methylthio)-ethyl]-1,3-dithiolane, 4,5-dimethyl-2-(methylthiomethyl)-1,3-dithiolane, and 4-methyl-2-(methylthiomethyl)-1,3-dithiolane, hop oil, and citrus oils (e.g., lemon oil, orange oil, lime oil, and grapefruit oil). DETAILED DESCRIPTION OF THE INVENTION

[0050] The inventors of the present invention have discovered a method for preparing a high solids content protein colloidal dispersion containing an active ingredient, the dispersion having a controlled particle size and low viscosity, allowing stable microcapsules to form upon drying. The resulting microcapsules are stable in liquid product formulations and are also stable under the conditions necessary to prepare the liquid product formulations (e.g., pasteurization conditions in the case of beverage formulations).

[0051] The present invention relates to a method for preparing biodegradable microcapsules, the method comprising:

[0052] (a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the plant-based protein and a second co-solvent decreases the solubility of the plant-based protein;

[0053] (b) inducing the plant-based protein in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel;

[0054] (c) subjecting the plant-based protein hydrogel to a shearing treatment to form a plant-based protein hydrogel slurry;

[0055] (d) dispersing an active ingredient in the plant-based hydrogel slurry to form a composition; and

[0056] (e) drying the composition to form microcapsules.

[0057] Any suitable plant-based protein can be used in the present invention. In a preferred method of the present invention, the plant-based protein is obtained from broad beans, mung beans, peas, rice, potatoes, rapeseed, lentils, chickpeas, sunflower seeds, pumpkin seeds, flax, chia, canola, lupins, alfalfa, moringa, wheat, corn zein or sorghum; preferably, the plant protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, broad 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, the plant-based protein is pea protein and / or potato protein. Such proteins are considered to be hypoallergenic proteins.

[0058] Suitable plant-based proteins also include:

[0059] - Brassica: includes Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongata: long mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St Hilarion cabbage, Brassica juncea: Indian mustard, brown mustard and leaf mustard, Sarepta mustard, Brassica napus: rapeseed, canola, rutabaga, Brassica narinosa: broadbeaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, kale, 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;

[0060] -Solanaceae: includes tomatoes, potatoes, eggplants, bell peppers and hot peppers;

[0061] - Cereals: including corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and fonio;

[0062] - Pseudocereals: including amaranth (love-lies-bleeding, red amaranth, prince-of-Wales-feather), breadnut, buckwheat, chia, cockscomb (also called quail grass or soko), pitseed goosefoot, quinoa Quinoa and wattleseed (also called acacia seed);

[0063] -Fabaceae: including Acacia alata (winged acacia), Acacia decipiens, Acacia saligna (which has many common names,These include coojong, golden wreath wattle, orange wattle, blue-leaved wattle), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus laburnum (common names include chain flower, golden chain or golden rain), Cytisus supinus, Dolichios lablab (common names include magpie bean, lablab-bean bonavist bean / pea, cow 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 (lathyrus clymenum), Lathyrus odoratus)(Lathyrus staivus)(Lathyrus staivus)(Lathyrus silvetris ... sativum (pea), Trifolium hybridum (clover), Trifolium pretense (red clover), Vicia faba (broad bean), Vicia sativa (wild pea), Vigna unguiculate (cowpea);

[0064] - Non-leguminous plants: including: 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), Brassicahirta (Eurasian mustard), Citrullus vulgaris (Watermelon), Citrus aurantiaca (Citrus), Cucurbita maxima (Squash), Buckwheat (Fagopyrum esculentum)(knotweed), Gossypium barbadense)(extra long staple cotton), Heianthus annuus)(sunflower), Nicotiana sp.)(tobacco plant), Prunus avium)(cherry), Prunus cerasus)(sour cherry), Prunus domestica)(plum), Prunus amygdalus)(almond), Ricinus communis)(castor bean / castor oil plant), Sasamumindicum)(sesame), Sinapis alba)(white mustard), Terlfalrea pedata(pomace fruit).

[0065] For the avoidance of doubt, the plant-based microcapsules of the present invention do not comprise plants in their natural state, for example, naturally occurring plant cells, organelles or vesicles are not the plant-based microcapsules of the present invention.

[0066] In step (a), the first cosolvent increases the solubility of the plant-based protein. The first cosolvent can be considered a solubility cosolvent. One or more solubility cosolvents can be present, and these solubility cosolvents can completely or partially dissolve the plant-based protein. The cosolvent can be added in step (a) in a highly concentrated form or in a diluted form.

[0067] Examples of solubilizing co-solvents are organic acids. Organic acids are organic compounds with acidic properties. Preferably, the organic acid is derived from natural plant-based or bio-based raw materials.

[0068] In a preferred method of the invention, the first cosolvent is an organic acid. Preferably, the organic acid is acetic acid, lactic acid, formic acid, gluconic acid, propionic acid, alpha-hydroxy acid and / or beta-hydroxy acid. Preferred alpha-hydroxy acids include glycolic acid, lactic acid, acetic acid, malic acid, citric acid and / or tartaric acid, preferably lactic acid or acetic acid. Preferred beta-hydroxy acids may include beta-hydroxypropionic acid, beta-hydroxybutyric acid, beta-hydroxy beta-methylbutyric acid, 2-hydroxybenzoic acid and carnitine. In a particularly preferred method of the invention, the organic acid is acetic acid and / or lactic acid.

[0069] The use of organic acids can achieve solubilization of plant proteins and also allow for mild hydrolysis of the proteins. For example, without wishing to be bound by theory, solubilization of plant-based proteins in organic acids is possible due to: i) protonation of the proteins and ii) the presence of an anionic solvation layer, which helps to reduce hydrophobic interactions. After initial solubilization in organic acids, the protonation of plant-based proteins can help stabilize them in their non-solvents (e.g., water).

[0070] In step (a), the second co-solvent reduces the solubility of the plant-based protein compared to the first co-solvent. The second co-solvent may be considered a desolubilizing co-solvent. One or more desolubilizing co-solvents may be present.

[0071] In a preferred process of the present invention, the second co-solvent is selected from water, ethanol and / or ethyl acetate, more preferably water and / or ethanol, even more preferably water.

[0072] In preferred methods of the invention, the solvent system comprises a cosolvent ratio of the first cosolvent to the second cosolvent of about 10-90% v / v, preferably about 20-90% v / v, preferably about 20-80% v / v, preferably about 20-60% v / v, about 25-55% v / v, about 30-50% v / v, about 20% v / v, about 30% v / v, about 40% v / v, about 50% v / v or about 60% v / v, most preferably about 30-50% v / v.

[0073] In a preferred method of the invention, the first co-solvent is present in the solution of step (a) at a concentration equal to or greater than the concentration of the protein on a weight percent basis.

[0074] In a preferred process of the present invention, the pH of the plant-based protein solution in step (a) is at least 0.5 pH unit below the isoelectric point of the plant-based protein, more preferably at least 1.0 pH unit below.

[0075] In a preferred method of the present invention, the concentration of the plant-based protein in the solvent system is 25-200 mg / ml, more preferably 50-150 mg / ml.

[0076] The ratio of organic acid may vary depending on the protein concentration, for example, as the protein concentration increases, a higher ratio of organic acid is used.

[0077] In a preferred method of the invention, the extent of protein hydrolysis (i.e., the percentage of cleaved peptide bonds in the protein hydrolysate) is controlled to alter the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation increases the extent of protein hydrolysis. A higher degree of protein hydrolysis results in the formation of a less rigid hydrogel.

[0078] In a preferred method of the present invention, the extent of protein hydrolysis is from 0.1% to 10%, preferably from 0.1% to 5%, even more preferably from 0.1% to 2.5%.

[0079] In order to form a solution comprising one or more plant-based proteins, it may be necessary to apply a physical stimulus to the protein / solvent system mixture to enable the protein to dissolve. Suitable physical stimuli include heating, ultrasonic treatment, stirring, high shear mixing, high shear homogenization or other physical techniques. The preferred technique is heating, optionally followed by ultrasonic treatment.

[0080] Preferably, the protein / solvent system mixture is subjected to a physical stimulus of heating, wherein the solution is heated to about 70° C. or above. More preferably, the protein / solvent system mixture is heated to about 75° C. or above, about 80° C. or above, about 85° C. or above, or about 90° C. Even more preferably, the protein / solvent system mixture is heated to 85° C.

[0081] Preferably, the protein / solvent system mixture is subjected to physical stimulation by heating for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more than 30 minutes. Optionally, the heated protein / solvent system mixture is subsequently subjected to ultrasonic treatment.

[0082] 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 reduced to a second temperature below the sol-gel transition temperature of the one or more plant-based protein solutions to form a hydrogel.

[0083] The protein solution is heated so that the liquid solution remains above the sol-gel transition of the protein. The sol-gel transition temperature of the protein can be changed by changing the solvent system (e.g., by selecting an organic acid, a ratio of an organic acid to another solvent, or by another means). By appropriately selecting conditions, the sol-gel transition of the protein can be finely controlled, thereby controlling the formation of the hydrogel.

[0084] Preferably, the protein solution is heated to about 70° C. or above. More preferably, the protein is heated to about 75° C. or above, about 80° C. or above, about 85° C. or above, or about 90° C. Even more preferably, the protein is heated to 85° C.

[0085] The protein solution can be kept at an elevated temperature for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes or 1 hour. The preferred period is at least 30 minutes to allow the protein to dissolve completely. The protein solution can be kept at an elevated temperature for a longer period of time.

[0086] After the protein solution is heated to a temperature higher than the sol-gel transition temperature, the temperature of the protein solution can be reduced to a second temperature lower than the sol-gel transition temperature to promote the formation of a hydrogel. The second temperature can be room temperature. The second temperature can range from 5°C to 25°C, preferably from 10°C to 20°C. The protein solution can be kept at a reduced temperature for a long period of time (e.g., several days, weeks) and then subjected to the shearing treatment in step (c). The protein solution can be kept at a reduced temperature for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or about 30 minutes. A specific shortened period of time is about 5 minutes.

[0087] The specific temperature will depend on the characteristics of the protein source, the solvent conditions used and thus the sol-gel transition temperature. Alternatively, the elevated temperature and the lowered temperature can be relatively fixed (e.g., about 85°C and then about room temperature), and the conditions of the co-solvent mixture can be adjusted to ensure a suitable sol-gel transition temperature for the selected plant-based protein.

[0088] Without wishing to be bound by theory, it is believed that when the vegetable protein is added to the solvent system, the vegetable protein forms a dispersion of insoluble colloidal protein aggregates. Aggregate size can be measured by dynamic light scattering (DLS). A suitable instrument for measuring aggregate size is the Zetasizer Nano S (Malvern).

[0089] It is believed that when a protein solution is heated to above the sol-gel transition temperature in the presence of a cosolvent system, the plant protein partially unfolds, resulting in the exposure of hydrophobic amino acids that were originally buried in the native structure of the protein. Once partially unfolded, the cosolvent is able to interact with the unfolded protein molecules. For example, organic acids more easily protonate the amino acid residues and enable the formation of anionic salt bridges that stabilize the hydrophobic interactions. In addition, when heated at elevated temperatures, protein-protein non-covalent intermolecular contacts are destroyed.

[0090] In addition, it is believed that applying mechanical agitation (e.g., ultrasonic treatment) destroys large-sized colloidal protein aggregates into smaller aggregates, and destroys protein molecular interactions. Using this method, before gelation, the size of protein aggregates can be significantly reduced to a particle diameter lower than 100nm. Preferably, the above method includes a protein aggregate with an average size of less than 200nm, preferably less than 150nm, less than 125nm, less than 100nm, less than 90nm, less than 80nm, less than 70nm, less than 60nm, less than 50nm, less than 40nm or less than 30nm. Therefore, it is believed that at this stage of the method, plant-based protein has a protein secondary structure, and the protein secondary structure has a high level of alpha-helix and random coil.

[0091] In addition, it is believed that when the protein solution is cooled below the sol-gel transition temperature, protein-protein non-covalent intermolecular contacts can be achieved, thereby promoting the self-assembly of plant protein molecules into a hydrogel of interconnected protein aggregates.

[0092] After gelation, the aggregates may be in the form of thin chains. The aggregates may have a median average length of 50nm to 500nm. The aggregates may have an average length of 50nm to 500nm. 80% of the aggregates may have an average length of 50nm to 500nm. The aggregates may have a median height of 5nm to 50nm. The aggregates may have an average height of 5nm to 50nm. 80% of the aggregates may have an average height of 5nm to 50nm. In a preferred embodiment, the aggregates have a median average length of 50nm to 500nm and / or a median average height of 5nm to 50nm.

[0093] It is believed that the method of the present invention allows plant proteins to aggregate into supramolecular structures maintained by intermolecular hydrogen bonding interactions, and especially between beta strands.

[0094] The method of the present invention is capable of forming a material in which there is a high level of beta-sheet intermolecular interactions. Thus, in the method of the present invention, the plant-based protein has a protein secondary structure having at least 40% intermolecular beta sheets, at least 50% intermolecular beta sheets, at least 60% intermolecular beta sheets, at least 70% intermolecular beta sheets, at least 80% intermolecular beta sheets, or at least 90% intermolecular beta sheets, wherein the percentage of intermolecular beta sheets is measured by FTIR (Fourier Transform Infrared Spectroscopy).

[0095] In a preferred process of the present invention, between step (b) and step (c), the plant-based protein hydrogel is subjected to a solvent reduction step, preferably a solubilizing solvent reduction step.

[0096] The solubilizing solvent refers to a solvent or solvent mixture in which the plant-based protein hydrogel dissolves. Examples include organic acids such as acetic acid, lactic acid, formic acid, propionic acid, α-hydroxy acids and / or β-hydroxy acids. The α-hydroxy acids may preferably be selected from glycolic acid, acetic acid, lactic acid, malic acid, citric acid and / or tartaric acid. The β-hydroxy acids may preferably be selected from β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy β-methylbutyric acid, 2-hydroxybenzoic acid and carnitine.

[0097] In a preferred method of the present invention, the solvent reduction step comprises:

[0098] (i) contacting the plant-based protein hydrogel with a non-dissolving solvent;

[0099] (ii) separating the plant-based hydrogel from the non-dissolving solvent to provide a washed plant-based protein hydrogel; and

[0100] (iii) optionally repeating steps (i) and (ii).

[0101] Step (i) involves contacting the plant-based protein hydrogel with a non-dissolving solvent. A non-dissolving solvent refers to a solvent or a solvent mixture in which the plant-based protein hydrogel is not soluble. Examples include water or a mixture of water and ethanol.

[0102] In a preferred method of the present invention, the shearing treatment comprises one step (ie a single shearing step). The single shearing step may be a higher shearing step. Preferably, the single shearing step involves breaking the plant-based protein hydrogel into pieces.

[0103] In a preferred method of the invention, the fragments produced in a single shearing step have a d of 0.5 to 50 μm, preferably 1 to 40 μm, preferably 2 to 30 μm, as determined by laser diffraction. 50 .

[0104] In a preferred method of the invention, the single shearing step involves ultrasonic treatment (for example using equipment such as Bandelin HD4200 or Hielscher UIP1000hdT), high shear mechanical stirring (for example using equipment such as a Silverson rotor-stator high shear mixer), high pressure homogenization or cavitation, preferably ultrasonic treatment.

[0105] In a preferred method of the invention, the single shearing step is performed at a temperature below the sol-gel transition temperature of the plant-based protein solution. In a preferred method of the invention, the first shearing step is performed for a duration of at least 5 minutes, more preferably for a duration of at least 1 minute.

[0106] In a preferred method of the invention, the shearing process comprises two steps. Preferably, the shearing process comprises a first shearing step followed by a second shearing step. The first shearing step may be a lower shearing step and the second shearing step may be a higher shearing step.

[0107] In a preferred method of the present invention, the first shearing step involves breaking the plant-based protein hydrogel into fragments. Preferably, at least 50 wt% of the fragments produced in the first shearing step have a particle size ranging from 1 mm to 100 mm, preferably from 1 mm to 50 mm, preferably from 5 mm to 30 mm, more preferably from 10 mm to 30 mm. More preferably, at least 80 wt% of the fragments produced in the first shearing step have a particle size ranging from 1 mm to 100 mm, preferably from 1 mm to 50 mm, preferably from 5 mm to 30 mm, more preferably from 10 mm to 30 mm. This can be measured by optical microscopy or by vision.

[0108] In a preferred method of the invention, the first shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-based protein solution.

[0109] In a preferred method of the present invention, the first shearing step involves mechanical cutting. Mechanical cutting refers to cutting using a blade (eg, a knife, an extruder blade, etc.).

