Biodegradable microcapsules and methods for their preparation

Abstract

The present invention relates to methods for preparing microcapsules and methods for preparing microcapsule compositions. The invention also relates to the microcapsules and the microcapsule compositions themselves. The invention also relates to uses of the microcapsules and methods involving the microcapsules, including preparing formulated products. The invention also relates to the formulated products themselves.

Description

[Technical Field]

[0001] The present invention relates to methods for preparing biodegradable microcapsules and methods for preparing biodegradable microcapsule compositions. The invention also relates to biodegradable microcapsules and biodegradable microcapsule compositions themselves. The invention also relates to uses of the biodegradable microcapsules and methods involving the biodegradable microcapsules, including preparing formulated products. The invention also relates to the formulated products themselves. [Background technology]

[0002] Encapsulation techniques, which involve embedding an active ingredient in an external matrix, can be used to protect the active ingredient from its external environment (e.g., exposure to chemicals, air, light, etc.) and also to protect the active ingredient itself from the external environment, such as when handling is hazardous. For example, this allows the active ingredient to be protected when added to a product formulation (e.g., a beverage formulation, a fabric conditioner formulation, etc.), meaning that the active ingredient in the product formulation has an acceptable shelf life and / or does not become prematurely activated. This also means that the external phase of the product formulation is not adversely affected by the active ingredient. The active ingredient is then released when needed (e.g., upon rupture of the encapsulate or enzymatic degradation of the encapsulate).

[0003] Many commonly available encapsulation techniques are based on the use of synthetic polymers to form a protective shell around active ingredients through a polymerization process.However, synthetic polymers may not be suitable as shell materials in pharmaceutical and food applications, or the acceptable exposure level in cosmetic applications may be limited.In addition, synthetic polymer shell materials are inherently poorly biodegradable, and may also lead to the formation of microplastics, which are harmful to the environment.

[0004] Drying techniques allow for the large-scale preparation of microcapsules, and spray drying of plant proteins has been attempted to achieve the production of biodegradable microcapsules or food-grade microcapsules. However, to achieve robust microcapsules of sufficiently small size via drying techniques, the material must have low viscosity at high shear rates so that it can be sprayed (e.g., the material must be shear-thinning to a sufficiently low viscosity for successful spray drying) and also have a sufficiently high protein solids content. Traditional methods for preparing plant protein-based materials suitable for spray drying involve hydrolyzing the protein (e.g., via treatment with acid or alkali or enzymes) to break it down into lower molecular weight fragments. However, the resulting spray-dried microcapsules are not structurally stable in water or under acidic or alkaline conditions, at high temperatures, and / or under high shear forces, meaning that they are not suitable for incorporation into product formulations, particularly liquid aqueous product formulations, via common manufacturing processes. Furthermore, many oils are unstable under alkaline conditions, so the processes involved can adversely affect the encapsulated material.

[0005] The use of protein-containing hydrogels in microencapsulation processes is known, for example, from International Publication No. 2022 / 221710, which describes a process using hydrogels to create microcapsules containing active substances, typically flavors. As a basic part of this process, the active substance is first emulsified with an emulsifier (e.g., gum arabic) and a "filler" (e.g., maltodextrin). This emulsion is then mixed with a protein, such as broad bean protein. The mixture is then heated to at least 50°C for a long period of time (at least 30 minutes) to form a hydrogel in situ that encapsulates the emulsified active substance. The mixture is then cooled and dried to form particles.

[0006] However, the process described in WO 2022 / 221710 is not always well suited to temperature-sensitive or highly volatile active substances. This is because the mixture containing the active substance must be heated for a long time to form a hydrogel. Examples of active substances that may be particularly temperature-sensitive include volatile fragrances, vitamins, and probiotics, which may be prone to thermal decomposition. The temperature experienced during spray drying is typically not very critical because the time scale involved is very short. A microencapsulation process that avoids the need to subject active substances to long-term high-temperature processes would be advantageous. It would also be advantageous to have a process for producing microcapsules that can be used in a wide range of products, such as liquid products, especially liquid aqueous products.

[0007] The present invention achieves this by forming a protein hydrogel slurry with the active dispersed therein, rather than forming a hydrogel in situ around droplets of the active. The protein hydrogel slurry is formed by first treating the plant protein with a solvent and / or adjusting the pH, such as below the isoelectric point, followed by shearing. Subsequent incorporation of the active into the protein hydrogel slurry can then be carried out at room temperature, thereby avoiding limitations due to the heat sensitivity of the active.

[0008] The hydrogels described and claimed in WO 2022 / 221710 contain protein, but the use of emulsifiers and "fillers" is also necessary along with the protein. The examples describe the use of gum arabic and glucose at levels approximately equal to or higher than those of broad bean protein. The resulting hydrogels and particles formed therefrom have very different properties from those of the present invention, particularly with regard to their solubility. It is interesting to note that the examples in WO 2022 / 221710 relate to dry products.

[0009] Microcapsules with significant wall solubility are clearly less suitable for use in liquid products, particularly liquid aqueous products: partial dissolution and / or solubilization of the microcapsule wall material very typically results in rapid and high active substance leakage and reduced product stability.

[0010] It is well known to use more soluble plant protein materials, such as highly hydrolyzed plant protein, as the encapsulation material for active substances in spray drying.The use of partially or completely water-soluble materials as the encapsulation material for immiscible active substances in drying processes, such as spray drying, is typically beneficial to create high-integrity, low-porosity microcapsule walls that can retain active substances.During the drying process, soluble materials typically dry to create lower-porosity solids, because dissolved species gradually fill and then block the small holes and gaps formed in the microcapsule walls.The use of such soluble materials in encapsulation and spray drying processes can help form high-integrity microcapsules that can better retain active substances during processing.

[0011] Such microcapsules are typically suitable for use in many dry products, such as protein powders and other food products. Examples are described, for example, in WO 2021 / 165289 and EP 3042571. However, the use of soluble wall materials typically results in microcapsules that are not very stable in liquid aqueous compositions, with rapid and high loss of active substance.

[0012] The use of preformed protein hydrogel slurries as encapsulation materials for dispersed active agents, as described in this invention, breaks this paradigm. The deformable nature of the protein hydrogel fragments allows high-integrity microcapsule walls to be formed from poorly soluble materials, resulting in microcapsules suitable for use in liquid products. The invention also allows for the use of more heat-sensitive materials in these applications.