[0110] In an optional preferred method of the present invention, the first shearing step involves extrusion. For example, the plant-based protein solution formed in step (a) can be extruded into a non-dissolving solvent (e.g., water) to form a plant-based protein hydrogel in the form of large discrete fragments, for example, the large discrete fragments can take the form of an extrudate having a thread or rope form. In this way, the fragments can be directly subjected to a solvent reduction step, as further described below. A first shearing step of this nature is more suitable for large-scale processing. In this case, the first shearing step can reduce at least one dimension of the large fragments (e.g., the diameter of the extrudate) to 1mm to 100mm. Preferably, at least 50wt% of the fragments produced in the first shearing step have at least one internal dimension ranging from 1mm to 100mm, preferably 1mm to 50mm, preferably 5mm to 30mm, and more preferably 10mm to 30mm. More preferably, at least 80wt% of the fragments produced in the first shearing step have at least one internal dimension ranging from 1mm to 100mm, preferably 1mm to 50mm, preferably 5mm to 30mm, and more preferably 10mm to 30mm. This can be measured by light microscopy or visually.

[0111] In a preferred method of the present invention, the second shearing step involves further fragmenting the plant-based protein hydrogel. Preferably, the fragments produced in the second shearing step have a d of 0.5 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns as measured by laser diffraction. 50 .

[0112] In a preferred method of the present invention, the particle size distribution of the hydrogel fragments in the plant-based protein hydrogel slurry can be adjusted by changing the nature and intensity of the second shearing step. In another preferred method, the particle size distribution of the hydrogel fragments in the plant-based protein hydrogel slurry can be adjusted by mixing or combining two or more different hydrogel slurries that have undergone different second shearing steps and have different particle size distributions.

[0113] In a preferred method of the present invention, the second shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-based protein.

[0114] In a preferred method of the present invention, the second shearing step is carried out at a temperature below the protein denaturation temperature of the plant-based protein.

[0115] In a preferred method of the invention, said second shearing step is performed for a duration of at least 5 minutes, more preferably at least 1 minute.

[0116] In a preferred method of the invention, the second shearing step involves ultrasonic treatment (for example using equipment such as Bandelin HD4200 or Hielscher UIP1000hdT), high shear mechanical stirring (for example using equipment such as a Silverson rotor-stator high shear mixer), high pressure homogenization or cavitation, preferably ultrasonic treatment.

[0117] In a preferred method of the present invention, step (c) further comprises subjecting the plant-based protein hydrogel slurry to a solvent reduction step, preferably a solubilizing solvent reduction step, between the first shearing step and the second shearing step.

[0118] In a preferred method of the present invention, the solvent reduction step comprises the following steps:

[0119] (i) contacting the fragments of the plant-based hydrogel slurry with a non-dissolving solvent;

[0120] (ii) separating the fragments of the plant-based hydrogel slurry from the non-dissolving solvent to provide a washed plant-based protein hydrogel; and

[0121] (iii) optionally repeating steps (i) and (ii).

[0122] The dissolving solvent is as defined above.

[0123] Step (i) involves contacting the fragments of the plant-based protein hydrogel slurry with a non-dissolving solvent. The non-dissolving solvent is as defined above.

[0124] In a preferred method of the invention, step (ii) involves screen filtration or centrifugation. More preferably, step (ii) involves screen filtration using a plurality of screens of decreasing size.

[0125] The skilled person will appreciate that if the fragments produced in the first shearing step are too small, then the solvent reduction step may be difficult because the fragments may eventually clog the mesh or the collected yield may be low. However, if the fragments produced in the first shearing step are too large, then the solvent reduction step may take too long due to the slow mass transfer of solvent from the core of the fragments.

[0126] Without wishing to be bound by theory, it is believed that due to the porous nature of the hydrogel, the solvent reduction step may remove some or all of the solvent (eg, organic acid) from the hydrogel by solvent exchange.

[0127] The strength of protein hydrogels can be altered by changing the concentrations of protein and organic acid, as well as other variables.

[0128] The strength of the hydrogel used to form the hydrogel slurry is useful within certain limits. This can be measured by oscillatory rheology. A suitable measure of hydrogel strength is the storage modulus G' of the hydrogel. Suitable test conditions are 1% strain at 20°C and an oscillation frequency of 1 Hz. A suitable apparatus is an Anton Paar MCR 92 rheometer with a 50 mm diameter, 1 degree cone and plate measurement geometry.

[0129] Therefore, in a preferred method of the present invention, before washing, the plant-based protein hydrogel has a storage modulus (G') at 10 rad / s of greater than 1000 Pa, preferably greater than 2000 Pa, more preferably greater than 5000 Pa, even more preferably greater than 6000 Pa, and most preferably greater than 8000 Pa. The skilled person will understand that 2πrad / s is equivalent to 1 Hz.

[0130] In a preferred method of the invention, the plant-based protein hydrogel has a storage modulus (G') of less than 20,000 Pa, preferably less than 15,000 Pa, more preferably less than 10,000 Pa at 10 rad / s before washing.

[0131] In a preferred method of the present invention, the plant-based protein hydrogel has a storage modulus (G') of about 1000 to 20,000 Pa, preferably about 2000 to 15,000 Pa, more preferably about 2000 to 10,000 Pa at 10 rad / s before washing.

[0132] Furthermore, in a preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') at 10 rad / s of greater than 200 Pa, preferably greater than 250 Pa, more preferably greater than 300 Pa, even more preferably greater than 350 Pa, more preferably greater than 400 Pa, more preferably greater than 450 Pa.

[0133] In a preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') of less than 5000 Pa, preferably less than 2500 Pa, preferably less than 1000 Pa, preferably less than 950 Pa, more preferably less than 900 Pa, more preferably less than 850 Pa, more preferably less than 800 Pa, more preferably less than 750 Pa at 10 rad / s.

[0134] In a preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') at 10 rad / s of about 200 to 5000 Pa, about 200 to 2500 Pa, about 200 to 1000 Pa, about 250 to 950 Pa, about 300 to 900 Pa, about 350 to 850 Pa, about 400 to 800 Pa, or about 450 to 750 Pa.

[0135] The preferred method of the present invention further comprises adding additional ingredients to the plant-based protein hydrogel slurry in step (c). Preferably, the additional ingredients are selected from plasticizers, surfactants, rheology modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers and nanoparticles, or mixtures thereof.

[0136] In a preferred method of the invention, the additional ingredient is a plasticizer. Preferably, the plasticizer is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oils, lecithin, waxes and amino acids.

[0137] The preferred method of the present invention further comprises the step of changing the pH of the plant-based protein hydrogel slurry so that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

[0138] Preferably, the step of changing the pH of the plant-based protein hydrogel slurry is performed after step (c). Alternatively, the step of changing the pH of the plant-based protein hydrogel slurry is performed sequentially with step (c).

[0139] During the adjustment of the pH of the plant-based protein hydrogel slurry, the slurry is likely to pass through the isoelectric point of the protein. Due to the lack of charge repulsion at the isoelectric point, the dispersed protein fragments in the plant-based protein hydrogel slurry can coagulate quickly. In order to avoid this, a pH adjusting substance can be used to quickly change the pH and thereby minimize the time the slurry is at the isoelectric point. 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.

[0140] Therefore, in a preferred method of the present invention, the step of changing the pH of the plant-based protein hydrogel slurry involves adding a pH adjusting substance to the plant-based protein hydrogel slurry. Preferably, the pH adjusting substance is a solution containing monovalent metal ions, divalent metal ions or ammonium ions, preferably an alkaline aqueous solution containing monovalent metal ions, divalent metal ions or ammonium ions. More preferably, the pH adjusting substance is an aqueous hydroxide solution, preferably an aqueous sodium hydroxide, potassium hydroxide or ammonium hydroxide solution.

[0141] In a preferred method of the present invention, after said step of altering the pH of the plant based protein hydrogel slurry, the pH of the plant based protein hydrogel slurry is at least 1 pH unit below the isoelectric point of the plant based protein.

[0142] In a preferred method of the present invention, after said step of altering the pH of the plant based protein hydrogel slurry, the pH of the plant based protein hydrogel slurry is at least 1 pH unit higher than the isoelectric point of the plant based protein.

[0143] The skilled person will appreciate that the addition of additional ingredients to the plant-based protein hydrogel slurry in step (c) may affect the pH of the slurry. Thus, the step of altering the pH of the plant-based protein hydrogel slurry preferably occurs after the addition of any additional ingredients.

[0144] In a preferred process of the invention, the composition formed in step (d) is a shear thinning composition.

[0145] In a preferred method of the present invention, the composition formed in step (d) has a -1 Viscosity in the range of 1 to 10000 cP, preferably at 20°C and 50s -1 The viscosity ranges from 10 to 7500 cP, more preferably at 20°C and 50s -1 Viscosity range of 15 to 5000 cP.

[0146] In a preferred method of the present invention, the composition formed in step (d) has a protein solids content ranging from 1 wt% to 25 wt%, preferably 2 wt% to 20 wt%, more preferably 4 wt% to 15 wt%, even more preferably 5 wt% to 12 wt%, based on the total weight of the composition. The term "protein solids" refers to the dry dispersed hydrogel solids derived from the plant protein added in step (a). The plant protein is typically a plant protein isolate, which mainly contains substances that can be chemically identified as proteins, but also contains small amounts of other insoluble plant substances, such as fibers. The percentage of protein solids is measured according to the method described herein.

[0147] In a preferred method of the present invention, the active ingredient is selected from vitamins, minerals, flavors, fragrances, pro-flavours, pro-fragrances, flavor enhancers, malodour counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antimicrobials, antivirals, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments and binders, or combinations thereof.

[0148] In a particularly preferred method of the present invention, the active ingredient is at least one aroma substance or flavor substance. Preferably, at least one aroma substance or flavor substance is selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds and Schiff bases.

[0149] The aroma substances and flavor substances used in the present invention can be of natural origin (that is, they are extracted from natural sources and are not synthetically modified in any way). Preferred aroma substances or flavor substances of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, citrus oil, orange flower extract, cedar, vetiver, eye-catching lavender, ylang-ylang extract, tuberose extract, sandalwood oil, bergamot oil, rosemary oil, spearmint oil, peppermint oil, lemon oil, lavender oil, citronella oil, chamomile oil, clove oil, sage oil, neroli oil, labdanum oil, eucalyptus oil, verbena oil, mimosa extract, narcissus extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil or a combination thereof. Preferably, the aroma substances or flavor substances of natural origin are of plant origin. The aroma substances or flavor substances of natural origin can be used alone or in combination, or in combination with synthetic aroma substances.

[0150] In a preferred method of the invention, the at least one aroma or flavor substance has a vapor pressure greater than or equal to 0.00001 Torr at 25°C.

[0151] In a preferred method of the invention, the at least one aroma or flavor substance has a logP greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0.

[0152] In a preferred method of the present invention, at least one aroma or flavor substance has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) less than 8, and hydrogen bonding (δH) of 2.5 to 11.

[0153] In a preferred method of the invention, the at least one aroma or flavoring substance is part of a aroma or flavoring agent.

[0154] Preferably, the aroma or flavor comprises at least 20 wt% aroma or flavor having a logP greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0, based on the total weight of the aroma or flavor.

[0155] Preferably, the aroma or flavor comprises at least 40 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0156] Preferably, the aroma or flavor comprises at least 50 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0157] Preferably, the aroma or flavor comprises at least 60 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0158] Preferably, the aroma or flavor comprises at least 10 wt%, preferably at least 30 wt%, more preferably at least 50 wt%, more preferably at least 70 wt% of aroma or flavor substances of natural origin based on the total weight of the aroma or flavor.

[0159] Preferably, the fragrance or flavoring agent contains at least 10 wt% of fragrance or flavoring agent, and the fragrance or flavoring agent has a biodegradation percentage of 60% to 100%, more preferably 65%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, measured according to ISO-14851 version 2019 based on O2 consumption after 28 days. Due to its low water solubility, the fragrance or flavoring agent can be added to the biodegradation test on an inert support according to the method in ISO 10634 version 2018 (water quality-preparation and treatment of poorly soluble organic compounds in water) to subsequently evaluate its biodegradability in an aqueous medium. In addition, any inhibitory effect of the fragrance or flavoring agent on microorganisms can be checked as indicated in the method.

[0160] Preferably, the fragrance or flavoring agent comprises at least 80 wt%, preferably at least 90 wt%, more preferably at least 95 wt% of fragrance or flavoring substances based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substances have at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) of less than 8 and hydrogen bond (δH) of 2.5 to 11.

[0161] Preferably, the fragrance or flavor contains only low levels of substances with alcohol functionality (e.g. primary alcohol functionality). In a preferred method of the invention, the fragrance or flavor contains less than 40 wt%, more preferably less than 20 wt%, of alcohol-containing substances based on the total weight of the fragrance or flavor. In a particularly preferred method of the invention, the fragrance or flavor does not contain alcohol-containing substances. Without wishing to be bound by theory, it is believed that alcohols (especially primary alcohols with linear alkyl groups) can easily diffuse through the shell of the microcapsule due to their structure, which means that they may be difficult substances to encapsulate.

[0162] Preferably, the fragrance or flavor substance has a high odor impact. This is advantageous because it ensures that even low levels of fragrance released from the microcapsules can be perceived.

[0163] In an optional particularly preferred method of the present invention, the active ingredient is a vitamin or a mineral. Preferably, the active ingredient is selected from the following vitamins or minerals: vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, magnesium, sodium, potassium, zinc, iron, calcium, iodine, linseed oil, ω-3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus or a mixture thereof. More preferably, the active ingredient is vitamin D.

[0164] The vitamins and minerals used in the present invention may be derived from vitamin-containing and / or mineral-containing materials. An example of such a material is seaweed.

[0165] In an alternative preferred method of the invention, the active ingredient is an agrochemical such as a pesticide, herbicide, fertilizer, fungicide, insecticide, animal repellent, or a combination thereof.

[0166] Preferably, the agrochemical is a natural alternative to synthetic materials and is based on plant extracts and / or plant essential oils (EO) or components of essential oils (e.g. thymol). Preferably, the agrochemical is a biopesticide. Preferably, the agrochemical is suitable for use in formulations for plant care and production, which can be certified as organic by organizations such as the USDA (United States Department of Agriculture) or the European Ecocert.

[0167] In a preferred method of the invention, the active ingredient is part of a composition comprising the active ingredient and an active carrier phase. Preferably, the active carrier phase is a solvent, a fat or a wax.

[0168] In a preferred method of the invention, the active carrier phase is a solvent. Preferably, the solvent is a solvent with low volatility (e.g., having a vapor pressure of less than 0.1 Torr at 25°C, preferably having a vapor pressure of less than 0.01 Torr at 25°C, preferably having a vapor pressure of less than 0.001 Torr at 25°C).

[0169] Preferably, the solvent has low or no odor.

[0170] Preferably, the solvent has at least two Hansen solubility parameters selected from the group consisting of atomic dispersion forces (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 forces (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0171] Preferably, the solvent has a mass greater than 1.07 g / cm 3 Solvents having this property are advantageously able to prevent creaming of the encapsulated material (e.g., in the final product formulation).

[0172] Preferably, the solvent contains only low levels of substances with alcohol functionality (e.g. primary alcohol functionality). In a preferred process of the invention, the solvent contains less than 40 wt%, more preferably less than 20 wt% of alcohol-containing substances based on the total weight of the solvent. In a particularly preferred process of the invention, the solvent does not contain alcohol-containing substances.

[0173] In a preferred method of the present invention, the active carrier phase is a solvent selected from the group consisting of carboxylic acid esters, fatty acid esters, phthalic acid esters, triols, diols, rosin resins, isoparaffins, terpenes and vegetable oils or combinations thereof.

[0174] 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 propanediol or a combination thereof, preferably 812N.

[0175] Preferably, the solvent is a vegetable oil selected from 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 provided in CTFA Cosmetic Ingredient Handbook, JM Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Vegetable oil is an oil from a plant source. Alternatively, the solvent is derived from vegetable oil.

[0176] In a preferred process of the invention, the active carrier phase is a fat or wax having a melting point below 60°C, preferably below 45°C, preferably below 25°C.

[0177] Preferably, the wax is selected from 100. 142 and 154, or a combination thereof, preferably 100.

[0178] In the method of the present invention, the method for forming the composition in step (d) is not particularly limited. For example, step (d) may involve membrane emulsification, mechanical stirring (e.g., low or high shear mechanical stirring), ultrasonic treatment, high shear mechanical stirring, high pressure homogenization and / or cavitation.

[0179] In a preferred process of the present invention, step (d) is carried out at a temperature in the range of 30°C to 50°C, preferably 35°C to 45°C.

[0180] In a preferred method of the present invention, the composition formed in step (d) comprises droplets of the active ingredient or comprises droplets of the composition comprising the active ingredient and an active carrier phase, the droplets having a d θ of 0.5 μm to 50 μm, preferably 1 μm to 40 μm, preferably 2 μm to 30 μm, as determined by laser diffraction. 50 .

[0181] In a preferred method of the invention, in step (d), an additive, preferably a water-soluble additive, is added to the composition.

[0182] Preferably, the additive is selected from plasticizers, surfactants, rheology modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers and nanoparticles, or mixtures thereof.

[0183] In a preferred method of the present invention, the additive is a plasticizer. Preferably, the additive is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oil, lecithin, wax and amino acid.

[0184] In a preferred method of the invention, the additive is a polysaccharide, preferably maltodextrin or OSA starch. The use of a polysaccharide additive allows the composition of the matrix of the resulting microcapsules to be controlled. For example, the use of a polysaccharide means that the amount of plant-based protein can be controlled.

[0185] In a preferred method of the invention, the water-soluble additive is a water-soluble vitamin, preferably vitamin C. The presence of an additive such as a water-soluble vitamin means that the final dried microcapsules are able to deliver the additive in addition to the encapsulated active ingredient during final use (e.g. when the plant-based protein hydrogel matrix is ​​enzymatically degraded in the oral cavity or digestive tract of a human or animal).

[0186] In a preferred method of the present invention, the weight ratio of the plant-based protein to the composition comprising the active ingredient and the active carrier phase in the composition formed in step (d) ranges from 20:1 to 1:20.

[0187] In a preferred process of the present invention, the composition formed in step (d) is a homogeneous composition.

[0188] In a preferred process of the invention, the composition formed in step (d) is a two-phase system (eg an aqueous two-phase system).

[0189] In the method of the present invention, step (e) involves drying the composition formed in step (d) to form microcapsules or microcapsule agglomerates or larger objects. The skilled person will be familiar with drying techniques and will know how to select the necessary conditions to obtain dried microcapsules or larger dried bodies. Suitable drying techniques include spray drying, electrostatic spray drying, spray freeze drying and spray vacuum drying as used herein, wherein the slurry is sprayed into the chamber under reduced pressure, so that the solvent can be removed at a lower temperature, thereby avoiding the problem of thermal degradation. Advantageously, in the method of the present invention, step (e) can be carried out at a relatively low temperature (e.g., at a temperature below 100° C.) under spray conditions to form fine slurry droplets. This facilitates the formation of small-sized microcapsules and also prevents the loss or degradation of any volatile or sensitive materials encapsulated.

[0190] Optional or additional drying techniques that can be used independently or in combination with spray drying to evaporate solvent from the composition of step (d) include fluidized bed drying, thin film drying, drum drying, belt drying, conduction drying, infrared drying or a combination thereof. The composition of step (d) can be dispersed and at least partially dried by spray drying to form microcapsules, which are then further dried by a secondary process (e.g., fluidized bed drying). The typical extended drying time of fluidized bed drying generally allows the use of lower drying temperatures. Some designs of spray dryers incorporate this two-stage drying process as an integral part of their design. When larger capsules are required, some fluidized bed drying processes are preferred, which generally include spraying or dispersing the composition of step (d) onto or into a fluidized bed of particles (e.g., capsules of composition (d) that have been dried). The composition can be sprayed onto or into a fluidized bed. Depending on the process conditions, the composition spray will agglomerate and / or form a coating (and thus accumulate) when passing through the microcapsules. An example of a fluidized bed coating and drying process is a Wurster type powder coater. The skilled person will be able to readily define the conditions for controlling the size of the package in a fluidized bed drying process.

[0191] Other drying methods may be selected depending on the desired form of the dried composition from step (e). Aesthetic shapes comprising the dried composition (d) may be formed using thin film drying, in which the composition is cast as a thin film onto a heated belt or drum. Belt drying using conductive heating or infrared heating may be used in combination with other drying techniques to accomplish drying under controlled conditions.

[0192] During the drying period of step (e), the plant-based protein hydrogel slurry forms a plant-based protein carrier that encapsulates the active ingredient in the form of microcapsules. During the drying period of step (e), at least a portion of the first cosolvent and the second cosolvent are evaporated to enable the microcapsules to form. This evaporation does not occur at the same rate for the two cosolvents, so that in the process, the slurry will usually become more concentrated in the least volatile cosolvent. The microcapsules formed during the drying period may still contain the first cosolvent and the second cosolvent trapped in the dried microcapsules, and their ratios may be different from their initial ratios in the solution of step (a).

[0193] In a preferred method of the present invention, the percentage of biodegradation of the plant-based protein carrier based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand is 60% to 100%, more preferably 65% ​​to 10%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100%, measured after 28 days based on O2 consumption according to ISO-14851 version 2019. ISO-14851 version 2019 describes a method that determines the extent of biodegradation of a substance in a natural aqueous environment using biological oxygen demand in a closed respirometer. This is achieved by exposing the substance to an inoculum from unadapted activated sludge that has not been pre-exposed in an aqueous standard test medium under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and the test is valid if its percentage of biodegradation is greater than 60% at the end of the test.

[0194] In a preferred method of the present invention, the percentage of biodegradation of the microcapsules based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand is 60% to 100%, more preferably 65% ​​to 10%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100%, measured based on O2 consumption according to ISO-14851 version 2019 after 28 days. ISO-14851 version 2019 describes a method for determining the extent of biodegradation of a substance in a natural aqueous environment using biological oxygen demand in a closed respirometer. This is achieved by exposing the substance to an inoculum from unadapted activated sludge without prior exposure in an aqueous standard test medium under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and the test is valid if its percentage of biodegradation is greater than 60% at the end of the test.

[0195] In a preferred process of the invention, step (e) involves electrostatic spray drying.

[0196] In a preferred process of the present invention, the vegetable-based protein residue formed during step (e) but not incorporated into the microcapsules is recycled, preferably added back to step (a).

[0197] In a preferred method of the invention, the microcapsules have a d value of less than or equal to 250 μm, less than or equal to 200 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. 50 .

[0198] In preferred methods of the invention, the microcapsules have a diameter determined by optical microscopy of less than or equal to 250 μm, less than or equal to 200 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.

[0199] The preferred method of the present invention further comprises a post-treatment step for the microcapsules. Preferably, the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step or a coating formation step.

[0200] In a preferred method of the present invention, the post-treatment step comprises a non-covalent cross-linking step. Preferably, the non-covalent cross-linking step comprises treating the microcapsules with a non-covalent cross-linking agent selected from the group consisting of sodium tripolyphosphate (NaTPP), sodium hexametaphosphate and phenolic compounds (e.g. tannic acid, caffeic acid, etc.).

[0201] 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 cross-linking resins or combinations thereof.

[0202] In a preferred method of the present invention, the post-treatment step includes a coating formation step.

[0203] In a preferred method of the present invention, the coating forming step comprises treating the microcapsules with a metal compound. Preferably, the metal compound is selected from a silver compound or a gold compound, preferably a silver compound.

[0204] In a preferred method of the present invention, the coating forming step comprises a step of subjecting the microcapsules to complex coacervation using a polysaccharide. Preferably, the polysaccharide is selected from xanthan gum, gellan gum and chitosan, or a combination thereof.

[0205] 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 salts, calcium salts, phosphates, carbonates, titanium salts or zinc salts or a combination thereof.

[0206] The skilled person will appreciate that 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 formation step (e.g., using chitosan) and optionally an additional non-covalent cross-linking step (e.g., using NaTPP).

[0207] The preferred method of the present invention further comprises adding a flow aid to the microcapsules after step (e). Preferably, the flow aid is fumigated silica. Adding a flow aid can prevent the newly formed microcapsules from agglomerating into agglomerates.

[0208] A preferred method of the invention comprises further drying the microcapsules after step (e). Preferably, the further drying is selected from fluidized bed drying and / or tray drying.

[0209] The preferred method of the present invention further comprises resuspending the microcapsules in an external phase, which is preferably an external aqueous phase, more preferably hard water or an acidic buffer solution.

[0210] Preferred methods of the invention further comprise sieving the microcapsules, for example for size classification.

[0211] The present invention also provides a biodegradable microcapsule obtained or obtainable by the method described above.

[0212] The present invention also provides a method for preparing a biodegradable microcapsule composition, the method comprising:

[0213] (a) preparing biodegradable microcapsules according to the method described above; and

[0214] (b) suspending biodegradable microcapsules in the external phase.

[0215] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0216] A preferred process of the invention further comprises adding a suspending agent to the external phase.

[0217] Preferably, the suspending agent is selected from gum arabic, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, type 1 hydrogenated vegetable oil, magnesium aluminum silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, polyvinyl pyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers (e.g., cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylate and cross-linked polyvinyl pyrrolidone), and swelling clays (e.g., bentonite and laponite).

[0218] The present invention also provides a biodegradable microcapsule composition obtained or obtainable by the method described above.

[0219] The present invention also provides a biodegradable microcapsule comprising an active ingredient and a plant-based protein carrier containing a plant-based protein, wherein the plant-based protein carrier encapsulates the active ingredient, and wherein the plant-based protein has a solubility of less than 50% when measured at a protein concentration of 5% w / w in an aqueous solution at 25° C. and pH 7.

[0220] Protein solubility is determined using the following protocol: a known amount of dried microcapsules is added to an aqueous solution, followed by centrifugation to separate the soluble and insoluble portions. After centrifugation, the liquid supernatant (i.e., the soluble portion) is taken out without obtaining any solids precipitated at the bottom (i.e., the insoluble portion). The resulting supernatant is analyzed for nitrogen content. The protein content in the supernatant is then calculated based on the nitrogen content using a factor of 6.25. Protein solubility is defined as the amount of protein in the supernatant divided by the amount of protein in the entire aqueous solution.

[0221] In preferred biodegradable microcapsules of the present invention, the plant-based protein has a solubility of less than 30%, more preferably less than 10%, when measured at a protein concentration of 5% w / w in aqueous solution at 25°C and pH 7.

[0222] In the preferred biodegradable microcapsules of the present invention, at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the initially encapsulated active ingredient is still present inside the microcapsules after incubation in water at 20° C. for 10 days as determined by high performance liquid chromatography (HPLC). In the optional preferred biodegradable microcapsules of the present invention, at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the initially encapsulated active ingredient is still present inside the microcapsules after incubation in water at 20° C. for 10 days as determined by gas chromatography (GC).

[0223] More preferably, the water has been acidified so that it has a pH less than 4.0, more preferably less than 3.0, more preferably less than 2.0, more preferably less than 1.0. Therefore, the biodegradable microcapsules of the present invention are insoluble in water and are also stable under strongly acidic conditions. This has the following advantages, that is, the biodegradable microcapsules of the present invention can be used in various applications where such harsh conditions are prevalent. For example, biodegradable microcapsules can be added to beverage preparations that are generally acidic to deliver active ingredients (such as vitamins or minerals) to consumers. The acid stability of the biodegradable microcapsules of the present invention also means that the microcapsules can withstand the harsh acidic conditions of the stomach after being ingested, so that the active ingredients are not released until they are in the small intestine and can be better absorbed. Therefore, the biodegradable microcapsules of the present invention can provide controlled release of encapsulated active ingredients. This is because the plant-based protein hydrogel matrix plays the role of enteric coating.

[0224] In the preferred biodegradable microcapsules of the present invention, the plant-based protein is obtained from broad bean, mung bean, pea, rice, potato, rapeseed, lentil, chickpea, sunflower seed, pumpkin seed, flax, chia, canola, lupin, alfalfa, moringa, wheat, corn zein or sorghum; preferably, the plant protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, broad 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, the plant-based protein is pea protein and / or potato protein. Such proteins are considered to be hypoallergenic proteins.

[0225] In the preferred biodegradable microcapsules of the present invention, the plant-based protein has been pretreated with an organic acid. Preferably, the organic acid is acetic acid, lactic 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.

[0226] Preferred alpha-hydroxy acids include glycolic acid, lactic acid, acetic acid, malic acid, citric acid and / or tartaric acid, more preferably lactic acid or acetic acid. Preferred beta-hydroxy acids include beta-hydroxypropionic acid, beta-hydroxybutyric acid, beta-hydroxy beta-methylbutyric acid, 2-hydroxybenzoic acid and carnitine.

[0227] In the preferred biodegradable microcapsules of the present invention, the active ingredients are selected from vitamins, minerals, flavors, fragrances, flavor precursors, fragrance precursors, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), drugs, antimicrobial agents, antiviral agents, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments and binders, or combinations thereof.

[0228] In the specific biodegradable microcapsules of the present invention, the active ingredient is at least one aroma substance or flavor substance. Preferably, at least one aroma substance or flavor substance is selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds and Schiff bases.

[0229] The aroma substances and flavor substances used in the present invention can be natural origin (that is, they are extracted from natural sources and are not synthetically modified in any way). Preferred aroma substances or flavor substances of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, citrus oil, orange flower extract, cedar, vetiver, eye-catching lavender, ylang-ylang extract, tuberose extract, sandalwood oil, bergamot oil, rosemary oil, spearmint oil, peppermint oil, lemon oil, lavender oil, citronella oil, chamomile oil, clove oil, sage oil, neroli oil, labdanum oil, eucalyptus oil, verbena oil, mimosa extract, narcissus extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil or a combination thereof. Preferably, the aroma substances or flavor substances of natural origin are plant-derived. The aroma substances or flavor substances of natural origin can be used alone or in combination, or in combination with synthetic aroma substances.

[0230] In the preferred biodegradable microcapsules of the present invention, at least one aroma or flavor substance has a vapor pressure greater than or equal to 0.00001 Torr at 25°C.

[0231] In the preferred biodegradable microcapsules of the present invention, at least one aroma or flavor substance has a logP greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0.

[0232] In the preferred biodegradable microcapsules of the present invention, at least one aroma or flavor substance has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) of less than 8, and hydrogen bonding (δH) of 2.5 to 11.

[0233] In the preferred biodegradable microcapsules of the present invention, the at least one aroma or flavor substance is part of an aroma or flavor.

[0234] Preferably, the aroma or flavor comprises at least 20 wt% aroma or flavor having a logP greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0, based on the total weight of the aroma or flavor.

[0235] Preferably, the aroma or flavor comprises at least 40 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0236] Preferably, the aroma or flavor comprises at least 50 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0237] Preferably, the aroma or flavor comprises at least 60 wt% of an aroma or flavor having a logP greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0, based on the total weight of the aroma or flavor.

[0238] Preferably, the aroma or flavor comprises at least 10 wt%, preferably at least 30 wt%, more preferably at least 50 wt%, more preferably at least 70 wt% of aroma or flavor substances of natural origin, based on the total weight of the aroma or flavor.

[0239] Preferably, the fragrance or flavoring agent contains at least 10 wt% of fragrance or flavoring agent, and the fragrance or flavoring agent has a biodegradation percentage of 60% to 100%, more preferably 65%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, measured according to ISO-14851 version 2019 based on O2 consumption after 28 days. Due to its low water solubility, the fragrance or flavoring agent can be added to the biodegradation test on an inert support according to the method in ISO 10634 version 2018 (water quality-preparation and treatment of poorly soluble organic compounds in water) to subsequently evaluate its biodegradability in an aqueous medium. In addition, any inhibitory effect of the fragrance or flavoring agent on microorganisms can be checked as indicated in the method.

[0240] Preferably, the fragrance or flavoring agent comprises at least 80 wt%, preferably at least 90 wt%, more preferably at least 95 wt% of fragrance or flavoring substances based on the total weight of the fragrance or flavoring agent, wherein the fragrance or flavoring substances have at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) of less than 8 and hydrogen bond (δH) of 2.5 to 11.

[0241] Preferably, the fragrance or flavor contains only low levels of substances with alcohol functionality (e.g., primary alcohol functionality). In preferred biodegradable microcapsules of the present invention, the fragrance or flavor contains less than 40 wt%, more preferably less than 20 wt%, of alcohol-containing substances based on the total weight of the fragrance or flavor. In particularly preferred biodegradable microcapsules of the present invention, the fragrance or flavor does not contain alcohol-containing substances.

[0242] Preferably, the fragrance or flavoring substance has a high odor impact. This is advantageous because it ensures that even low levels of fragrance released from the microcapsules can be perceived.

[0243] In the optional particularly preferred biodegradable microcapsules of the present invention, the active ingredient is a vitamin or a mineral. Preferably, the active ingredient is selected from the following vitamins or minerals: vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, magnesium, sodium, potassium, zinc, iron, calcium, iodine, linseed oil, omega-3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus or mixtures thereof. More preferably, the active ingredient is vitamin D.

[0244] The vitamins and minerals used in the present invention may be derived from vitamin-containing and / or mineral-containing materials. An example of such a material is seaweed.

[0245] In the preferred biodegradable microcapsules of the present invention, the active ingredient is part of a composition comprising the active ingredient and an active carrier phase. Preferably, the active carrier phase is a solvent, a fat or a wax.