[0013] Therefore, there is a need for a method of preparing active ingredient-containing microcapsules that are small enough in size to be incorporated into liquid aqueous product formulations, and that are biodegradable and stable in the liquid aqueous product formulations (i.e., the microcapsules retain their structural integrity under either 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

[0014] Viewed from a first aspect, the present invention provides a method for the preparation of biodegradable microcapsules, comprising: (a) forming a mixture comprising one or more plant-derived proteins in a solvent system, the solvent system comprising miscible co-solvents, a first co-solvent increasing the solubility of the plant-derived protein and a second co-solvent decreasing the solubility of the plant-derived protein, the co-solvents being added to the mixture in either concentrated or diluted form, and the pH of the plant-derived protein mixture being at least 0.5 pH units below the isoelectric point of the plant-derived protein; (b) subjecting the plant-derived protein mixture to a shear treatment to form a plant-derived protein hydrogel slurry; (c) dispersing an active ingredient in the plant-derived hydrogel slurry to form a composition; (d) drying the composition to form microcapsules.

[0015] Viewed from a further aspect, the present invention provides biodegradable microcapsules obtained or obtainable by the above method.

[0016] Viewed from a further aspect, the present invention provides a method for the preparation of a biodegradable microcapsule composition, comprising: (a) preparing biodegradable microcapsules according to the method described above; (b) suspending the biodegradable microcapsules in the external phase.

[0017] Viewed from a further aspect, the present invention provides a biodegradable microcapsule composition obtained or obtainable by the above method.

[0018] Viewed from a further aspect, the present invention provides spray-dried biodegradable microcapsules comprising an active ingredient and a plant-derived protein carrier comprising a plant-derived protein, wherein the plant-derived protein carrier encapsulates the active ingredient and the plant-derived protein carrier has a solubility of less than 50% when measured at a protein concentration of 5% w / w in aqueous solution at pH 7 and 25°C.

[0019] Viewed from a further aspect, the present invention provides a composition comprising the biodegradable microcapsules described above and an external phase.

[0020] Viewed from a further aspect, the present invention provides a formulated product comprising the biodegradable microcapsules described above.

[0021] Viewed from a further aspect, the present invention provides a method of making a formulated product, comprising the steps of: (a) preparing biodegradable microcapsules according to the method described above; (b) mixing the biodegradable microcapsules with a product formulation.

[0022] Viewed from a further aspect, the present invention provides the use of the biodegradable microcapsules described above in a formulated product.

[0023] definition As used herein, the term "microcapsule" refers to any form of microparticle. For example, the term encompasses core-shell microcapsules (i.e., microcapsules having a central core containing an active ingredient, the core surrounded by a plant-derived protein hydrogel in the form of a shell). As will be appreciated by those skilled in the art, core-shell microcapsules may have a multi-core morphology (i.e., the core phase is in the form of multiple droplets) or a single-core morphology (the core phase is in the form of a single droplet). The term also encompasses matrix microcapsules (i.e., microcapsules composed of a plant-derived protein hydrogel matrix with the active ingredient dispersed throughout). Microcapsules have a diameter of 500 nm to 2 mm as measured by laser diffraction. 50 may have:

[0024] As used herein, the term "lower shear process" may refer to a process step in which low levels of mechanical energy are applied to a material, preferably by a cutting action, to break or fragment it primarily into large, separate fragments. "Low shear" typically refers to high velocity impacts, e.g., less than 2 ms -1 This does not include any comminution process that crushes or fragments a material by impact with a velocity difference greater than 1 mm. It also typically does not include cavitation-based comminution processes. In certain embodiments, during the low shear process, the hydrogel is fragmented to obtain fragments such that at least 80% by weight of the hydrogel fragments have a maximum dimension of between 1 mm and 100 mm as measured by optical microscopy.

[0025] As used herein, the term "higher shear step" may refer to a process step in which energy is applied to reduce a hydrogel into small fragments, such as to form a colloidal dispersion. In certain embodiments, during the higher shear step, the hydrogel is fragmented to a size of 0.2 to 50 microns, as measured by laser diffraction. 50The laser diffraction can be carried out according to the method defined herein to give fragments having particle sizes.

[0026] For the avoidance of doubt, the high shear step subjects the hydrogel to a higher level of shear than the low shear step. Where a process involves both a low shear step and a high shear step, the high shear step occurs after the low shear step (i.e., they are separate steps occurring in this particular order).

[0027] As used herein, the term "sol-gel transition temperature" refers to the temperature at which a plant-derived protein transforms from a liquid state to a hydrogel state. Thus, at temperatures above the sol-gel transition temperature, the plant-derived protein is in a liquid state, and at temperatures below the sol-gel transition temperature, the plant-derived protein is in a hydrogel state. As used herein, "fragrance" (used interchangeably with the term "fragrance") refers to a formulation ingredient that can impart or modify the odor of a product, such as a fabric conditioner or hair conditioner, or the odor of a substrate, such as fabric or hair. Fragrances are typically used to impart an overall pleasant odor or odor profile to a product to provide a pleasant experience, e.g., a fine fragrance, or to provide sensory cues regarding the benefits and functionality of the product, e.g., the calming effect of a lavender sleep aid, the notion of cleanliness in laundry products, or to mask unpleasant odors, such as in insect repellent products. A "fragrance" can be composed of one or more ingredients, which can be a single chemical, referred to herein as a "fragrance material" (used interchangeably with the term "perfume material"), or a mixture of different "fragrance materials." Fragrance materials can be created by synthetic processes or extracted from nature, particularly plants, to obtain naturally occurring plant essential oils and plant extracts, e.g., orange oil. Fragrance materials created by synthetic processes can be either entirely new chemicals or nature-identical fragrance materials. Synthetic and naturally derived fragrance materials can then be blended into fragrances by skilled perfumers (also called noses) for use in consumer products. Fragrance materials can be obtained from specialized fragrance suppliers, known as fragrance houses, as individual chemicals, natural blends, or proprietary specialty blends whose complete compositions are not disclosed.Individual fragrance materials, including known natural blends, can be found by consulting journals commonly used by those skilled in the art, such as "Perfume and Flavorist" or "Journal of Essential Oil Research," or journals listed in reference works such as S. Arctander, "Perfume and Flavor Chemicals," 1969, Montclair, New Jersey, USA, and more recently republished by Allured Publishing Corporation, Illinois (1994) and "Perfume and Flavor Materials of Natural Origin," S. Arctander, Ed., Elizabeth, NJ, 1960. For purposes of the present invention, "fragrance material" will be understood to include pro-fragrances such as acetal pro-fragrances, ketal pro-fragrances, ester pro-fragrances, hydrolyzable inorganic-organic pro-fragrances, and combinations thereof. The fragrance material can be released from the pro-fragrance in any number of ways, for example, by hydrolysis, by a shift in an equilibrium reaction, by a pH change, by enzyme release, or by UV radiation.

[0028] Fragrance materials can be described in terms of their odor intensity, detection threshold, odor saturation, and its characteristics. In fragrance encapsulations, it is preferred to use fragrance materials with low odor detection thresholds and high intensities to maximize detectability even when the level of encapsulated and released fragrance is low.