[0246] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent. Preferably, the solvent is a solvent with low volatility (e.g., a vapor pressure of less than 0.1 Torr at 25°C, preferably a vapor pressure of less than 0.01 Torr at 25°C, preferably a vapor pressure of less than 0.001 Torr at 25°C).

[0247] Preferably, the solvent has low or no odor.

[0248] Preferably, the solvent has at least two Hansen solubility parameters selected from the group consisting of atomic dispersion forces (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 forces (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0249] Preferably, the solvent has a mass greater than 1.07 g / cm 3 Solvents having this property are advantageously able to prevent creaming of the encapsulated material (e.g., in the final product formulation).

[0250] Preferably, the solvent contains only low levels of substances with alcohol functionality (e.g. primary alcohol functionality). In preferred biodegradable microcapsules of the present invention, the solvent contains less than 40 wt%, more preferably less than 20 wt% of alcohol-containing substances based on the total weight of the solvent. In particularly preferred biodegradable microcapsules of the present invention, the solvent does not contain alcohol-containing substances.

[0251] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent selected from carboxylic acid esters, fatty acid esters, phthalic acid esters, triols, diols, rosin resins, isoparaffins, terpenes and vegetable oils or combinations thereof.

[0252] 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 propanediol or a combination thereof, preferably 812N.

[0253] Preferably, the solvent is a vegetable oil selected from 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 provided in CTFA Cosmetic Ingredient Handbook, JM Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Optionally, the solvent is derived from a vegetable oil.

[0254] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a fat or wax having a melting point below 60°C, preferably below 45°C, preferably below 25°C.

[0255] Preferably, the wax is selected from 100. 142 and 154, or a combination thereof, preferably 100.

[0256] In the preferred biodegradable microcapsules of the present invention, the plant-based protein carrier further comprises additives, preferably water-soluble additives.

[0257] Preferably, the additive is selected from plasticizers, surfactants, rheology modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers and nanoparticles, or mixtures thereof.

[0258] In the preferred biodegradable microcapsules of the present invention, the additive is a plasticizer. Preferably, the additive is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oil, lecithin, wax and amino acid.

[0259] In the preferred biodegradable microcapsules of the present invention, the additive is a polysaccharide, preferably maltodextrin or OSA starch. The use of a polysaccharide additive allows the composition of the matrix of the resulting microcapsules to be controlled. For example, the use of a polysaccharide means that the amount of plant-based protein can be controlled.

[0260] In preferred biodegradable microcapsules of the present invention, the water-soluble additive is a water-soluble vitamin, preferably vitamin C. The presence of additives such as water-soluble vitamins means that the final microcapsules are able to deliver the additives in addition to the encapsulated active ingredients during final use (e.g. when the plant-based protein hydrogel matrix is ​​enzymatically degraded in the oral cavity or digestive tract of humans or animals).

[0261] In the preferred biodegradable microcapsules of the present invention, the microcapsules have a d value of less than or equal to 250 μm, less than or equal to 200 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 measured by laser diffraction. 50 .

[0262] In preferred biodegradable microcapsules of the present invention, the microcapsules have a diameter of less than or equal to 250 μm, less than or equal to 200 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 optical microscopy.

[0263] In the preferred biodegradable microcapsules of the present invention, the plant-based protein has 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 percentage content of intermolecular β-sheets is measured by FTIR.

[0264] Preferred biodegradable microcapsules of the present invention are free or substantially free of crosslinking agents.

[0265] In the optional, preferably biodegradable microcapsules of the present invention, the plant-based protein carrier has been non-covalently modified by a non-covalent cross-linking agent. Preferably, the non-covalent cross-linking agent is selected from sodium tripolyphosphate, sodium hexametaphosphate and phenolic compounds (such as tannic acid, caffeic acid, etc.).

[0266] In the optional, preferably biodegradable microcapsules of the present invention, the plant-based protein carrier has been covalently modified by a covalent crosslinking agent. Preferably, the non-covalent crosslinking agent is selected from the group consisting of genipin, epoxy compounds, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase and cross-linking resin, or a combination thereof.

[0267] In the preferred biodegradable microcapsules of the present invention, the plant-based protein carrier has a coating deposited thereon. Preferably, the coating is a metal coating, a coacervate coating or a mineral coating.

[0268] In the preferred biodegradable microcapsules of the present invention, the metal coating is a silver coating or a gold coating, preferably a silver coating.

[0269] In the preferred biodegradable microcapsules of the present invention, the coacervate coating is formed by a polysaccharide. Preferably, the polysaccharide is selected from xanthan gum, gellan gum and chitosan, or a combination thereof.

[0270] In the preferred biodegradable microcapsules of the present invention, the mineral coating is formed by a mineral aqueous solution. Preferably, the mineral aqueous solution comprises iron salt, calcium salt, phosphate, carbonate, titanium salt or zinc salt, or a combination thereof.

[0271] Preferred biodegradable microcapsules of the present invention have a coating deposited on a covalently modified or non-covalently modified plant-based protein matrix. Preferred features of the coating are as described above.

[0272] In particularly preferred biodegradable microcapsules of the present invention, the plant-based protein is pea protein and the active ingredient is vitamin D.

[0273] In the preferred biodegradable microcapsules of the present invention, the percentage of biodegradation of the plant-based protein carrier based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand is 60% to 100%, more preferably 65% ​​to 100%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100%, measured after 28 days based on O2 consumption according to ISO-14851 version 2019. ISO-14851 version 2019 describes a method for determining the extent of biodegradation of a substance in a natural aqueous environment using biological oxygen demand in a closed respirometer. This is achieved by exposing the substance to an inoculum from unadapted activated sludge without prior exposure in an aqueous standard test medium under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose was also tested and the test was valid if its percent biodegradation was greater than 60% at the end of the test.

[0274] In the preferred biodegradable microcapsules of the present invention, the percentage of biodegradation of the microcapsules based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand is 60% to 100%, more preferably 65% ​​to 100%, even more preferably 70% to 100%, even more preferably 75% to 100%, even more preferably 80% to 100%, even more preferably 85% to 100%, and most preferably 90% to 100%, measured after 28 days based on O2 consumption according to ISO-14851 version 2019. ISO-14851 version 2019 describes a method for determining the extent of biodegradation of a substance in a natural aqueous environment using biological oxygen demand in a closed respirometer. This is achieved by exposing the substance to an inoculum from unadapted activated sludge without prior exposure in an aqueous standard test medium under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose was also tested and the test was valid if its percent biodegradation was greater than 60% at the end of the test.

[0275] The present invention also provides a composition comprising the biodegradable microcapsules as described above and an external phase.

[0276] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0277] Preferred compositions of the present invention also comprise a suspending agent in the external phase.

[0278] Preferably, the suspending agent is selected from gum arabic, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, type 1 hydrogenated vegetable oil, magnesium aluminum silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, polyvinyl pyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers (e.g., cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylate and cross-linked polyvinyl pyrrolidone), and swelling clays (e.g., bentonite and laponite).

[0279] The present invention also provides a formulation product comprising the biodegradable microcapsules as described above.

[0280] The present invention also provides a method for manufacturing a formulated product, the method comprising:

[0281] (a) preparing biodegradable microcapsules according to the method described above; and

[0282] (b) mixing the biodegradable microcapsules with a product formulation.

[0283] Preferably, the formulated product is a food, beverage, cosmetic, home care product, personal care product, pharmaceutical, industrial product (eg, coating, adhesive, sandpaper, tape, etc.), medical device, biomaterial, or agrochemical.

[0284] The present invention also provides the use of the biodegradable microcapsules as described above in a formulated product.

[0285] Preferably, the formulated product is a food, beverage, cosmetic, home care product, personal care product, pharmaceutical, industrial product (eg, coating, adhesive, sandpaper, tape, etc.), medical device, biomaterial, or agrochemical.

[0286] The formulations described in the various aspects of the invention may take any form suitable for use. Preferably, the formulation is in the form of a steam spray, an aerosol, an emulsion, a lotion, a liquid, a cream, a gel, a stick, an ointment, a paste, a mousse, a powder, a granular product, a matrix or a semisolid. BRIEF DESCRIPTION OF THE DRAWINGS

[0288] Figure 1a and 1b The 10x optical microscope photographs are of the microcapsules of Examples 2B and 2E, respectively.

[0289] Figure 2a and 2b The 10-fold optical microscope photographs are respectively of the microcapsules of Examples 4A and 4B.

[0290] Figure 3 This is a 10x optical microscope image of the control of Example 5 in water.

[0291] Figure 4a and 4b The SEM images of the microcapsules of Example 6A are magnified by 80 and 300 times, respectively.

[0292] Figure 5a , 5b 5a and 5b are SEM images of the microcapsules of Example 6B at 60, 500 and 1250 times magnification, respectively.

[0293] Figure 6 This is a SEM image of the microcapsule of Example 7 at 1200 times magnification.

[0294] Figure 7a and 7b The microcapsules of Example 7 before being squeezed to release the oil ( Figure 7a ) and after ( Figure 7b ) at 10x optical microscope image.

[0295] Figure 8a , 8b8c are 10 times microscope images of the concentrated slurries of Examples 10A, 10B and Comparative Example 10C.

[0296] Fig. 9 is a 10x optical microscope image of the microcapsules of Example 15 in single-strength citrate buffer.

[0297] Fig.10 This is a photograph of four inverted tubes of Example 16. Example

[0298] Material

[0299] Pea protein isolate (PPI) (80% protein, 4 wt% carbohydrates - ProEarth P16109) was purchased from Cambridge Commodities Ltd.

[0300] Lactic acid (85%, food grade) was purchased from Fisher Scientific.

[0301] Acetic acid (glacial 99%) was purchased from Fisher Scientific.

[0302] Sodium benzoate was purchased from Fisher Scientific.

[0303] Vitamin D2 oil (1 MIU / g) was purchased from Prinova Europe, UK.

[0304] 812N was purchased from 1010 Leochemical.

[0305] 100 was purchased from 1010 Leochemical.

[0306] Vitamin D2 powder (VITAMIN D2 (ERGOCALCIFEROL) 100,000 IU / G SD, gum arabic based) was obtained from Prinova Europe, UK.

[0307] Robinsons Double Strength No Added Sugar Squash Apple & Blackcurrant (manufactured by Britvic Plc and commercially available in supermarkets in the UK).

[0308] Miwadi Single Concentrate Blackcurrant (manufactured by Britvic Plc and commercially available in supermarkets in the Republic of Ireland).

[0309] Thymol was purchased from Fisher Scientific, UK.

[0310] Calcium sulfate dihydrate (CaSO4·2H2O) was purchased from Fisher Scientific.

[0311] Dry magnesium sulfate (MgSO4) was purchased from Fisher Scientific.

[0312] Potassium chloride (Extra Pure, SLR, Eur. Ph., (KCl)) was purchased from Fisher Scientific.

[0313] Sodium bicarbonate (Certified AR (NaHCO3) for analysis) was purchased from Fisher Scientific.

[0314] Potassium hydroxide (KOH) (>85%) was purchased from Sigma Aldrich.

[0315] Ethanol absolute 99.8+% (EtOH) (certified AR for analysis) was purchased from Fisher Scientific.

[0316] Maltodextrin was purchased from Sigma-Aldrich Gillingham, UK.

[0317] Polysorbate 80 was purchased from Sigma-Aldrich Gillingham, UK.

[0318] All components for phosphate buffered saline (PBS) (pH 7.4) were purchased from Fisher Scientific, UK, and prepared in the following proportions: 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4.

[0319] Sodium acetate trihydrate was purchased from Alfar Aesar, UK.

[0320] Calcium chloride dihydrate was purchased from Fisher Scientific, UK.

[0321] Methylparaben was purchased from Alfar Aesar, UK.

[0322] Tannic acid was purchased from Fisher Scientific, UK.

[0323] Gellan gum (high acyl) was purchased from Special Ingredients Ltd, UK.

[0324] Linseed oil was purchased from Naissance, UK.

[0325] Aroma substances

[0326]

[0327]

[0328] Standard hard water and buffer composition

[0329] 1) Hard water

[0330] This was prepared in deionized (DI) water with the following salt concentrations: 192 mg / l NaHCO3, 120 mg / l CaSO4.2H2O, 120 mg / l MgSO4, 8 mg / l KCl. The pH was adjusted to 3 with 1 M HCl.

[0331] 2) Single strength buffer

[0332] Prepare 100 ml of single strength buffer solution as follows:

[0333] i. Weigh 1.904 g of citric acid monohydrate and 0.276 g of trisodium citrate dihydrate;

[0334] ii. Make up the mixture to 80 g using DI water;

[0335] iii. The pH of the solution was adjusted to 3 with 1M HCl and 1M NaOH solution, and the volume of the solution was made up to 100 g with DI water;

[0336] iv. Add 0.38 g CaCl2 dihydrate;

[0337] v. Add 0.2g sodium benzoate;

[0338] vi. Place the solution in an ultrasonic bath at 80°C for 5-10 min (ie, until complete dissolution is observed);

[0339] vii. Allow the solution to cool to 20°C.

[0340] 3) Double strength buffer

[0341] Prepare 100 ml of double strength buffer solution as single strength buffer, but with double the levels of all substances, but still make up the mixture to 80 g.

[0342] Measurement method

[0343] Vitamin D2 Analysis of Microencapsulated Dry Powder Samples: Version A

[0344] Vitamin D2 in the sample is extracted into dimethyl sulfoxide and then partitioned into hexane. Vitamin D2 is quantified using normal phase HPLC with UV detection. This method is suitable for determining high levels (usually 35 iu / g to 500,000 iu / g) of vitamin D2.

[0345] Vitamin D2 Analysis of Microencapsulated Dry Powder Samples: Version B

[0346] The powder sample (about 0.1 g) was suspended in 4 g of 50% w / v aqueous potassium hydroxide solution and 8 g of absolute ethanol. 0.1 g of sodium ascorbate was added. The sample was placed in an ultrasonic bath for 1 to 1.5 hours without heating, cooled on ice, and then centrifuged at 14,500 rpm for 10 minutes and then analyzed by HPLC on a reverse phase column using 100% methanol as eluent and UV detection at 265 nm.

[0347] Vitamin D2 analysis in dilute liquid systems containing microcapsules

[0348] The sample is cold saponified overnight with an alcoholic potassium hydroxide solution (containing pyrogallol). The unsaponified portion is separated by extraction into hexane. The extracted sample is then concentrated and injected onto a semi-preparative HPLC column. The eluate fraction containing vitamin D2 is collected, evaporated to dryness, dissolved in methanol, and vitamin D2 is separated using reversed-phase HPLC with UV detection. This method is suitable for determining lower levels of vitamin D2 (usually 0.5 iu / g to 100 iu / g). Lower levels can be quantified by further dilution.

[0349] Hydrogel slurry particle size

[0350] The particle size of the hydrogel slurry was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. The measurement was performed by diluting the hydrogel in an aqueous solution adjusted to the same pH with acetic acid or lactic acid within 1 hour of completion of the manufacture. The test material was diluted to the required concentration to ensure the absence of agglomerates and to have the required optical density for the measurement (typically 5-15% obscuration). The referenced d 50 is calculated for volume distribution by general analysis using Mie theory.

[0351] Droplet size of the composition for spray drying

[0352] The droplet size of the oil or wax containing the active ingredient in the composition for spray drying is measured using the Anton Paar laser diffraction particle size analyzer PSA 1190. The measurement is carried out by diluting the composition in an aqueous solution adjusted to the same pH with acetic acid or lactic acid within 1 hour of completion of the manufacture. The test material is diluted to the desired concentration to have the optical density required for the measurement (usually 5-15% obscuration). The referenced d50 is calculated for volume distribution by general analysis using Mie theory.

[0353] Protein solid content

[0354] The solid content of the hydrogel slurry is measured by the mass remaining after drying. About 5 g of the hydrogel slurry is pipetted into a small polypropylene dish and the mass is accurately recorded. The dish is placed in a 40 °C oven to dry overnight. The dry mass is measured immediately after the dish is removed from the oven, and the solid content of the protein hydrogel slurry is calculated as a percentage of the initial wet mass.

[0355] Viscosity of the composition for spray drying

[0356] The viscosity of the composition for spray drying was measured using an Anton Paar MCR 92 rheometer within 1 hour of completing the composition manufacture. 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 / s.

[0357] Particle size of spray-dried microcapsules

[0358] The particle size of the final microcapsule powder is measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. The powder is added to reverse osmosis water diluted to the required concentration to have the required optical density for the measurement (usually 5-15% obscuration). The dispersion should be checked by optical microscopy to determine if there are any larger microcapsule agglomerates. If these microcapsule agglomerates are visible, 0.5wt% acetic acid can be added to ensure that the primary particles are well dispersed. Referenced d 50 is calculated for volume distribution by general analysis using Mie theory.

[0359] Alternatively, particle size can be measured using optical microscopy (e.g., using an openFrame microscope equipped with a CellCam 200CR camera, an Aura Pro phase contrast illuminator, and 4x, 10x, and 20x universal flat fluorite objective). As required, microcapsule powder is added to reverse osmosis water or a single strength buffer. In this method, particle size is obtained from the average size measurement of 50 microcapsules of each corresponding sample. The optical microscope is then calibrated using the grid of the Hirschmann counting chamber (FuchsRosenthal). 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 duplicate capsules.

[0360] Scanning electron microscopy (SEM)

[0361] SEM images were acquired using a Hitachi TM3030.

[0362] Optical microscopy

[0363] Use the openFrame microscope that is equipped with CellCam 200CR camera, Aura Pro phase contrast illuminator and 4 times, 10 times and 20 times general plane fluorite objective lens to obtain optical microscopy image.Before microscopy imaging, by first dry microcapsules are rehydrated in hard water or single strength buffer for 5 minutes to prepare the sample for optical microscopy.Then cover glass is placed on the top of the sample of rehydration, and image is taken.Then slide is removed from the microscope stage, and cover glass is pressed hard with hand, then sample is put back on the stage, and the image of broken capsule is taken.

[0364] Total fragrance and fragrance load

[0365] The levels of flavorants and aroma substances in the spray-dried microcapsules can be determined by extracting the aroma substances and injecting them into a GC column. Quantification is achieved by using a calibration curve of a given aroma substance diluted in ethanol.

[0366] In order to measure the total level of aroma substances in the encapsulated sample, the capsule must first be broken by sonication. For example, 10-50 mg of dry encapsulated material or 0.3-0.5 g of wet encapsulated material is collected on a 40 μm cell filter in 3.0 g of deionized (DI) water and 10% KOH and vortexed to mix thoroughly. The solution is then sonicated for 1-2 minutes with a Bandelin mini-probe TS104, with an amplitude of 30% (a total of about 1.5-3.0 kJ). While sonicating, the temperature is maintained at less than 20° C. with ice to avoid any loss of aroma agents by evaporation. Visually check whether the capsule is completely broken using an optical microscope as described in detail above. If not completely broken, repeat the sonication step. Add ethanol to the mixture, then mix and centrifuge. Collect the supernatant, then dilute appropriately, and inject GC for analysis.

[0367] For samples containing benzyl salicylate, it was quantified by measuring the benzyl alcohol formed by its hydrolysis.

[0368] Total thymol loading

[0369] The thymol level in the spray dried microcapsules was determined by extracting the thymol and injecting it into a GC column following the total flavor loading method herein.

[0370] Olfactory evaluation of microcapsule slurry

[0371] For any given package, the total fragrance level is quantified by GC as described herein. Based on the package loading, the material is diluted to deliver the appropriate level of fragrance for olfactory evaluation. Optionally, preservatives and suspending agents are added to the diluted package samples.

[0372] Use a low shear micropipette tip to pipette 50 μl of the diluted encapsulant onto a standard microscope slide, covering most of the surface area and leaving the edges sharp for easy handling of the slide. Repeat this at least four times.

[0373] Also transfer 50 μl of the diluted encapsulant slurry onto a rectangular flavor evaluation card blotter paper (approximately 9 cm x 5 cm). Repeat this operation at least twice.

[0374] Slides and blotters were assessed by trained assessors at different time points:

[0375] 1) Immediately after application;

[0376] 2) Place at 20-25°C overnight to dry;

[0377] 3) Leave to dry overnight at 20-25°C, and then rub the slide 10 times (in either direction) with a gloved index finger;

[0378] 4) Leave to dry overnight at 20-25°C, and then press the slide with another slide for 10 seconds and remove.

[0379] 3) Leave to dry overnight at 20-25°C, and then rub the absorbent paper 10 times (in either direction) with another clean absorbent paper.

[0380] Evaluators rated the intensity and commented on the characteristics of the fragrances according to the following scale:

[0381] Odor intensity describe 5 Very strong 4 powerful 3 medium 2 weak 1 Very weak 0 No odor

[0382] Example 1 - Preparation of protein hydrogel slurry with acetic acid

[0383] Preparation of protein hydrogels

[0384] 800 ml of a mixture consisting of 12.5% ​​(w / v) pea protein isolate (PPI) in 30% (v / v) acetic acid solution was prepared.

[0385] The mixture was then heated in a water bath at 85°C, followed by a sonication step to break up large colloidal aggregates (Hielscher UIP) and obtain a clear solution. The energy applied was 200 kJ.

[0386] The solution was then poured into a container with a depth of 1 cm and cooled at 5 °C for 28 h to obtain a free-standing protein hydrogel.

[0387] Applying shear to protein hydrogels

[0388] Shear was then applied to the hydrogel as follows. The protein hydrogel was cut into approximately 1 cm cubes by a low shear cutting step. The cubes were placed in a 75 micron filter bag, which was then submerged in a bucket containing 10 L of reverse osmosis water. This formed a crude protein hydrogel slurry within the filter bag. The hydrogel cubes were kept submerged, with occasional gentle stirring. This step was performed to reduce the concentration of acetic acid in the hydrogel by diffusion into the continuous aqueous phase.

[0389] The filtered gel cubes were transferred to a bottle supplemented with 4 wt% sodium benzoate and then exposed to high shear and subsequent ultra-high shear (probe sonication to 200 kJ with a Hielscher sonicator) on ice to form a homogenous low viscosity protein hydrogel slurry.

[0390] Example 2 - Preparation of spray-dried microcapsules containing vitamin D2

[0391] Preparation of wax / vitamin D2 mixture

[0392] By Vitamin D2 oil (1 MIU / g) in 100% wax was made into a 22 wt% (220,000 IU / g) and 11 wt% (110,000 IU / g) mixture. The wax was melted at above 40°C, the vitamin oil was added, and the mixture was homogenized by shaking by hand. The mixture was kept above 40°C until needed.

[0393] Example 2A: Acid-treated hydrogel slurry solids to wax ratio 5:1, with addition of vitamin D2 by sonication

[0394] 1500 g of protein hydrogel slurry was prepared according to Example 1, wherein the PPI and acetic acid mixture was heated for 20 minutes and the gel cubes were soaked for 2 hours, then high sheared at 15,000 rpm for 15 minutes, and then ultra high sheared at 0.25 kj / ml to form a hydrogel slurry.

[0395] Preparation of composition

[0396] The protein hydrogel slurry was heated to 35°C and 22 wt% of the wax / vitamin D2 molten mixture was added to a weight ratio of 98.3:1.7 for the protein hydrogel slurry to the wax / vitamin D2. The resulting mixture was mechanically high sheared at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser and then ultra-high sheared on ice using a Hielscher sonicator with an energy of 0.1 kJ / ml. This resulted in a viscosity of 27.3 cP and an average droplet size of d 50 A composition of 0.67 microns.

[0397] Spray drying of the composition

[0398] The composition was spray dried according to the process settings in Table 1 to obtain a 23.8 micron average particle size d measured by laser diffraction. 50 The concentration of vitamin D2 in the microcapsule powder was measured using method version A described herein and was found to be 100,250 ug / 100 g.

[0399] Example 2B: Acid-treated hydrogel slurry solids to wax ratio of 2:1, vitamin D2 added by mechanical mixing

[0400] 1500 g of protein hydrogel was prepared according to Example 1, wherein the PPI and acetic acid mixture was heated for 20 minutes and the gel cubes were soaked for 2 hours, then high sheared at 1,5000 rpm for 15 minutes, and then ultra high sheared at 0.25 kj / ml to form a hydrogel slurry.

[0401] Preparation of composition

[0402] The protein hydrogel was heated to 35°C and 11 wt% of the wax / vitamin D2 molten mixture was added to achieve a weight ratio of protein hydrogel slurry to wax / vitamin D2 of 96:4. The resulting mixture was mechanically high sheared at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This resulted in a viscosity of 31.4 cP and an average droplet size of d 50 A composition of 4.6 microns.

[0403] Spray drying of the composition

[0404] The slurry was spray dried according to the process settings in Table 1 to obtain a slurry having an average particle size d of 35.9 μm as measured by laser diffraction. 50 The concentration of vitamin D2 in the microcapsule powder was measured using method version A described herein and was found to be 94,750 ug / 100 g.

[0405] Example 2C: Acid-treated hydrogel slurry solids to wax ratio of 5:1, vitamin D2 added by mechanical mixing

[0406] 1500 g of protein hydrogel slurry was prepared according to Example 1, wherein the PPI and acetic acid mixture was heated for 20 minutes, and the gel cubes were soaked for 2 hours and then high sheared at 15,000 rpm for 15 minutes, and then ultra high sheared at 0.25 kj / ml to form a hydrogel slurry.

[0407] Preparation of composition

[0408] The protein hydrogel slurry was heated to 35°C and 22 wt% of the wax / vitamin D2 molten mixture was added to a weight ratio of 96:4 for the protein hydrogel slurry to wax / vitamin D2. The mixture was mechanically high sheared at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This resulted in a viscosity of 35.8 cP and an average droplet size of d 50 A composition of 6.2 microns.

[0409] Spray drying of the composition

[0410] The composition was spray dried according to the process settings in Table 1. Two different settings were used to produce samples Ci and Cii. The Ci and Cii microcapsules were each measured to have an average particle size of 31.3 microns by laser diffraction measurement. 50 The concentration of Vitamin D2 in the Cii microcapsule powder was measured using Method Version A described herein and found to be 95,500 ug / 100 g.

[0411] Comparative Example 2D: Sonicated PPI (in 5 wt % acetic acid) with a protein solids to wax ratio of 5:1, with vitamin D2 added by mechanical mixing

[0412] Preparation of PPI mixture

[0413] A mixture of 8% wt% pea protein isolate in 5% wt% (v / v) acetic acid solution was prepared to give a solids concentration comparable to the protein hydrogel slurry prepared in Example 1. The mixture was ultra-high sheared to an energy input of 1 kJ / ml using a Hielscher probe sonicator. This produced an average particle size of d 50 The pH of the protein mixture was measured to be 3.4.

[0414] Preparation of composition

[0415] The protein mixture was heated to 35°C and 22 wt% of the wax / vitamin D2 molten mixture was added to give a weight ratio of protein mixture to wax / vitamin D2 of 98.3:1.7. The mixture was mechanically high sheared at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This resulted in a viscosity of 5.0 cP and an average droplet size of 4.4 microns. 50 composition.

[0416] Spray drying of the composition

[0417] The composition was spray dried according to the process settings in Table 1 to obtain a particle size of 20.2 μm as measured by laser diffraction. 50 The concentration of vitamin D2 in the microcapsule powder was measured using method version A described herein and was found to be 86,500 ug / 100 g.

[0418] Comparative Example 2E: Sonicated PPI with a protein solids to wax ratio of 5:1 and vitamin D added by mechanical mixing

[0419] Preparation of PPI mixture

[0420] A slurry of 8 wt% pea protein isolate in reverse osmosis water was prepared to give a solids concentration comparable to the protein hydrogel slurry prepared in Example 1. The mixture was ultra-high sheared to an energy input of 1 kJ / ml using a Hielscher probe sonicator. This produced a particle size of 1.4 microns. 50 The pH of the protein mixture was measured to be 6.85.

[0421] Preparation of composition

[0422] The protein mixture was heated to 35°C and 22 wt% of the wax / vitamin D2 molten mixture was added to give a weight ratio of protein mixture to wax / vitamin D2 of 98.3:1.7. The mixture was mechanically high sheared at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This resulted in a viscosity of 4.9 cP and an average droplet size of d 50 A composition of 4.6 microns.

[0423] Spray drying of the composition

[0424] The composition was spray dried according to the process settings in Table 1 to obtain a 300 μm particle size having an average particle size d of 40.0 μm as measured by laser diffraction. 50The concentration of vitamin D2 in the microcapsule powder was measured using method version A described herein and was found to be 93,000 ug / 100 g.

[0425] Spray Drying Conditions for Examples 2A to 2E

[0426] All compositions prepared in Examples 2A to 2E were spray dried in a concurrent spray dryer with a 1 m diameter chamber and a spray system FloMax nozzle. The inlet air flow was maintained at 1.5 kg / min and the slurry feed addition rate was adjusted within the specified range to maintain the outlet temperature. The feed characteristics and spray drying conditions are summarized in Table 1 below.

[0427]

[0428] Table 1

[0429] The conditions in Table 1 show that although the feed viscosity of inventive Examples 2A, 2B and 2C was higher than that of Controls 2D and 2E, the compositions were successfully spray dried using standard equipment and process conditions. This is believed to be due to the shear thinning properties of the compositions of inventive Examples 2A, 2B and 2C.

[0430] All compositions were successfully spray dried to produce free-flowing powders with an average particle size of d 50 20 microns to 40 microns. Figure 1a and 1b 10X optical microscope pictures of the spray-dried microcapsules of Examples 2B and 2E, respectively, showing the visual similarity of the microcapsule powders produced. To achieve this, a much higher energy level was used in preparing the protein dispersion of Example 2E compared to Example 2B to obtain similar emulsion droplet and microcapsule sizes.

[0431] Example 3 - Preparation of protein hydrogel slurry with lactic acid

[0432] Preparation of protein hydrogels

[0433] 50.0 g of pea protein isolate was added to 212.5 g of reverse osmosis water in a bottle. The bottle was shaken to disperse the protein and 187.5 ml of 85% (v / v) lactic acid was mixed in. The mixture was placed in an 85° C. water bath for 30 minutes and then sonicated with a Bandelin sonicator until 100 kJ was delivered. The hot protein solution was poured into a culture dish and cooled at 5° C. overnight to obtain a self-supporting protein hydrogel.

[0434] Applying shear to protein hydrogels

[0435] The hydrogel was cut into 1 cm cubes and placed in a 75 micron nylon mesh bag in a bucket. 5 L of reverse osmosis water was added and the cubes were soaked for about 1.5 hours with occasional gentle stirring. The water was then drained from the bag and the same soaking procedure was repeated three more times until the pH was above 3.0.

[0436] The hydrogel was then drained and added to a 5 wt% sodium benzoate solution (pH adjusted to 4-5 with lactic acid) at 20 ml per kg of gel. The mixture was sonicated using a Bandelin sonicator until 0.1 kJ / ml was applied. The solids content of the hydrogel slurry was 5.1 wt% as measured by the method described herein.

[0437] Example 4 - Preparation of spray-dried microcapsules containing vitamin D2

[0438] Preparation of composition

[0439] Vitamin D2 oil (1 MIU / g) was added 812N or The granules were diluted to a concentration of 6,000 ug / g in 100% wax. This concentration was chosen to produce spray-dried particles containing 1,000 ug / g of vitamin D2. In the case of 100% wax, melt the wax at above 40°C, add the vitamin oil and homogenize the mixture by shaking by hand. Keep the mixture above 40°C until required.

[0440] The desired mass of oil or wax and vitamin D2 mixture was then added to the protein hydrogel slurry prepared in Example 3 to achieve a mass ratio of hydrogel slurry solids to vitamin D2: wax / oil mixture of 5: 1. The composition was formed by mixing with an IKA Ultra-Turrax at 5000 rpm for 15 minutes followed by probe sonication to 0.1 kJ / ml with a Bandelin sonicator.

[0441] Spray drying conditions

[0442] Spray drying was performed using a ProCept spray dryer with its three-column setup. This provided a column height of 1.8 m and a column diameter of 0.15 m. A two-fluid nozzle with a tip size of 0.8 mm was used. The atomizing air flow rate was 10 ml / ml. The fluid was pumped into the spray dryer at 6 ml / min using a syringe pump.

[0443] The other settings are given in the following Table 2. An additional air flow of 100 l / min was set for the cyclone separator.

[0444]

[0445] Table 2

[0446] The results show that protein hydrogel slurries formed using lactic acid can also be spray dried using standard equipment and under standard conditions. The results also show that a range of different carrier materials for the active ingredient (here vitamin D2) can be used (e.g. oils and waxes).

[0447] The spray drying experiments produced two free-flowing powders with an average particle size of d for Example 4A as measured by laser diffraction. 50 is 15.8 microns and for Example 4B, the average particle size d 50 The concentration of vitamin D2 in the microcapsule powder was measured using method version A described herein and was found to be 89,000 μg / 100 g and 86,500 μg / 100 g in samples 4A and 4B, respectively. Figure 2a and 2b 10X optical microscopy pictures of the spray-dried microcapsules of Examples 4A and 4B, respectively, showing the visual similarity of the powders produced, where differences in particle size can be observed.