[0029] To impart an odor, fragrance materials must be volatile, even if only slightly, because their molecules must be airborne and enter the nose, where they attach to specific neural receptors and trigger signals within the olfactory system. Fragrance materials can be classified according to their volatility. Preferably, fragrance materials are liquid at 20°C and atmospheric pressure, but in some cases they may be solid and blended with other liquid fragrance materials or solvents. Typically, the fragrance industry refers to volatility and persistence by broadly classifying materials into one of three categories: base notes, which are the least volatile and most persistent; heart notes, which are medium volatile and persistent; and top notes, which are the most volatile and least persistent. This is based on the perception of the material's odor and is entirely subjective. One way to objectively classify the volatility of fragrance materials is by their vapor pressure.

[0030] As used herein, the term "vapor pressure" refers to the partial pressure of a given chemical species in air at a specified 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 greater the proportion of the material that resides in a closed headspace at equilibrium. It is also related to the evaporation rate of a fragrance material, defined as the rate at which the material leaves the system in an open environment. Vapor pressure can be readily determined according to the reference program ACD / Percepta Desktop Software, Version 14.0 (Build: Aug / 26 / 2021), Advanced Chemistry Development, Inc. (ACD / Labs), Toronto, Canada, www.acdlabs.com.

[0031] A physical parameter relevant to the encapsulation of a fragrance material is its hydrophobicity, which can be defined in terms of its partition coefficient, P. As used herein, the term "partition coefficient" refers to the ratio between the equilibrium concentration of that substance in n-octanol and that in water, and is a measure of the differential solubility of that substance between these two solvents. As used herein, the term "logP" refers to the base 10 logarithm of the partition coefficient, P. logP can be readily determined according to the reference program ACD / Percepta Desktop Software, Version 14.0 (Build: Aug. 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 material 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 carbon isolation. logP GALAS is based on an algorithm that takes into account a database of training sets of compounds and adjusts values ​​with data from structurally similar compounds. Consensus logP is a model based on the previous two algorithms and can be expressed as Consensus logP = a × logP Classic + b × logP GALAS, where a and b are coefficients of the model. The latter value, Consensus logP, is the logP value presented herein.

[0032] Another aspect related to the encapsulation of fragrance materials is their Hansen Solubility Parameter (HSP). The term HSP refers to the solubility parameter approach proposed by Charles Hansen, which was first used to predict polymer solubility in a given solvent, as described in *The Three Dimensional Solubility Parameter and Solvent Diffusion Coefficient* by Charles Hansen, Danish Technical Press (Copenhagen, 1967). This approach has since been reapplied to many other molecules. A fragrance material (or flavor material or solvent) and its interaction with its environment are defined by three forces: atomic dispersion forces, molecular permanent dipole forces, and molecular hydrogen bond forces. Materials with similar HSP parameters are more likely to be miscible. These forces can be quantified by three values: δD, the Hansen dispersion value related to van der Waals interactions (intermolecular forces); δP, the Hansen polarity value related to dipole moments (charges); and δH, the Hansen hydrogen bond ("h-bond") value. Solubility parameter δ (MPa 1 / 2 ) is 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. HSP values ​​for a given material can be obtained in two main different ways from the HSPiP (Hansen Solubility Parameters in Practice) software, available at www.hansen-solubility.com. These values ​​can be retrieved from a Master Dataset containing over 20,000 materials by searching by name or CAS number, or they can be predicted using the Y-MB (Yamamoto-Molecular Breaking) method by entering the SMILE string of a given molecule in the DIY section of the software. Furthermore, determining the HSP sphere for a given fragrance material is a good way to predict the solubility preference within a blend of fragrance materials. The radius of the HSP sphere, Ro, is defined as Ro = Ra / RED, where Ra is the radius of Ra. 2 =4(δ D1 -δ D2 ) 2 +(δ P1 -δ P2 ) 2 +(δ H1 -δ H2 ) 2 where RED is the relative energy difference between two molecules (1 and 2) and RED is the HSP distance between the two molecules (1 and 2). This RED value can also be extracted or predicted from HSPiP software, and a good solvent for a given material should exhibit a RED value of 1 or less, while a solvent exhibiting a RED value greater than 1 should be considered a poor solvent for the given material.

[0033] The fragrance materials may be selected from alcohols, aldehydes, ketones, esters, ethers, acetates, alkenes, nitriles, nitrogen heterocycles, sulfur heterocycles, and Schiff bases.

[0034] Preferred aldehyde fragrance materials include α-amylcinnamaldehyde, anisaldehyde, decyl aldehyde, lauric aldehyde, methyl n-nonylacetaldehyde, methyl octylacetaldehyde, nonyl aldehyde, benzenecarboxaldehyde, neral, geranial, 1,1-diethoxy-3,7-dimethylocta-2,6-diene, 4-isopropylbenzaldehyde, 2,4-dimethyl-3-cyclohexene-1 -carboxaldehyde, α-methyl-p-isopropyldihydrocinnamaldehyde, 3-(3-isopropylphenyl)butanal, α-hexylcinnamaldehyde, 7-hydroxy-3,7-dimethyloctan-1-al, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, octylaldehyde, phenylacetaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, hexanal, 3,7-dimethyloctanal, 6,6-dimethylbis(2-methyl-4-methyl-2 ... Chloro[3.1.1]hept-2-ene-2-butanal, nonanal, octanal, 2-nonenal, undecenal, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexenyl-1)-2-butenal, 2,6-dimethyloctanal, 3-(p-isopropylphenyl)propionaldehyde, 3-phenyl-4-pentenal, citronellal, o / p-ethyl-α,α,9-decenal, dimethyldihydrocinnamaldehyde, p-isobutyl-α-methylhydrocinnamaldehyde aldehyde, cis-4-decen-1-al, 2,5-dimethyl-2-ethenyl-4-hexenal, trans-2-methyl-2-butenal, 3-methylnonanal, α-sinensal, 3-phenylbutanal, 2,2-dimethyl-3-phenylpropionaldehyde, m-tert-butyl-α-methyldihydrocinnamic aldehyde, geranyloxyacetaldehyde, trans-4-decen-1-al, methoxycitronellal, and mixtures thereof.