[0448] Example 5 - Accelerated stability test of vitamin D2 microcapsules in beverage formulations

[0449] Sample preparation

[0450] The microcapsules of Examples 4A and 4B were each added to Robinson's double concentrated sugar-free apple and blackcurrant juice drinks. More specifically, 45 mg of microcapsules were added to 1 liter of juice drink to provide a concentration of 4.5 ug / 100 ml of vitamin D in the product (assuming a vitamin D concentration of 100,000 ug / 100 g in the microcapsules). A control was also prepared by adding 9 mg of Prinova spray-dried vitamin D2 to the same juice drink sample. Prinova spray-dried vitamin D2 is a commercially available powdered vitamin D2 substance. The three mixtures were heated to 95°C on a hot plate while stirring continuously, and then kept at 95°C for 30 seconds to simulate the temperature profile in a flash pasteurization process. The mixture was then cooled to 20°C.

[0451] Accelerated stability testing

[0452] 200 ml clear plastic PET bottles were filled with each of the three juice beverage samples and placed in a Binder KBFP 240-230V chamber set up for accelerated ambient light stability studies with UV-A set to 1.1 W / m 2, temperature was 20°C, and laboratory humidity (typically 50% relative humidity (RH)) was used. The initial vitamin D2 loading in the juice beverage was measured using the method described herein, and then measured again after 4 weeks in the Binder chamber. The percentage reduction in vitamin D2 content over 4 weeks was calculated and recorded in Table 3. It is expected that vitamin D2 will be significantly degraded by exposure to UV during this period.

[0453]

[0454] Table 3

[0455] As can be seen from Table 3, after 4 weeks in the fruit juice beverage, the commercially available powdered vitamin D2 was almost completely degraded, with 95% of the initial vitamin D2 being degraded.

[0456] Figure 3 is a 10x microscope image of a powdered control vitamin D2 added to cold tap water showing the presence of a fine dispersion of material but no microcapsules. Without wishing to be bound by theory, it is believed that the carrier material in the powder is highly soluble in water, so it decomposes in water and thus fails to protect the vitamin D2 in the accelerated stability test.

[0457] However, for Example 4A, after 4 weeks in the juice beverage, the level of vitamin D2 was still about half of the original level, indicating that the microcapsules with a plant-based protein carrier and an oil-based active carrier phase protected it from exposure to UV and thereby reduced degradation. The presence of an insoluble plant-based protein carrier significantly helped to protect the encapsulated vitamin D2 compared to the commercially available materials tested in the control experiment. In addition, for Example 4B, after 4 weeks in the juice beverage, the level of vitamin D2 was essentially unchanged compared to the original level, indicating that the microcapsules with a plant-based protein carrier and a wax-based active carrier phase were able to fully protect the vitamin D2 from degradation. Without wishing to be bound by theory, it is believed that the use of a solid wax carrier for vitamin D2 means that the material surrounding the vitamin is more opaque and therefore provides even better UV protection for the vitamin.

[0458] Example 6 - Preparation of Spray-dried Fragrance Microcapsules

[0459] Preparation of fragrance / solvent mixtures

[0460] Preparation of δ-damascone by gentle mixing 812 (weight ratio is 80:20).

[0461] Preparation of spray-dried microcapsules

[0462] The protein hydrogel slurry was prepared according to Example 1, wherein the PPI and acetic acid mixture was heated for 30 minutes, the gel cubes were soaked for 1 hour, and then high sheared at 25,000 rpm for 15 minutes using an IKA Ultra-Turrax, and then ultra-high sheared to 1.0 kJ / ml using sonication (Hielscher sonicator) to form a hydrogel slurry.

[0463] The average particle size of the hydrogel slurry 50 This was measured as described herein and found to be 2.5 microns. The average solids content of the hydrogel slurry was measured as described herein and found to be 8.6 wt%.

[0464] Two samples with the compositions shown in Table 4 were prepared by pouring the fragrance / solvent mixture into the hydrogel slurry and mixing with an Ultra-Turrax at 15,000 rpm for 5-15 seconds to obtain the desired dispersed droplet size of 5 μm. Table 4

[0465]

[0466] Table 4

[0467] Example 6A phase separated easily to produce a clear liquid top layer and a hydrogel fragment bottom layer, but could be made homogenous again by vigorous mixing before spray drying.The well mixed slurry had a viscosity of 150 cP at 50 / sec.

[0468] A 1.5 mg droplet of Example 6A was suspended on a thin wire connected to a microbalance and placed in the middle of a tube with an inner diameter of 2.5 cm. Air at a temperature of 97°C and a velocity of 0.85 m / s was passed around the droplet and the weight of the droplet was recorded over 11 minutes. At the end of the experiment, the dried droplet was carefully separated from the wire and placed on a Hitachi TM3030 SEM mounting pad for examination.

[0469] like Figure 4a and 4b As shown in the SEM images, the obtained particles have an internal matrix structure and an external smooth skin.

[0470] Example 6B was homogeneous, viscous and semisolid with a viscosity of 1000 cP at 50 / sec.

[0471] A 2.5 mg droplet of Example 6B was suspended on a thin wire connected to a microbalance and placed in the middle of a tube with an inner diameter of 2.5 cm. Air at a temperature of 85°C and a velocity of 0.85 m / s was passed around the droplet and the weight of the droplet was recorded over 3.5 minutes. At the end of the experiment, the dried droplet was carefully separated from the wire and placed on a Hitachi TM3030SEM mounting pad for examination.

[0472] like Figure 5a , 5b As shown in the SEM images of 5 and 5c, the obtained particles have a matrix structure and a porous surface.

[0473] Example 7 - Preparation of Spray-dried Fragrance Microcapsules

[0474] Preparation of fragrance / solvent mixtures

[0475] Preparation of δ-damascone by gentle mixing 812 (volume ratio of 80:20).

[0476] Preparation of spray-dried microcapsules

[0477] A protein hydrogel slurry was prepared according to Example 1. The hydrogel slurry was diluted with acetic acid to an average protein solids content of 8.0 wt%. The 221 g batch was stirred at 8000 rpm for 2 minutes using an UltraTurrax laboratory mixer. Then 131 g of a 1% ethanol solution containing 80% delta-damascone and 20% 812 Oil Fragrance: Oil Mixture. The composition is given in Table 5 below.

[0478]

[0479]

[0480] Table 5

[0481] The batch was subjected to high shear for 5 minutes using an UltraTurrax laboratory mixer set at 8000 rpm to produce a miniemulsion. The droplet size was expected to be approximately 5 microns.

[0482] The sample was then spray dried using a ProCepT R&D spray dryer with its three-column setup. This provided a column height of 1.8 m with a column diameter of 0.15 m. The air inlet temperature was 130°C and the flow rate was 550 l / min. A two-fluid nozzle with a tip size of 1.0 mm was used. The atomizing air flow rate was 10 l / min. The fluid was pumped into the spray dryer at 6 ml / min using a syringe pump. An additional air flow of 140 l / min was introduced below the drying chamber to deliver air to the cyclone separator.

[0483] The dried microcapsule powder formed in Example 7 was collected from the cyclone separator, and was also collected from the inner surface of the device to which it adhered (eg, in a transfer pipe leading to the cyclone separator).

[0484] The microcapsules of Example 7 were examined under Hitachi TM3030 SEM, and it was observed that there were mainly spherical flavorant encapsulations ( Figure 6 ). The microcapsules appeared to have a pockmarked surface, indicating that there was very little free flavorant present and that the sample was reasonably dry.

[0485] The microcapsules of Example 7 were rehydrated in hard water and optical microscope photographs were taken ( Figure 7a The same capsules are then squeezed, causing them to burst and release the oil within ( Figure 7b ). The oil droplets around the ruptured microcapsules are clearly visible, with a very well defined black interface between the oil and water.

[0486] The microcapsules of Example 7 were analyzed by the method described herein to determine their total fragrance loading. It was found to be 28.8%.

[0487] Example 8 - Preparation of protein hydrogel slurry with acetic acid

[0488] Preparation of protein hydrogels

[0489] 1120 g of reverse osmosis (RO) water was added to a 2-liter stainless steel container and 216 g of pea protein isolate was added. The container was placed in a 92°C water bath and mixed with an overhead stirrer at 1500 rpm. After stirring for 3 minutes, 480 grams of glacial acetic acid were added. The mixture was stirred at 1500 rpm for 15 minutes and then at 1200 rpm 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 about 10 mm and left overnight at room temperature.

[0490] Applying shear to protein hydrogels

[0491] Then apply shear to the hydrogel as follows. The protein hydrogel is cut into approximately 1 cm cubes by a low shear cutting step. The cubes are divided into two 75 micron filter bags, and each filter bag is then immersed in a bucket containing 16L RO water. This forms a crude protein hydrogel slurry in the filter bag. The hydrogel cubes are soaked for 90-150min and stirred with an overhead stirrer at 600-800rpm. This step is performed to reduce the concentration of acetic acid in the hydrogel by diffusion to the continuous aqueous phase. The pH of the wash water is then measured, and if it is higher than 3.2, soak for another 30 minutes. If it is lower than 2.9, half of the water is drained and replaced with fresh RO water, and then soak for another 30 minutes. The filter bag is then suspended above the bucket to drain for 5 minutes. The washed gel from the two filter bags is transferred to a 5-liter beaker and homogenized with a Silverson mixer at 5000rpm for 5 minutes, at 6000rpm for 5 minutes, and at 7000rpm for 5 minutes. The smooth slurry was then transferred to 1 L Nalgene bottles (800 g per bottle) and exposed to high shear sonication (Hielscher UP500Hdt) with cooling on ice until 250 kJ was applied, with shaking every 75 kJ. The hydrogel slurry was then passed through a 200 micron sieve before use.

[0492] Comparative Example 9 - Preparation of a protein slurry without acid

[0493] 450 grams of reverse osmosis water were added to a 1 liter stainless steel container and 50g of pea protein isolate was added. The container was placed in a 92°C water bath and mixed with an overhead stirrer at 1500rpm. 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 cooled to ambient temperature and, contrary to the experiment using acetic acid, no gel was formed. Shear was then applied at 8000rpm with a Silverson mixer for 10 minutes and then processed with an ultrasonic processor (Hielscher UP500Hdt) until 156kJ was applied. For comparison, the protein slurry prepared with organic acid used 0.3125kJ / g. The mixture was then passed through a 200 micron sieve before use.

[0494] Example 10 - Preparation of spray-dried microcapsules containing vitamin D2

[0495] Preparation of composition

[0496] Vitamin D2 oil (1 MIU / g) was added 100% wax was diluted to a concentration of 6000 ug / g. This concentration was chosen with the goal of producing spray-dried particles containing 1,000 ug / g of vitamin D2. The wax was melted at above 40°C, the vitamin oil was added, and the mixture was homogenized by shaking by hand. The mixture was kept above 40°C until needed.

[0497] The desired mass of the wax and vitamin D2 mixture was then added to the protein hydrogel slurry of Example 8 and mixed at 7000 rpm for 10 minutes using a Silverson L5M-A homogenizer to form a composition containing 11.9% by weight of total solids (including vitamin D2 and wax). The composition was then further diluted with 3.0% aqueous acetic acid to produce 2 samples: Example 10A, wherein the total solids content including vitamin D2 and wax was 10.0% (8.9% by weight of protein solids); and Example 10B, wherein the total solids content including vitamin D2 and wax was 5.0% (4.4% by weight of protein solids).

[0498] The desired mass of the wax and vitamin D2 mixture was also added to the protein slurry of Comparative Example 9 and mixed with a Silverson L5M-A homogenizer at 7000 rpm for 10 minutes to form a composition. The composition was then further diluted with a 3.0% aqueous acetic acid solution to produce Comparative Example 10C, wherein the total solids content including vitamin D2 and wax was 11.7% by weight (10 wt% protein solids).

[0499] Spray drying conditions

[0500] For Examples 10A and 10B, spray drying was carried out using a ProCept spray dryer in its three-column setup. This provided a column height of 1.8 m and a column diameter of 0.15 m.

[0501] For Example 10A, a two-fluid nozzle with a tip size of 1 mm was used. The atomizing air flow rate was 10 l / min. The fluid was pumped into the spray dryer at 6 ml / min using a syringe pump. An additional air flow of 150 l / min was set for the cyclone separator.

[0502] For Example 10B, an ultrasonic nozzle was used. The atomizing air flow rate was 10 l / min. The fluid was pumped into the spray dryer at 6 ml / min using a syringe pump. An additional air flow of 150 l / min was set for the cyclone separator.

[0503] For Comparative Example 10C, spray drying was performed using a Buchi B290 spray dryer using a two-fluid nozzle with a tip size of 1.4 mm. The aspirator flow rate was set to 100% and the Q-flow was set to 40. The fluid was pumped into the spray dryer using a peristaltic pump at a speed setting of 35% (about 10 ml / min). The dry microcapsule powder formed was collected from a collection tank.

[0504] Other process settings are given in Table 6 below.

[0505]

[0506] Table 6

[0507] The spray drying experiments produced three free-flowing powders. For Examples 10A, 10B and Comparative Example 10C, the average particle size d was measured by laser diffraction according to the method described therein. 50 28.9 microns, 40.8 microns and 9.1 microns respectively. It should be noted that for the acid-treated vegetable protein encapsulation, the more dilute the composition dried with the ultrasonic nozzle, the larger the microcapsules produced. The non-acid-treated vegetable protein powder with similar solid content has a much smaller average particle size.

[0508] The vitamin D2 concentration in the microcapsule powder was measured using method version B described herein and found to be 963 μg / g, 919 μg / g, and 1149 μg / g in Examples 10A, 10B, and Comparative Example 10C, respectively. This is close to the expected target of 1000 μg / g microcapsules, indicating that there is little loss of vitamin D2 during the spray drying process.

[0509] Example 11 - Accelerated stability test of vitamin D2 microcapsules in beverage formulations

[0510] Preparation of beverage samples

[0511] The microcapsules of Examples 10A, 10B and Comparative Example 10C were prepared into concentrated slurries respectively, so that they could be subsequently added to Miwadi black currant single concentrate juice beverage.

[0512] 0.2 g of each microcapsule was first suspended in 30 g of boiling water, shaken vigorously, and then vortexed at 2000 rpm for 1 minute to simulate the temperature profile during flash pasteurization. It should be noted that the concentrated slurry prepared with Comparative Example 10C was more turbid and appeared finer than Examples 10A and 10B. Figure 8a , 8b8c are 10x microscope images of the concentrated slurries of Examples 10A, 10B and Comparative Example 10C. The microcapsules of Examples 10A and 10B can be seen and they clearly withstood the treatment. For Comparative Example 10C, the image is notable for the lack of intact microcapsules, indicating that heating and shearing thereof resulted in rapid disintegration of the microcapsules. Instead, smaller protein aggregates and very small wax and vitamin D2 particles can be observed.

[0513] Accelerated stability testing

[0514] The calculated mass of each concentrated slurry was added to 2 liters of the juice beverage and mixed with an overhead mixer at 800 rpm for 5 minutes to ensure homogeneity before transferring to bottles.

[0515] A 200 ml clear plastic PET bottle was filled with each of the three juice drink samples and the initial vitamin D2 loading was measured using the method described herein. Table 7 gives the initial vitamin D loading of the beverages of Examples 10A and 10B. However, in Comparative Example 10C, the vitamin D2 loading of the beverage initially measured was not detected. Without wishing to be bound by theory, it is believed that this is because in the concentrated slurry preparation, the microcapsules are suspended and heated, and they are destroyed to release the vitamin D2. The vitamin D2 oil floats on the top of the bottle without being sampled.

[0516] The filled PET bottles of Examples 10A and 10B were then placed in a Binder KBF P240-230V chamber set up for accelerated ambient light stability studies with the UV-A setting at 1.1 W / m 2 , temperature was 20°C, and laboratory humidity (typically 50% relative humidity (RH)) was used. After 4 weeks in the Binder chamber, the vitamin D loading was measured again. The percentage reduction in vitamin D2 content was calculated and recorded in Table 7.

[0517]

[0518] Table 7

[0519] After 4 weeks in the juice beverage, the vitamin D2 in the encapsulates of the present invention was protected from degradation, so that only 22.5% of the initial vitamin D2 was lost for Example 10A, and only 5.5% of the initial vitamin D2 was lost for Example 10B. It is expected that unencapsulated vitamin D2 would degrade significantly when exposed to UV during this period of time, and this low level of loss demonstrates the protection provided by the microcapsules of the present invention. It can also be seen that the larger the average encapsulate size, the lower the loss. Without wishing to be bound by theory, it is believed that the larger the average particle size of the encapsulate, the less surface area available per mass of vitamin D2 when exposed to UV, and therefore the less degradation.

[0520] Example 12 - Encapsulation of natural agrochemical active substances

[0521] Example 12A: Thymol protein microcapsule formulation

[0522] Preparation and application of shear to protein hydrogels

[0523] Protein hydrogel shells were prepared and sheared as described in Example 8.

[0524] The protein solids content was measured to be 9.3 wt%. Dilute acetic acid (3 wt% in DI water) was added to reduce the protein solids content to 8.0%.

[0525] Preparation of spray-dried microcapsules

[0526] The diluted dispersion was homogenized with a Silverson L5M-A high shear mixer (homogenized at 8000 rpm for 2 minutes). 16.81 g of thymol diluted at 40 wt% in Miglyol 812N was added to 103.25 g of the diluted dispersion. The mixture was homogenized for an additional 5 minutes at 8000 RPM with a Silverson. The droplet size was expected to be about 5 microns.

[0527] The sample was then spray dried using a Buchi B290 spray dryer. The air inlet temperature was 120°C and the Q-flow was set to 40. A two-fluid nozzle with a tip size of 1.4 mm was used. The aspirator flow rate was set to 100%. The fluid was pumped into the spray dryer using a peristaltic pump at a speed setting of 13% (4-5 ml / min).

[0528] The resulting dry microcapsule powder was collected from a collection tank. The microcapsules were analyzed to determine their total thymol loading. The total thymol loading was found to be 4.9 wt%. The particle size was measured by laser diffraction and was found to have a d of 23 microns. 50 .

[0529] Example 12B: Comparative maltodextrin thymol microcapsule formulation

[0530] 45 g of maltodextrin was dissolved in 105 g of deionized water and mixed with a magnetic stirrer until a uniform slurry was formed. In addition to 0.14 g of polysorbate 80, 15.23 g of thymol (diluted at 40 wt % in Miglyol 812N) was added to the slurry. The mixture was homogenized at 8000 rpm for 5 minutes using a Silverson L5M-A high shear mixer.

[0531] The sample was then spray dried using a Buchi B290 spray dryer. The air inlet temperature was 130°C and the Q-flow was set to 40. A two-fluid nozzle with a tip size of 1.4 mm was used. The aspirator flow rate was set to 100%. The fluid was pumped into the spray dryer using a peristaltic pump at a speed setting of 13% (4-5 ml / min).

[0532] The resulting dry microcapsule powder was collected from a collection jar. The microcapsules were analyzed to determine their total thymol loading. The total thymol loading was found to be 3.4 wt%. The particle size of these microcapsules could not be measured because they were too fragile. When placed on a microscope slide with a cover glass, it was observed that the weight of the cover glass was sufficient to break them.

[0533] Example 12C: Aqueous Environment Release Test

[0534] The thymol microcapsule powder of Example 12A was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben and sodium acetate and calcium chloride solutions according to Table 8, so that the sample contained 4 mg thymol. Replicate samples were prepared so that samples could be analyzed at incubation times of t=0, 24 hours, 72 hours, 1 week, and 2 weeks.

[0535]

[0536] Table 8 - Composition of test samples

[0537] The samples were mixed on day 0 and placed in a dark incubator at 37°C except for the sample at t=0, 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 dilute the supernatant sample 10 times. The concentration of thymol in the supernatant was determined by gas chromatography, which was used to calculate the amount of thymol released from the microcapsules.

[0538] 2.9ml DI water and 0.1ml 10% KOH solution were added to the pelleted material after each sample centrifugation. The mixture was subjected to ultrasonic treatment (Bandelin Sonopuls HD4200, using probe TS104) for 2 minutes with 30% amplitude, vortexed and centrifuged at 4900rpm for 5 minutes. The 100 μl supernatant from each sample was diluted 10 times with ethanol, and the concentration of thymol was then measured by gas chromatography. This was used to calculate the amount of thymol remaining in the sample at the end of the experiment, which was considered to be the amount of thymol that was not initially released in the first supernatant. The results are shown in Table 9.

[0539]

[0540] Table 9

[0541] This release study showed that over 2 weeks, most of the active ingredient (over 90%) remained encapsulated. This slurry is suitable for storage prior to application to fields and crops.

[0542] Example 12D: Comparative maltodextrin-thymol release test

[0543] The thymol microcapsule powder of Comparative Example 12B was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben according to Table 10, so that the sample contained 4 mg thymol, the same level as in Example 12A. There were only minor differences in the suspension composition due to the absence of low levels of NaOAc and CaCl2 (which were insignificant in this example).

[0544]

[0545] Table 10

[0546] Prepare the sample, mix and incubate for one hour. Centrifuge the sample at 4900 RPM for 10 minutes and transfer the supernatant to a 50 ml Falcon tube. Add ethanol to dilute the supernatant sample 10 times. The concentration of thymol in the supernatant was determined by gas chromatography, which was used to calculate the amount of thymol released from the capsules in buffered water.

[0547] 2.9 ml of deionized water and 0.1 ml of 10% KOH solution were added to the pelleted material after the sample was centrifuged. The mixture was ultrasonically treated (Bandelin Sonopuls HD4200, using probe TS104) for 2 minutes with 30% amplitude, vortexed and centrifuged at 4900 rpm for 5 minutes. 100 μl of supernatant was diluted 10 times with ethanol, and the concentration of thymol was then measured by gas chromatography. This was used to calculate the amount of thymol remaining in the sample at the end of the experiment, which was considered to be the amount of thymol that was not initially released in the first supernatant. The results are shown in Table 11.

[0548]

[0549] Table 11

[0550] This release study shows that for maltodextrin microcapsules, in Comparative Example 12B, most of the active ingredient (more than 40%) was released in the aqueous buffer after 1 hour. This slurry is not suitable for storage before application to fields and crops. After being formulated into an aqueous composition, or in the presence of water (e.g., rainwater) in the environment, it releases the active ingredient prematurely, showing no controlled release properties.

[0551] Example 13 - Preparation of Spray-Dried Microcapsules Containing Fragrance Oil

[0552] Fragrance / Solvent Mixtures

[0553] Two different flavors (13A and 13B) were prepared according to Table 12. Each flavor was then mixed with one of the two solvents at a weight ratio of 80:20 flavor:solvent: 812N or isopropyl myristate (IPM).

[0554] Fragrance 13A contains only moderately volatile fragrance materials with similar volatility, ranging from very low to very high hydrophobicity. Fragrance 13B contains materials with similar hydrophobicity, with a full range of volatility, including low, medium and high volatility materials. Both fragrances have fragrance materials with the following range of HSP parameters: δD of 15 to 20, δP of 1 to 7 and δH of 2.5 to 8.

[0555]

[0556] Table 12

[0557] Preparation of spray-dried microcapsules

[0558] The protein hydrogel slurry was prepared according to Example 8.

[0559] The first batch of flavor composition was prepared as follows. A batch of 100 to 120 g of the protein hydrogel slurry of Example 8 was diluted 20% with 3% acetic acid solution to obtain a slurry having a protein solids content of 8.0 wt%. The diluted slurry was then stirred in a 400 ml Nalgene beaker with a Silverson laboratory mixer at 8000 rpm for 2 minutes. Then 31.5 wt% (based on the weight of the slurry) of flavor The 812N mixture was added to the slurry and further emulsified using a Silverson mixer at 8000 rpm for 5 minutes to form a mini-emulsion. The mini-emulsion was aged at room temperature for about 2 days and then spray dried. This mixture was used for the second round of TTB.

[0560] A second batch of flavor composition was prepared as described above, except that the flavor mixture was emulsified by gentle hand shaking rather than using a Silverson mixer just prior to spray drying. This mixture was used for the third round of TTB.

[0561] The third batch of fragrance composition was prepared according to the method of the first batch, except that isopropyl myristate (IPM) was used instead of 812N to form a flavor: solvent mixture. The flavor and slurry mixture was further emulsified using a Silverson mixer at 8000 rpm for 5 minutes to form a mini-emulsion. The mini-emulsion was aged at room temperature for about 2 days and then spray dried. This mixture was used for the 6th round of TTB.

[0562] A fourth batch of flavor composition was prepared with the same composition and in the same manner as the materials used in the 6th round of TTB and was used in the 7th round of CPB and the 8th round of TTB.

[0563] The samples were spray dried using a ProCepT R&D spray dryer in its three-column setup. This provided a column height of 1.8 m with a column diameter of 0.15 m.

[0564] Change the air inlet temperature to obtain dry powder. Keep the air inlet temperature as low as possible to reduce the loss of volatile aroma substances. Keep the drying inlet air flow rate constant at 0.4m 3 / min. A two-fluid nozzle from Spray Systems was used with a tip size of 1.0 mm. The atomizing air flow rate varied between runs as shown in Table 13 below. For all runs, the fluid was pumped into the spray dryer at 4 ml / min using a syringe pump. An additional air flow of 150 l / min was introduced below the drying chamber to deliver air to the cyclone separator.

[0565] The formed dry microcapsule powder was collected in a collection tank and was also collected from the inner surface of the equipment to which it adhered (eg in a transfer pipe leading to a cyclone separator).The particle size of the microcapsules was measured by laser diffraction.

[0566] Flavor 13B was successfully encapsulated as shown in Table 13.

[0567]

[0568] Table 13

[0569] When drying at a lower temperature (inlet temperature about 69 ° C), use Fresh coarse emulsion prepared with 812N as solvent produced a maximum particle size of 30.1 microns. The flavor solvent in the aged miniemulsion was changed from Changing 812N to isopropyl myristate had little effect on particle size.

[0570] Reducing the atomizing air flow rate resulted in larger particles (compare 8th round TT to 7th round CP).

[0571] The aroma composition of these microcapsules was analyzed using the aroma loading quantification method via GC described herein. The results are given in Table 14.

[0572]

[0573] Table 14

[0574] Samples dried at lower air inlet temperatures generally had higher total aroma loadings than samples dried at higher air inlet temperatures.

[0575] When dried at lower temperatures (inlet temperature about 69°C), for the miniemulsions, isopropyl myristate resulted in a higher 812 N higher total loading. Also, under these drying conditions, the coarse emulsion resulted in higher loading.

[0576] When drying at higher temperatures (at least 80°C at the inlet) and the atomizing air flow rate is reduced, higher total loadings are obtained than with high air atomization.

[0577] Fragrance 13B demonstrates that for fragrances of similar hydrophobicity, a range of volatilities can be encapsulated. However, as expected, the least volatile fragrances were enriched in the encapsulates, with benzyl salicylate accounting for 48.4% to 56.3% of the fragrance, whereas it accounted for 20% of the fragrance before encapsulation. Drying at lower temperatures and using a coarse emulsion improved the encapsulation of more volatile fragrances (e.g., limonene) from 0.6% to 1.5% of the fragrance.

[0578] Flavor composition 13A was also successfully encapsulated as shown in Table 15.

[0579] The first batch of flavor composition was prepared as follows. A batch of 100 to 120 g of the protein hydrogel slurry of Example 8 was diluted 20 wt% with 3% acetic acid solution to obtain a slurry having a protein solids content of 8.0 wt%. The diluted slurry was then stirred in a 400 ml Nalgene beaker with a Silverson laboratory mixer at 8000 rpm for 2 minutes. Then 28.3 wt% (based on the weight of the slurry) of the composition The 812N mixture was added to the slurry and further emulsified using a Silverson mixer at 8000 rpm for 5 minutes to form a mini-emulsion. The mini-emulsion was aged at room temperature for about 2 days and then spray dried. This mixture was used for the first round of TTA.

[0580] A second batch of fragrance composition was prepared as described above. Immediately before use, 120 g of the emulsion was diluted with 60 g of 3% acetic acid solution while gently stirring. This mixture was used for the 9th round of TTA.

[0581] A third batch of flavor composition was prepared in the same manner as the first batch described above, except that the flavor mixture was sheared and emulsified rather than aged prior to spray drying. This mixture was used in a third round of TTA.

[0582] A fourth batch of flavor composition was prepared as described above, except that the flavor mixture was emulsified by gentle hand shaking rather than using a Silverson mixer just prior to spray drying. This mixture was used for the second round of TTA.

[0583] A fifth batch of fragrance composition was prepared in the same manner as the first batch, but now using isopropyl myristate (IPM) instead of 812 N. This mixture was used for the 4th round of TTA.

[0584] A sixth batch of flavor composition was prepared in the same manner as the fifth batch, and this mixture was used in the fifth CPA run.

[0585] The same equipment as in the previous run was used, and the drying air flow rate, emulsion flow rate, and transfer air flow rate were the same as for the fragrance composition 13B. The dry microcapsule powder formed was also collected in a collection tank, and was also collected from the inner surface of the equipment to which it adhered (e.g., in the transfer pipe leading to the cyclone separator). The particle size of the microcapsules was measured by laser diffraction.

[0586]

[0587]

[0588] Table 15

[0589] Before spray drying The fresh coarse emulsion prepared with 812N as the flavor solvent produced the largest particle size.

[0590] by Aged miniemulsion with 812N as the fragrance solvent produced the smallest microcapsule particle size. Dilution of the aged miniemulsion and drying at lower temperatures did not affect the particle size. Changing 812N to isopropyl myristate had little effect on particle size.

[0591] After aging the spray dried miniemulsion had a smaller particle size than the same freshly prepared emulsion.

[0592] The aroma composition of these microcapsules was analyzed using the aroma loading quantification method via GC described herein. The results are given in Table 16.

[0593]

[0594]

[0595] Table 16

[0596] All samples had similar total fragrance loading, but the aged miniemulsion sample with IPM, sprayed at a lower air atomization rate, had the highest loading of 30%. This was significantly lower than fragrance composition 13B, which had the highest total fragrance loading of 51%. Without wishing to be bound by theory, this may be due to the presence of very low logP fragrance materials in fragrance 13A.

[0597] Fragrance composition 13A comprises 4 kinds of fragrance materials with similar medium volatility and different hydrophobicity, and shows that materials with a range of hydrophobicity can be encapsulated. For all embodiments, the composition of the encapsulated fragrance material is rich in the fragrance material with the strongest hydrophobicity, dodecanitrile. This is least obvious in the crude emulsion sample.

[0598] Delta damascone was the least enriched aroma compound, although it had a higher logP than geraniol and methyldecenol. Both alcohols (especially geraniol) were well encapsulated, indicating that these relatively hydrophilic aroma compounds can still be encapsulated in fragrances.

[0599] Olfactory evaluation of spray-dried microcapsules

[0600] Prior to preparing the diluted preparations for olfactory evaluation, a concentrated slurry of the microcapsules from round 3 of TTA was prepared. Some single strength buffer solution with preservative was added dropwise to the dried microcapsules in a 50 ml Falcon tube, vortexing at 2000 rpm between drops, to obtain a slurry paste. When the paste appeared to be homogenous, more buffer solution was added to dilute further, still using vortexing as a means of ensuring uniform dispersion of the microcapsules. The suspension was allowed to stand for 1 hour to allow for possible swelling of the microcapsules and then filtered through a 40 μm cell strainer for 5 minutes. Any excess moisture was absorbed using a paper towel.

[0601] The total fragrance level was 12.6 wt % by GC analysis according to the method herein. This is lower than the total fragrance level of the spray dried microcapsules. Without wishing to be bound by theory, it is believed that hydration and swelling of the hydrogel shell occurred, resulting in an increase in the mass of the shell. In addition, a small amount of surface fragrance may have been lost in the aqueous phase.

[0602] Prepare a dilute slurry of microcapsules containing gellan gum to contain 0.1wt% of total flavor for olfactory evaluation. Gellan gum helps to keep the microcapsules well suspended. Prepare a gellan gum solution by mixing 9.5mg gellan gum with 15g deionized water in a 50ml Falcon tube. The mixture is heated to above 90°C for 20 minutes and stirred at 500rpm using an Eppendorf Thermomixer C set at 100°C. After cooling to room temperature, 15g of double strength buffer solution is added. Use a micropipette with a low shear tip to add the concentrated encapsulated material slurry to the gellan gum solution, close the sample tube, and repeatedly invert to mix.

[0603] The pH of the diluted slurry was 3.05. The odor of the diluted slurry was evaluated according to the method described herein and the results are shown in Table 17.

[0604]

[0605] Table 17

[0606] Upon initial application, there was a strong characteristic odor of the fragrance, indicating that some fragrance oil was associated with the surface of the hydrogel shell. After drying for 24 hours, the odor was very faint, indicating that most of the surface fragrance had evaporated. When rubbed or pressed on glass, a strong characteristic odor was felt as the force caused the microcapsules to rupture, releasing the fragrance. When rubbed on card, the odor was of medium intensity and the fragrance characteristics were slightly different from those on glass, indicating that the encapsulated fragrance was only partially released.

[0607] Example 14 - Cross-linking of Spray-dried Fragrance Microcapsules

[0608] Spray-dried microcapsules were prepared as described in Example 13, 3rd round of TTA.