[0035] Preferred ester fragrance materials include allyl cyclohexane propionate, allyl heptanoate, allyl amyl glycolate, allyl caproate, amyl acetate (n-pentyl acetate), amyl propionate, benzyl acetate, benzyl propionate, benzyl salicylate, cis-3-hexenyl acetate, citronellyl acetate, citronellyl propionate, cyclohexyl salicylate, dihydroisojasmonate, Dimethylbenzylcarbinyl acetate, ethyl acetate, ethyl acetoacetate, ethyl butyrate, ethyl 2-methylbutyrate, ethyl 2-methylpentanoate, fenchyl acetate (1,3,3-trimethyl-2-norbornanyl acetate), tricyclodecenyl acetate, tricyclodecenyl propionate, geranyl acetate, cis-3-hexenyl isobutyrate, hexyl acetate, cis-3-hexenyl salicylate, n- Hexyl salicylate, isobornyl acetate, linalyl acetate, para-tert-butylcyclohexyl acetate, (-)-L-menthyl acetate, ortho-tert-butylcyclohexyl acetate, methyl benzoate, methyl dihydroisojasmonate, α-methylbenzyl acetate, methyl salicylate, 2-phenylethyl acetate, prenyl acetate, cedryl acetate, cyclabute, phenethylphenyl acetate, tetrahydrofuran Lupinyl formate, citronellyl anthranilate, ethyl tricyclo[5.2.1.0-2,6]decane-2-carboxylate, n-hexylethyl acetoacetate, 2-tert-butyl-4-methylcyclohexyl acetate, formic acid, 3,5,5-trimethylhexyl ester, phenethyl crotonate, cyclogeranyl acetate, geranyl crotonate, ethyl geranate, geranyl isobutyrate, 3,7-dimethylethyl 2-noninoate-2,6-Octadienoic acid methyl ester, citronellyl valerate, 2-hexenyl cyclopentanone, cyclohexyl anthranilate, L-citronellyl tiglate, butyl tiglate, pentyl tiglate, geranyl caprylate, 9-decenyl acetate, 2-isopropyl-5-methylhexyl-1-butyrate, n-pentyl benzoate, 2-methylbutyl benzoate (and mixtures thereof with pentyl benzoate), dimethylbenzyl carbinyl propionate, dimethylbenzyl carbinyl Nyl acetate, trans-2-hexenyl salicylate, dimethylbenzylcarbinyl isobutyrate, 3,7-dimethyloctyl formate, rosinyl formate, rosinyl isovalerate, rosinyl acetate, rosinyl butyrate, rosinyl propionate, cyclohexyl ethyl acetate, neryl butyrate, tetrahydrogeranyl butyrate, myrcenyl acetate, 2,5-dimethyl-2-ethenylhex-4-enoic acid methyl ester, 2,4-dimethylcyclohexane-1-methyl Acetate, ocimenyl acetate, linalyl isobutyrate, 6-methyl-5-heptenyl-1-acetate, 4-methyl-2-pentyl acetate, n-pentyl 2-methylbutyrate, 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)- Methyl 3-pentenoate, 3-methoxy-3-methylbutyl acetate, n-pentyl crotonate, n-pentyl isobutyrate, propyl formate, furfuryl butyrate, methyl angelate, methyl pivalate, prenyl caproate, furfuryl propionate, diethyl maleate, isopropyl 2-methylbutyrate, dimethyl malonate, bornyl formate, styrallyl acetate, 1-(2-furyl)-1-propanone, l-citronellyl acetate, 3,7-dimethyl-1,Examples of suitable hydroxybenzoates include, but are not limited to, 6-nonadien-3-yl acetate, neryl crotate, dihydromyrcenyl acetate, tetrahydromyrcenyl acetate, lavandulyl acetate, 4-cyclooctenyl isobutyrate, cyclopentyl isobutyrate, 3-methyl-3-butenyl acetate, allyl acetate, geranyl formate, cis-3-hexenyl caproate, and mixtures thereof.

[0036] Preferred alcohol fragrance materials include benzyl alcohol, β-γ-hexenol (2-hexen-1-ol), cedrol, citronellol, cinnamic alcohol, p-cresol, cumin alcohol, dihydromyrcenol, 3,7-dimethyl-1-octanol, dimethylbenzylcarbinol, eucalyptol, eugenol, fenchyl alcohol, geraniol, hydratropic alcohol, and the like. alcohol), isononyl alcohol (3,5,5-trimethyl-1-hexanol), linalool, methyl chavicol (estragole), methyl eugenol (eugenyl methyl ether), nerol, 2-octanol, patchouli alcohol, phenylhexanol (3-methyl-5-phenyl-1-pentanol), phenethyl 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, ethylnorbornylcyclohexanol, β-methylcyclohexaneethanol, 3,7-dimethyl-(2),6-octene(adiene)-1-ol, trans-2-undecen-1-ol, 2-ethyl-2-prenyl-3 -Hexenol, isobutylbenzyl carbinol, dimethylbenzyl carbinol, ocimenol, 3,7-dimethyl-1,6-nonadien-3-ol (cis & 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-heptenol 2-Hexenol, 3,7,9-trimethyl-1,6-decadien-3-ol, 3,7-dimethyl-6-nonen-1-ol, 3,7-dimethyl-1-octyn-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,Examples of suitable methyl alcohols include, but are not limited to, p-methylphenylethanol, 2-methyl-5-phenyl-1-pentanol, 3-methylphenethyl alcohol, para-methyldimethylbenzylcarbinol, methylbenzylcarbinol, p-methylphenylethanol, 3,7-dimethyl-2-octen-1-ol, 2-methyl-6-methylene-7-octen-4-ol, and mixtures thereof.

[0037] Preferred ketone fragrance materials include oxacycloheptadec-10-en-2-one, benzylacetone, 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, ethanone, octahydro-2,3,8,8-tetramethyl-2-acetonaphthalene, α-Iron, 1-(5,5-dimethyl-1-cyclohexen-1-yl)-4-penten-1-one, 3-nonanone, ethylhexyl ketone, menthone, 4-methyl-acetophenone, γ-methylionone, methylpentyl ketone, methylheptenone (6-methyl-5-hepten-2-one), methylheptyl ketone, methylhexyl ketone, δ-muscenone, 2-octanone, 2-pentyl-3-methyl cyclopenten-1-one, 2-heptylcyclopentanone, α-methylionone, 3-methyl-2-(trans-2-pentenyl)-cyclopentenone, octenylcyclopentanone, n-amylcyclopentenone, 6-hydroxy-3,7-dimethyloctanoic acid lactone, 2-hydroxy-2-cyclohexen-1-one, 3-methyl-4-phenyl-3-buten-2-one, 2-pentyl-2,5,5-trimethylcyclopentanone, 2-cyclopentylcyclopentanol-1,5-methylhexan-2-one, γ-dodecalactone, δ-dodecalactone, γ-nonalactone, δ-nonalactone, γ-octalactone, δ-undecalactone, γ-undecalactone, α-damascone, β-damascone, γ-damascone, δ-damascone, and mixtures thereof.

[0038] Preferred ether fragrance materials include, but are not limited to, diphenyl oxide, p-cresyl 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-hexamethylindan (phantolide), 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (tonalide), 2-phenylethyl-3-methylbut-2-enyl ether, ethyl geranyl ether, phenylethyl isopropyl ether, and mixtures thereof.

[0039] Preferred alkene fragrance materials include, but are not limited to, allo-ocimene, camphene, β-caryophyllene, cadinene, diphenylmethane, d-limonene, limolene, β-myrcene, para-cymene, 2-α-pinene, β-pinene, α-terpinene, γ-terpinene, terpineolene, 7-methyl-3-methylene-1,6-octadiene, and mixtures thereof.

[0040] Preferred nitrile fragrance materials include, but are not limited to, 3,7-dimethyl-6-octenenitrile, 3,7-dimethyl-2(3),6-nonadienenitrile, (2E,6Z)-2,6-nonadienenitrile, n-dodecanenitrile, and mixtures thereof.

[0041] Preferred Schiff base fragrance materials include, but are not limited to, citronellyl nitrile, nonanal / methyl anthranilate, N-octylidene-anthranilic acid methyl ester, hydroxycitronellal / methyl anthranilate, cyclamen aldehyde / methyl anthranilate, methoxyphenylpropanal / methyl anthranilate, ethyl p-aminobenzoate / hydroxycitronellal, citral / methyl anthranilate, 2,4-dimethylcyclohex-3-enecarbaldehyde methyl anthranilate, hydroxycitronellal-indole, and mixtures thereof.

[0042] As used herein, the term "flavor" refers to a formulation ingredient that can impart or modify the taste and smell of a product, such as toothpaste or food. Flavors are typically used to impart an overall pleasant taste and smell or taste and smell profile to a product, either to provide a simply pleasant experience, such as in foods, or to mask unpleasant tastes and smells, such as in pharmaceuticals. A flavor or flavoring material can be described in terms of its aroma intensity, detection threshold, and quality. A "flavor" can be composed of one or more components, which can be a single chemical, referred to herein as a "flavoring material," or a mixture of different "flavoring materials." Flavoring materials can be created by synthetic processes or can be naturally occurring, particularly extracted from plants to create naturally occurring plant and animal oils and exudates, such as vanilla. Synthetic and naturally derived flavoring materials can then be blended by skilled perfumers into flavors for use in consumer products. Flavoring materials can be obtained from specialty flavor suppliers known as flavor houses as individual chemicals, natural blends, or proprietary specialty blends whose complete composition is not disclosed.Individual flavor materials, including known natural blends, can be found by consulting journals commonly used by those skilled in the art, such as "Perfume and Flavorist" or "Journal of Essential Oil Research," or journals listed in reference works such as S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA, and more recently, journals republished by Allured Publishing Corporation, Illinois (1994), "Perfume and Flavor Materials of Natural Origin," S. Arctander, Ed., Elizabeth, NJ, 1960, and "Flavorings," E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998. It will be understood that flavors can be volatile or can have volatile components that are detected by the nose, similar to fragrances. Therefore, flavor materials can also be classified according to their physical properties, such as volatility and hydrophobicity, using the methods described above for fragrance materials. Flavor materials can also be described according to their Hansen Solubility Parameters using the method described above for fragrance materials.

[0043] Sources of flavoring materials include essential oils, concretes, absolutes, resins, resinoids, balsams, and tinctures. Preferred flavoring materials 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, y-butyryllactone, 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, g-hexenyl lactone, 2,4-decadienal, 2,4-heptadienal, methylthiazole alcohol (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, polysulfides, dipropyl disulfide, methylbenzyl disulfide, alkylthiophene, 2,3-dimethylthiophene, 5-methylfurfural, acetylfuran, 2,4-deca Dienal, Guacol, phenylacetaldehyde, b-decalactone, d-limonene, acetoin, amyl acetate, maltol, ethyl butyrate, levulinic acid, piperonal, ethyl acetate, n-octanal, n-pentanal, 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, propylpropenyl disulfide, propylpropenyl trisulfide, diallyl disulfide, diallyl trisulfide, dipropenyl disulfide, dipropenyl 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 oil, such as lemon, orange, lime, and grapefruit oil. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present inventors have discovered a method for preparing high solids protein colloidal dispersions of controlled particle size and low viscosity containing an active ingredient that allows for the formation of stable microcapsules upon drying. The resulting microcapsules are stable in liquid product formulations and also under the conditions required to prepare such liquid product formulations (e.g., pasteurization conditions in the case of beverage formulations).

[0045] The present invention provides a method for the preparation of biodegradable microcapsules, comprising: (a) forming a mixture comprising one or more plant-derived proteins in a solvent system, the solvent system comprising miscible co-solvents, a first co-solvent increasing the solubility of the plant-derived protein and a second co-solvent decreasing the solubility of the plant-derived protein, the co-solvents being added to the mixture in either concentrated or diluted form, and the pH of the plant-derived protein mixture being at least 0.5 pH units below the isoelectric point of the plant-derived protein; (b) subjecting the plant-derived protein mixture to a shear treatment to form a plant-derived protein hydrogel slurry; (c) dispersing an active ingredient in the plant-derived hydrogel slurry to form a composition; (d) drying the composition to form microcapsules.

[0046] Any suitable plant-derived protein can be used in the present invention. In a preferred method of the present invention, the plant-derived 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, and 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-derived protein is pea protein and / or potato protein. Such proteins are considered to be hypoallergenic proteins.

[0047] Suitable plant-derived proteins further include: Brassica: for example, Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongata: elongated mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St. Hilarion's cabbage, Brassica juncea: Indian mustard, brown mustard and mustard greens, Sarepta mustard, Brassica napus: rapeseed, canola, rutabaga, Brassica narinosa: broadbeaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, Chinese broccoli, Brussels sprouts, kohlrabi, Brassica perviridis: tender greens, mustard spinach, Brassica rapa (also known as B. campestris): Chinese cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii: Asian mustard - Solanaceae: for example, tomato, potato, eggplant, bell and pepper; - Cereals: e.g., corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio - Quasi-cereals: such as amaranth (also called amaranth, red amaranth, prince-of-Wales-feather), breadfruit, buckwheat, chia, cockscomb (also called quail grass or soko), pitseed goosefoot, qaniwa, quinoa and wattleseed (also called acacia seed); - legumes, such as Acacia alata (Winged Wattle), Acacia decipiens, Acacia saligna (commonly known by various names including coojong, golden wattle, orange wattle, and blue-leafed wattle), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus laburnum (common laburnum, golden chain, or golden rain), Cytisus supinus, Dolichos lablab (commonly known as lablab bean, lablab-bean, bonavist bean / pea, dolichos bean, seim bean); bean), love love bean, Egyptian bean, Indian bean, bataw, and Australian pea), Ervum lens (lentil), Genista tinctorial (common names include dyer's whin, waxen woad, and waxen wood), Glycine max (soybean), Lathyrus clymenum (pea vine or lathyrus), Lathyrus odoratus (pea vine or lathyrus), Lathyrus staivus (pea vine or lathyrus), Lathyrus silvetris (pea vine or lathyrus), Lotus tetragonolobus (asparagus-pea or winged pea), Lupinus albus (lupine), Lupinus angustifolius (lupine), Lupinus luteus (lupine), Lupinus polyphyllus (lupine), Medicago sativa (alfalfa), Phaseolus aureus (mung bean), Phaseolus coccineus (runner bean), Phaseolusnanus (green bean / French bean), Phaseolus vulgaris (green bean / French bean), Pisum sativum (pea), Trifolium hybridum (clover), Trifolium pretense (red clover), Vicia faba (broad bean), Vicia sativa (broad bean), Vigna unguiculate (cowpea) - Non-legumes: for example: Acanshosicyos horrida, Aesculus hyppocastanum, Anacardium occidentale, Balanites aegyptica, Bertholletia excels, Beta vulgaris, Brassica napus, Brassica juncea, Brassica nigra, Brassica hirta, Cannabis sativa, Citrullus vulgaris, Citrus aurantiaca, Cucurbita maxima, Fagopyrum esculentum, Gossypium barbadense (extra-long staple cotton), Heianthus annuus (sunflower), Nicotiana sp. (tobacco), Prunus avium (cherries), Prunus spp. (sour cherries), Prusus domestica (plums), Prusus amygdalus (almonds), Rricinus communis (castor bean / castor oil plant), Sasamum indicum (sesame), Sinapis alba (white mustard), and Terlfalrea pedata (oyster nuts).