[0609] Hard water adjusted to pH 3 was added dropwise to 0.8 g of dried microcapsules in a 50 ml Falcon tube, vortexing at 2000 rpm between additions to obtain a slurry paste. When the paste appeared to be uniform, more pH 3 hard water was added to further dilute, still using vortexing as a means to ensure uniform dispersion of the microcapsules. The suspended microcapsules were further diluted in a total of 390 ml of hard water and the pH was adjusted to 3.0 ± 0.5.

[0610] Shake by hand and use Fisherbrand TM A 10 wt% tannic acid solution was prepared by vortexing with a ZX4 IR vortex mixer for a few seconds followed by immersion in an ultrasonic bath set at 50°C for 5-10 minutes until complete dissolution.

[0611] 10.6 g of tannic acid solution was added to the microcapsule suspension at 50°C with mechanical stirring at 170 rpm and stirred for 5.5 hours. After this time, the suspension was stored in a refrigerator for about 36 hours. The cross-linked microcapsule suspension was poured onto a 38 μm sieve, washed with hard water at pH 3, and resuspended in hard water at pH 3 in a clean separatory funnel. The decanted capsules were collected a second time on a 38 μm sieve, washed with a single strength buffer solution with a preservative, and resuspended in the buffer solution in a separatory funnel. The capsules were collected as a concentrated slurry after decantation and appeared as brown microcapsules, indicating that cross-linking had occurred.

[0612] The cross-linked capsule slurry was directly filtered through a 40 μm cell strainer for 5 minutes and any excess water was absorbed with a paper towel. The total fragrance level was analyzed by gas chromatography according to the method herein and was 4.1 wt%. This is lower than the total fragrance level of the microcapsules before cross-linking. Without wishing to be bound by theory, it is believed that this is due to the removal of surface and loosely bound fragrance by the washing process during cross-linking. The particle size was measured by laser diffraction and obtained a D of 47.9 μm. 50 This is larger than the particle size of the original spray-dried encapsulate, indicating that the hydrogel shell has hydrated and the encapsulate has expanded.

[0613] Olfactory evaluation of spray-dried and cross-linked microcapsules

[0614] A diluted slurry of microcapsules with gellan gum was prepared to contain 0.1 wt% total flavor. Gellan gum helps keep the microcapsules well suspended. This was done according to the method in Example 13. The pH of the diluted slurry was 3.04. The odor of the diluted slurry was evaluated according to the method described herein, and the results are shown in Table 18.

[0615]

[0616]

[0617] Table 18

[0618] Upon initial application, there was a moderate characteristic odor of the fragrance, indicating that a small amount of the fragrance was associated with the surface of the hydrogel shell. After drying for 24 hours, the odor was very faint, indicating that most of the surface fragrance had evaporated. When rubbed or pressed on glass, a strong characteristic odor was felt as the force caused the microcapsules to rupture, releasing the fragrance. When rubbed on card, a moderate odor was felt, and the fragrance profile was similar to that on glass.

[0619] Example 15 - Preparation of spray-dried microcapsules containing linseed oil

[0620] Preparation and application of shear to protein hydrogels

[0621] A protein hydrogel slurry was prepared as described in Example 8. The protein solids content was measured to be 9.5 wt%.

[0622] Preparation of spray-dried microcapsules

[0623] Flaxseed oil is a dietary supplement used due to its high levels of omega-3 fatty acids, which are also a vegetarian alternative to fish oil. The slurry was homogenized with a Silverson L5M-A high shear mixer at 8000 rpm for 2 minutes. Flaxseed oil was added to the slurry at a 1:1 oil:protein solids weight ratio, and the mixture was further homogenized with a Silverson at 8000 RPM for 5 minutes to create a fine emulsion.

[0624] The slurry was spray dried on the day of preparation using a Buchi B290 spray dryer using a two-fluid nozzle with a tip size of 1.4 mm. The aspirator flow rate was set to 100% and the Q-flow was set to 40. The fluid was pumped into the spray dryer with a peristaltic pump at a speed setting of 13% (4-5 ml / min). The air inlet temperature was varied between 183°C and 180°C, and the air outlet temperature was varied between 126°C and 122°C.

[0625] A dry microcapsule powder was formed, indicating that the oil was efficiently encapsulated. The particle size was measured by laser diffraction according to the method herein and was found to have a d of 45.3 microns. 50 The powder had no rancid off-odor, indicating that the temperatures in the dryer had not degraded the linseed oil. It had a faint nutty flavor and a very slight vinegar flavor, indicating only low levels of surface oil.

[0626] Fig. 9A 10x optical microscope image of Example 15 suspended in single strength citrate buffer is shown, from which it can be seen that the encapsulation remains intact.

[0627] The microcapsule powder of Example 15 was also suspended in Miwadi Blackcurrant Single Concentrate by vortexing at 2000 rpm for 1 minute at a level of 12.5 g / L. The encapsulate did not impart any negative aroma to the characteristic red fruit flavor of the original product, indicating that the encapsulate was insoluble and that the linseed oil remained encapsulated and therefore had very little impact on the product flavor. In contrast, the same level of linseed oil could not be incorporated into the beverage without an emulsifier and provided a very noticeable nutty aroma and characteristic impact on the taste of the beverage.

[0628] Example 16 - Demonstration of protein gel formation

[0629] Reverse osmosis (RO) water and pea protein isolate were added to a 1 liter stainless steel container and placed in a 92°C water bath and mixed with an overhead stirrer at 1300 rpm. After stirring for 3 minutes, glacial acetic acid was added. The mixture was stirred at 1300 rpm for a total of 45 minutes, ensuring that the temperature remained above 80°C. The mixture was then high sheared with a Silverson L5M-A homogenizer at 8000 rpm for 10 minutes. A 20 ml liquid aliquot was poured into a 50 ml falcon tube, sealed, and refrigerated overnight in a normal upright position.

[0630] Different levels of organic acids were tested, as shown in Table 19.

[0631] Example 16A 16B 16C 16D Mass of glacial acetic acid (g) 0 5 50 150 Mass of pea protein isolate (g) 50 50 50 50 Water mass (g) 450 455 400 300 wt% acetic acid 0 1 10 30 Weight ratio of acetic acid: water 0 1:91 1:8 1:2 Weight ratio of acetic acid:protein solids 0 1:10 1:1 3:1

[0632] Table 19

[0633] After refrigeration for at least 12 hours, gel formation was assessed by inverting the vials. Fig.10 As can be seen in Figure 2, the use of 30% and 10% glacial acetic acid resulted in the formation of a solid gel that did not fall under gravity. The use of 1% glacial acetic acid and no acid did not form a gel and fell into the lid of the inverted Falcon tube.

Claims

1. A method for preparing biodegradable microcapsules, the method comprising: (a) forming a solution comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises a miscible co-solvent; wherein a first cosolvent increases the solubility of the plant-based protein and a second cosolvent decreases the solubility of the plant-based protein; and wherein the cosolvent is added to the solution in concentrated or diluted form; preferably, wherein the pH of the plant-based protein solution is at least 0.5 pH units lower than the isoelectric point of the plant-based protein; (b) inducing the plant-based protein in the solution to undergo a sol-gel transition to form a plant-based protein hydrogel; (c) subjecting the plant-based protein hydrogel to a shearing treatment to form a plant-based protein hydrogel slurry; (d) dispersing an active ingredient in the plant-based hydrogel slurry to form a composition; and (e) drying the composition, thereby evaporating at least a portion of the first co-solvent and the second co-solvent to form microcapsules.

2. The method according to claim 1, wherein the plant-based protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, broad 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.

3. The method of claim 1 or claim 2, wherein the first co-solvent is an organic acid, preferably, wherein the organic acid is acetic acid, lactic acid, formic acid, gluconic acid, propionic acid, α-hydroxy acid and / or β-hydroxy acid, more preferably lactic acid or acetic acid, and wherein the first co-solvent is present in the solution of step (a) at a concentration equal to or greater than the concentration of protein solids on a weight percentage basis.

4. The method according to any one of claims 1 to 3, wherein the second co-solvent is selected from water, ethanol and / or ethyl acetate, more preferably water and / or ethanol, even more preferably water.

5. The method according to any one of claims 1 to 4, 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 reduced to a second temperature below the sol-gel transition temperature of the one or more plant-based protein solutions to form a hydrogel.

6. The method according to any one of claims 1 to 5, wherein the shearing treatment comprises a single shearing step involving breaking the plant-based protein hydrogel into fragments, preferably wherein the fragments have a d θ of 0.5 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns as measured by laser diffraction. 50 .

7. The method according to any one of claims 1 to 5, wherein the shearing treatment comprises a first shearing step followed by a second shearing step.

8. The method according to claim 7, wherein the first shearing step involves breaking the plant-based protein hydrogel into fragments, preferably, wherein at least 80 wt% of the fragments produced in the first shearing step have a particle size in the range of 1 mm to 100 mm, preferably 1 mm to 50 mm, preferably 1 mm to 30 mm, more preferably 10 mm to 30 mm, more preferably 15 mm to 30 mm, even more preferably 20 mm to 30 mm as determined by optical microscopy.

9. The method according to claim 7 or claim 8, wherein the second shearing step involves further fragmentation of the plant-based protein hydrogel, preferably wherein the fragments produced in the second shearing step have a d of 0.5 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns as measured by laser diffraction. 50 .

10. The method according to any one of claims 7 to 9, wherein step (c) further comprises subjecting the plant-based protein hydrogel slurry to a solvent reduction step, preferably a solubilizing solvent reduction step, between the first shearing step and the second shearing step.

11. The method according to claim 10, wherein the solvent reduction step comprises the following steps: (i) contacting the fragments of the plant-based hydrogel slurry with a non-dissolving solvent; (ii) separating the fragments of the plant-based hydrogel slurry from the non-dissolving solvent to provide a washed plant-based protein hydrogel slurry; and (iii) optionally repeating steps (i) and (ii).

12. The method according to any one of claims 1 to 11, further comprising the step of changing the pH of the plant-based protein hydrogel slurry so that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

13. The method of claim 12, wherein the step of changing the pH of the plant-based protein hydrogel slurry is performed after step (c), or sequentially with step (c).

14. The method of any one of claims 1 to 13, wherein the composition formed in step (d) is a shear thinning composition.

15. The method according to any one of claims 1 to 14, wherein the composition formed in step (d) has a -1 The viscosity range of 1 to 10000 cP, preferably at 20 ° C and 50s -1 The viscosity ranges from 10 to 7500 cP, preferably at 20°C and 50s -1 Viscosity range of 15 to 5000 cP.

16. The method according to any one of claims 1 to 15, wherein the composition formed in step (d) has a protein solids content in the range of 1 wt% to 25 wt%, preferably 2 wt% to 20 wt%, more preferably 4 wt% to 15 wt%, even more preferably 5 wt% to 12 wt%, based on the total weight of the composition.

17. The method according to any one of claims 1 to 16, wherein the active ingredient is selected from vitamins, minerals, flavors, fragrances, flavor precursors, fragrance precursors, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antimicrobials, antivirals, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments, and adhesives, or combinations thereof.

18. The method according to claim 17, wherein the active ingredient is at least one aroma or flavor substance, and wherein: The at least one aroma or flavor substance has a vapor pressure greater than or equal to 0.00001 Torr at 25°C; and / or The at least one aroma or flavor substance has a logP greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.

0.

19. The method of claim 17, wherein the active ingredient is at least one aroma or flavoring substance, which is part of a fragrance or flavoring agent, preferably, wherein the fragrance or flavoring agent comprises at least 20 wt% of an aroma or flavoring substance having a logP greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0, based on the total weight of the aroma or flavoring agent.

20. The method according to claim 17, wherein the active ingredient is a vitamin or a mineral, preferably selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, magnesium, sodium, potassium, zinc, iron, calcium, iodine, linseed oil, omega-3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus or mixtures thereof, more preferably vitamin D.

21. The method according to any one of claims 1 to 20, wherein the active ingredient is part of a composition comprising the active ingredient and an active carrier phase.

22. The method according to any one of claims 21, wherein the active carrier phase is a solvent, a fat or a wax.

23. The method according to any one of claims 1 to 22, wherein the composition formed in step (d) comprises droplets of the active ingredient or comprises droplets of the composition comprising the active ingredient and an active carrier phase, the droplets having a d θ of 0.5 to 50 μm, preferably 1 to 40 μm, preferably 2 to 30 μm, as determined by laser diffraction. 50 .

24. A process according to any one of claims 1 to 23, wherein plant-based protein residues formed during step (e) but not incorporated into the microcapsules are recycled in the process, preferably added back to step (a).

25. The method according to any one of claims 1 to 24, further comprising a post-treatment step of the microcapsules, preferably, wherein the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step or a coating formation step.

26. A biodegradable microcapsule obtained or obtainable by the method of any one of claims 1 to 25.

27. A method for preparing a biodegradable microcapsule composition, the method comprising: (a) preparing biodegradable microcapsules according to the method according to any one of claims 1 to 25; and (b) suspending the biodegradable microcapsules in the external phase.

28. A biodegradable microcapsule composition obtained or obtainable by the method of claim 27.

29. A biodegradable microcapsule comprising an active ingredient and a plant-based protein carrier containing a plant-based protein, wherein the plant-based protein carrier encapsulates the active ingredient and wherein the plant-based protein has a solubility of less than 50% when measured at a protein concentration of 5% w / w in an aqueous solution at 25°C and pH 7.

30. The biodegradable microcapsule according to claim 29, wherein at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the initially encapsulated active ingredient is still present inside the microcapsule after incubation in water at 20°C for 10 days as determined by HPLC.

31. The biodegradable microcapsule according to claim 29 or claim 30, wherein the plant-based protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, broad 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.

32. The biodegradable microcapsule according to any one of claims 29 to 31, wherein the plant-based protein has been pretreated with an organic acid, preferably, wherein the organic acid is acetic acid, lactic acid, formic acid, propionic acid, α-hydroxy acid and / or β-hydroxy acid, more preferably lactic acid or acetic acid.

33. The biodegradable microcapsule according to any one of claims 29 to 32, wherein the active ingredient is selected from vitamins, minerals, flavors, fragrances, flavor precursors, fragrance precursors, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), drugs, antimicrobial agents, antiviral agents, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments, and adhesives, or combinations thereof.

34. The biodegradable microcapsule according to claim 33, wherein the active ingredient is at least one aroma substance or flavor substance, and wherein: The at least one aroma or flavor substance has a vapor pressure greater than or equal to 0.00001 Torr at 25°C; and / or The at least one aroma or flavor substance has a logP greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.

0.

35. The biodegradable microcapsule according to claim 33, wherein the active ingredient is at least one aroma or flavoring substance, which is part of a fragrance or flavoring agent, preferably, wherein the fragrance or flavoring agent comprises at least 20 wt% of an aroma or flavoring substance having a logP greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0, based on the total weight of the aroma or flavoring agent.

36. The biodegradable microcapsule according to claim 33, wherein the active ingredient is a vitamin or a mineral, preferably selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, magnesium, sodium, potassium, zinc, iron, calcium, iodine, linseed oil, omega-3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus or a mixture thereof, more preferably vitamin D.

37. The biodegradable microcapsule according to any one of claims 29 to 36, wherein the active ingredient is part of a composition comprising the active ingredient and an active carrier phase.

38. The biodegradable microcapsule according to claim 37, wherein the active carrier phase is a solvent, a fat or a wax.

39. The biodegradable microcapsule according to any one of claims 29 to 38, wherein the microcapsule has a d value of less than or equal to 250 μm, less than or equal to 200 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. 50 .

40. The biodegradable microcapsule according to any one of claims 29 to 39, wherein the plant-based protein has 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 percentage content of intermolecular β-sheets is measured by FTIR.

41. The biodegradable microcapsule according to any one of claims 29 to 40, wherein the plant-based protein carrier has been non-covalently modified by a non-covalent cross-linking agent, or the plant-based protein carrier has been covalently modified by a covalent cross-linking agent, or the plant-based protein carrier has a coating deposited thereon.

42. The biodegradable microcapsule according to any one of claims 29 to 41, wherein the plant-based protein is pea protein, and the active ingredient is vitamin D.

43. A composition comprising the biodegradable microcapsules according to any one of claims 29 to 42 and an external phase.

44. A formulated product comprising the biodegradable microcapsules according to any one of claims 29 to 42.

45. A method of making a formulated product comprising: (a) preparing biodegradable microcapsules according to the method according to any one of claims 1 to 25; and (b) mixing the biodegradable microcapsules with a product formulation.

46. ​​Use of the biodegradable microcapsules according to any one of claims 29 to 42 in a formulated product.

47. The method of claim 45 or the use of claim 46, wherein the formulated product is a food, a beverage, a cosmetic, a home care product, a personal care product, a pharmaceutical, an industrial product (e.g., a coating, an adhesive, sandpaper, tape, etc.), a medical device, a biomaterial, or an agrochemical.

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