[0048] For the avoidance of doubt, plant-derived microcapsules of the present invention do not encompass plants in their natural state, for example naturally formed plant cells, organelles or vesicles are not plant-derived microcapsules of the present invention.

[0049] In step (a), the first co-solvent increases the solubility of the plant-derived protein. The first co-solvent can be considered a solubilizing co-solvent. One or more solubilizing co-solvents can be present, and the solubilizing co-solvents can completely or partially solubilize the plant-derived protein. The co-solvents can be added in step (a) in a highly concentrated or diluted form.

[0050] An example of a solubilizing co-solvent is an organic acid. An organic acid is an organic compound that has acidic properties. Preferably, the organic acid is sourced from a natural plant-derived or bio-derived feedstock.

[0051] In a preferred method of the present invention, the first co-solvent is an organic acid. Preferably, the organic acid is acetic acid, lactic acid, formic acid, propionic acid, α-hydroxy acid, and / or β-hydroxy acid. Preferred α-hydroxy acids include glycolic acid, lactic acid, acetic acid, malic acid, citric acid, maleic acid, gluconic acid, and / or tartaric acid, preferably lactic acid or acetic acid. Preferred β-hydroxy acids include β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxyβ-methylbutyric acid, 2-hydroxybenzoic acid, and carnitine. In a particularly preferred method of the present invention, the organic acid is acetic acid and / or lactic acid.

[0052] The use of organic acids can solubilize plant proteins and also enable gentle hydrolysis of proteins.For example, without being bound by theory, the solubility of plant-derived proteins in organic acids is due to i) the protonation of proteins, and ii) the presence of an anionic solvation layer, which contributes to reducing hydrophobic interactions.When initially dissolved in organic acids, the protonation of plant-derived proteins can help stabilize them in their non-solvent, such as water.

[0053] In step (a), the second co-solvent reduces the solubility of the plant-derived protein compared to the first co-solvent. The second co-solvent can be considered a desolubilizing co-solvent. One or more desolubilizing co-solvents may be present.

[0054] In a preferred method 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.

[0055] In a preferred method of the present invention, the cosolvent ratio of the first cosolvent to the second cosolvent in the solvent system is 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%, about 30%, about 40%, about 50% or about 60% v / v, most preferably about 30-50% v / v.

[0056] According to the present invention, it may be preferred that the first co-solvent is present in the mixture of step (a) at a concentration on a weight percent basis that is equal to or greater than the concentration of the protein.

[0057] In a preferred method of the invention, the pH of the plant-derived protein mixture in step (a) is at least 0.5 pH units, more preferably at least 1.0 pH units, below the isoelectric point of the plant-derived protein.

[0058] In a preferred method of the present invention, the concentration of the plant-derived protein in the solvent system is 25 to 200 mg / mL, more preferably 50 to 150 mg / mL.

[0059] The ratio of organic acids may vary depending on the protein concentration, for example, a higher ratio of organic acids may be used with increasing protein concentration.

[0060] In a preferred method of the present invention, the degree of proteolysis (i.e., the percentage of broken peptide bonds in the protein hydrolysate) is controlled to modify the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation increases the degree of proteolysis. A higher degree of proteolysis results in the formation of a less rigid hydrogel.

[0061] In a preferred method of the present invention, the degree of protein hydrolysis is between 0.1 and 10%, preferably between 0.1 and 5%, even more preferably between 0.1 and 2.5%.

[0062] To form a mixture containing one or more plant-derived proteins, it may be necessary to apply a physical stimulus to the protein / solvent system mixture to allow interaction between the solvent and the protein. Suitable physical stimuli include heating, sonication, stirring, high-shear mixing, high-shear homogenization, or other physical techniques. A preferred technique is heating, optionally followed by sonication.

[0063] Preferably, the protein / solvent system mixture is subjected to a physical stimulus that is heating, and the mixture is heated to about 70° C. or higher. More preferably, the protein / solvent system mixture is heated to about 75° C. or higher, about 80° C. or higher, about 85° C. or higher, or about 90° C. Even more preferably, the protein / solvent system mixture is heated to 85° C.

[0064] Preferably, the protein / solvent system mixture is subjected to the physical stimulus of heating for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or more than 30 minutes. The heated protein / solvent system mixture is optionally subjected to subsequent sonication.

[0065] In a preferred method of the present invention, the protein mixture is heated, preferably between steps (a) and (b) or in step (b), to a first temperature higher than the sol-gel transition temperature of the one or more plant-derived protein mixtures, and then reduced to a second temperature lower than the sol-gel transition temperature of the one or more plant-derived protein mixtures to form a hydrogel.

[0066] The protein mixture is heated so that the liquid mixture is maintained above the sol-gel transition of the protein. By modifying the solvent system (e.g., through the choice of organic acid, the ratio of organic acid to additional solvent, or through additional means), it is possible to modify the sol-gel transition temperature of the protein. Through appropriate selection of conditions, it is possible to carefully control the sol-gel transition of the protein, and thereby control the formation of a hydrogel.

[0067] Preferably, the protein mixture 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.

[0068] The protein mixture can be held at elevated temperature for about 5, 10, 15, 20, 25, 30, 45 minutes, or 1 hour. A preferred time is at least 30 minutes to fully solubilize the protein. It is possible to hold the protein mixture at elevated temperature for longer periods of time.

[0069] After heating the protein mixture to a temperature above the sol-gel transition temperature, the temperature of the protein mixture can be reduced to a second temperature below the sol-gel transition temperature to facilitate the formation of a hydrogel. The second temperature can be room temperature. The second temperature can be in the range of 5 to 25°C, preferably 10 to 20°C. The protein mixture can be kept at a low temperature for an extended period of time, such as several days or weeks, before performing the shearing treatment in step (b). The protein mixture can be kept at a low temperature for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or about 30 minutes. A particular shortened time is about 5 minutes.

[0070] The particular temperature will depend on the properties of the protein source, the solvent conditions used, and therefore the sol-gel transition temperature. Alternatively, the high and low temperatures may be relatively fixed (e.g., about 85°C, then about room temperature), with the co-solvent mixture conditions adjusted to ensure a suitable sol-gel transition temperature for the selected plant-derived protein.

[0071] Without being bound by theory, it is believed that when plant proteins are added to a solvent system, they form a dispersion of insoluble colloidal protein aggregates. Aggregate size can be measured by dynamic light scattering (DLS). A suitable device for measuring aggregate size is the Zetasizer Nano S (Malvern).

[0072] When a protein mixture is heated above the sol-gel transition temperature in the presence of a cosolvent system, the plant protein is thought to partially unfold, exposing hydrophobic amino acids that were initially buried within the protein's native structure. Once partially unfolded, the cosolvent can interact with the unfolded protein molecule. For example, organic acids can not only facilitate the protonation of amino acid residues but also form anion-salt bridges that stabilize hydrophobic interactions. Furthermore, heating at high temperatures disrupts noncovalent protein-protein intermolecular contacts.

[0073] Furthermore, the application of mechanical agitation, for example sonication, is believed to break down large colloidal protein aggregates into smaller aggregates and also disrupt protein intermolecular interactions.

[0074] Furthermore, if the protein mixture is cooled below the sol-gel transition temperature, non-covalent protein-protein intermolecular contacts may be possible, thus promoting the self-assembly of plant protein molecules into a hydrogel of interconnected protein aggregates.

[0075] It is believed that the methods of the present invention allow plant proteins to aggregate into supramolecular structures held together by intermolecular hydrogen bonding interactions, particularly by interactions between beta strands.

[0076] The methods of the present invention allow for the formation of materials in which there are high levels of β-sheet intermolecular interactions, such that the plant-derived protein has a protein secondary structure with at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet, where the % intermolecular β-sheet content is measured by FTIR (Fourier Transform Infrared Spectroscopy).

[0077] In a preferred method of the present invention, the plant-derived protein hydrogel is subjected to a solvent reduction step, preferably a solubilizing solvent reduction step, preferably between steps (a) and (b) or in step (b).

[0078] The solubilizing solvent refers to a solvent or mixture thereof in which the plant-derived 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, maleic acid, gluconic acid, and / or tartaric acid. The β-hydroxy acids may preferably be selected from β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxyβ-methylbutyric acid, 2-hydroxybenzoic acid, and carnitine.

[0079] In a preferred method of the present invention, the solvent reduction step comprises: (i) contacting a plant-derived protein hydrogel with a non-solubilizing solvent; (ii) separating the plant-derived hydrogel from the non-solubilizing solvent to obtain a washed plant-derived protein hydrogel; (iii) optionally repeating steps (i) and (ii).

[0080] Step (i) involves contacting the plant-derived protein hydrogel with a non-solubilizing solvent. By non-solubilizing solvent, we mean a solvent or mixture thereof in which the plant-derived protein hydrogel does not dissolve. Examples include water or a mixture of water and ethanol.

[0081] In a preferred method of the present invention, the shearing treatment comprises one step (i.e., a single shearing step). The single shearing step may be a higher shearing step. Preferably, the single shearing step involves fragmenting the plant-derived protein hydrogel into fragments.

[0082] In a preferred method of the present invention, the fragments produced in a single shearing step have a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. 50 It has.

[0083] In preferred methods of the invention, the single shear step involves sonication (e.g., using equipment such as a Bandelin HD4200 or Hielscher UIP1000hdT), high shear mechanical agitation (e.g., using equipment such as a Silverson rotor-stator high shear mixer), high pressure homogenization, or cavitation, preferably involving sonication.

[0084] In a preferred method of the invention, the single shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-derived protein mixture, and in a preferred method of the invention, the first shearing step is carried out for at least 5 minutes, more preferably at least 1 minute.

[0085] In a preferred method of the present invention, the shearing treatment comprises two steps. Preferably, the shearing treatment comprises a first shearing step followed by a second shearing step. The first shearing step may be a low shearing step and the second shearing step may be a high shearing step.

[0086] In a preferred method of the present invention, the first shearing step involves fragmenting the plant-derived protein hydrogel into fragments. Preferably, at least 50% by weight 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 5 mm to 30 mm, more preferably 10 mm to 30 mm. More preferably, at least 80% by weight 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 5 mm to 30 mm, more preferably 10 mm to 30 mm. This can be determined by optical microscopy or visually.

[0087] In a preferred method of the present invention, the first shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-derived protein mixture.

[0088] In a preferred method of the present invention, the first shearing step involves mechanical cutting, which means cutting using a knife edge (e.g. knife, extruder blade, etc.).

[0089] In an alternative preferred method of the present invention, the first shearing step involves extrusion. For example, the plant-derived protein mixture formed in step (a) can be extruded into a non-solubilizing solvent (e.g., water) to form a plant-derived protein hydrogel in large, discrete fragments, e.g., the large, discrete fragments can take the form of an extrudate having the shape of threads or strings. In this manner, the fragments can be directly subjected to a solvent reduction step, as described in more detail 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 between 1 mm and 100 mm. Preferably, at least 50% by weight of the fragments produced in the first shearing step have at least one internal dimension in the range of between 1 mm and 100 mm, preferably between 1 mm and 50 mm, preferably between 5 mm and 30 mm, more preferably between 10 mm and 30 mm. More preferably, at least 80% by weight of the fragments produced in the first shearing step have at least one internal dimension in the range of 1 mm to 100 mm, preferably 1 mm to 50 mm, preferably 5 mm to 30 mm, more preferably 10 mm to 30 mm, which can be measured by optical microscopy or visually.

[0090] In a preferred method of the present invention, the second shearing step involves further fragmenting the plant-derived protein hydrogel. Preferably, the fragments produced in the second shearing step have a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. 50 It has.

[0091] In a preferred method of the present invention, the particle size distribution of the hydrogel fragments in the plant-derived protein hydrogel slurry can be adjusted by varying the nature and intensity of the second shearing step. In another preferred method, the particle size distribution of the hydrogel fragments in the plant-derived protein hydrogel slurry can be adjusted by blending or combining two or more different hydrogel slurries that have been subjected to different second shearing steps and have different particle size distributions.

[0092] In a preferred method of the invention, the second shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-derived protein.

[0093] 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-derived protein.

[0094] In a preferred method of the present invention, the second shearing step is carried out for at least 5 minutes, more preferably at least 1 minute.

[0095] In preferred methods of the invention, the second shearing step involves sonication (e.g., using equipment such as a Bandelin HD4200 or Hielscher UIP1000hdT), high shear mechanical agitation (e.g., using equipment such as a Silverson rotor-stator high shear mixer), high pressure homogenization, or cavitation, preferably involving sonication.

[0096] In a preferred method of the present invention, step (b) further comprises subjecting the plant-derived protein hydrogel slurry to a solvent reduction step, preferably a solubilizing solvent reduction step, between the first shearing step and the second shearing step.

[0097] In a preferred method of the present invention, the solvent reduction step comprises: (i) contacting the plant-derived hydrogel slurry fragments with a non-solubilizing solvent; (ii) separating the plant-derived hydrogel slurry fragments from the non-solubilizing solvent to obtain a washed plant-derived protein hydrogel; (iii) optionally repeating steps (i) and (ii).

[0098] The solubilizing solvent is as defined above.

[0099] Step (i) involves contacting the plant-derived protein hydrogel slurry fraction with a non-solubilizing solvent, which is as defined above.

[0100] In a preferred method of the invention, step (ii) involves mesh filtration or centrifugation. More preferably, step (ii) involves mesh filtration using multiple size-reducing meshes.

[0101] As will be appreciated by those skilled in the art, if the fragments produced in the first shearing step are too small, the solvent reduction step may be difficult because the fragments may eventually plug the mesh or the collection yield may be low. However, if the fragments produced in the first shearing step are too large, the solvent reduction step may take excessively long because of slow mass transfer of solvent from the center of the fragments.

[0102] Without wishing to be bound by theory, it is believed that due to the porosity of the hydrogel, the solvent reduction step may remove some or all of the solvent (e.g., organic acid) from the hydrogel via solvent exchange.

[0103] The strength of the protein hydrogel can be altered by varying the concentrations of the protein and organic acid, among other variables.

[0104] It is useful for the strength of the hydrogel used to form the hydrogel slurry to be within certain limits, which can be measured by oscillatory rheometry. A suitable measure of hydrogel strength is the storage modulus, G', of the hydrogel. Suitable test conditions are 1% strain at 20°C with an oscillatory frequency of 1 Hz. A suitable instrument is an Anton Paar MCR 92 Rheometer with a 50 mm diameter, 1 degree angle cone and plate measurement geometry.

[0105] Thus, in a preferred method of the present invention, before washing, the plant-derived 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. As will be appreciated by those skilled in the art, 2π rad / s is equal to 1 Hz.

[0106] In a preferred method of the invention, before washing, the plant-derived protein hydrogel has a storage modulus (G') at 10 rad / s of less than 20,000 Pa, preferably less than 15,000 Pa, more preferably less than 10,000 Pa.

[0107] In a preferred method of the present invention, before washing, the plant-derived 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.

[0108] Furthermore, in a preferred method of the present invention, the washed plant-derived 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.

[0109] In a preferred method of the present invention, the washed plant-derived protein hydrogel has a storage modulus (G') at 10 rad / s 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.

[0110] In a preferred method of the present invention, the washed plant-derived 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.

[0111] A preferred method of the present invention further comprises adding an additional ingredient to the plant-derived protein hydrogel slurry in step (b) or between steps (b) and (c), or to the composition in step (c). Preferably, the additional ingredient is selected from a plasticizer, a surfactant, a rheology modifier, an opacifier, a preservative, a pigment, a carbohydrate, a gum, a polymer, and a nanoparticle, or a mixture thereof.

[0112] In a preferred method of the invention, the additional ingredient is a plasticizer, preferably 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.

[0113] A preferred method of the present invention further comprises the step of altering the pH of the plant-derived protein hydrogel slurry so that it differs from the isoelectric point of the plant-derived protein by more than 1 pH unit.

[0114] Preferably, the step of changing the pH of the plant-derived protein hydrogel slurry is carried out after step (b), or alternatively, the step of changing the pH of the plant-derived protein hydrogel slurry is carried out consecutively with step (b).

[0115] During the pH adjustment of the plant-derived protein hydrogel slurry, the slurry may pass through the isoelectric point of the protein. Due to the lack of charge repulsion at the isoelectric point, dispersed protein fragments in the plant-derived protein hydrogel slurry may rapidly coagulate. To avoid this, a pH-modifying material can be used to rapidly change the pH to minimize the time the slurry is at the isoelectric point. The isoelectric point of a particular plant-derived protein can be measured using the method described in Helmick et al., Food Biophysics (2021) 16:474-483, or may be available in the literature, for example, Guldekin et al., Food Hydrocolloids (2023), 145:109029.

[0116] Therefore, in a preferred method of the present invention, the step of changing the pH of the plant-derived protein hydrogel slurry involves adding a pH-modifying material to the plant-derived protein hydrogel slurry. Preferably, the pH-modifying material is a solution containing monovalent metal ions, divalent metal ions, or ammonium ions, preferably an aqueous alkaline solution containing monovalent metal ions, divalent metal ions, or ammonium ions. More preferably, the pH-modifying material is an aqueous hydroxide solution, preferably sodium hydroxide, potassium hydroxide, or ammonium hydroxide.

[0117] In a preferred method of the present invention, the pH of the plant-derived protein hydrogel slurry after the step of altering the pH of the plant-derived protein hydrogel slurry is at least 1 pH unit below the isoelectric point of the plant-derived protein.

[0118] In a preferred method of the present invention, the pH of the plant-derived protein hydrogel slurry after the step of altering the pH of the plant-derived protein hydrogel slurry is at least 1 pH unit higher than the isoelectric point of the plant-derived protein.

[0119] As will be appreciated by those skilled in the art, the addition of additional ingredients to the plant-derived protein hydrogel slurry in step (b) or between steps (b) and (c), or to the composition in step (c), may affect the pH of the slurry. Thus, the step of altering the pH of the plant-derived protein hydrogel slurry is preferably performed after the addition of any additional ingredients.

[0120] In a preferred method of the present invention, the composition formed in step (c) is a shear thinning composition.

[0121] In a preferred method of the present invention, the composition formed in step (c) is heated at 20°C and 50 s -1 Viscosity in the range of 1 to 10,000 cP at 20°C and 50 s -1 Viscosity in the range of 10 to 7500 cP at 20°C and 50 s -1 It has a viscosity in the range of 15 to 5000 cP.

[0122] In a preferred method of the present invention, the composition formed in step (c) has a protein solids content ranging from 1% to 25% by weight, preferably 2% to 20% by weight, more preferably 4% to 15% by weight, and even more preferably 5% to 12% by weight, based on the total weight of the composition. The term "protein solids" refers to the dry dispersion hydrogel solids derived from the plant protein added in step (a). Plant protein isolates are typically composed primarily of material chemically identifiable as protein, but also contain low levels of other insoluble plant material, such as fiber. The % protein solids is measured according to the methods described herein.

[0123] In a preferred method of the present invention, the active ingredient is selected from vitamins, minerals, flavor materials, fragrance materials, pro-flavors, pro-fragrances, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antibacterial agents, antiviral agents, anti-inflammatory agents, insecticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioning agents, hair conditioning agents, dyes, pigments, and adhesives, or combinations thereof.

[0124] In a particularly preferred method of the present invention, the active ingredient is at least one fragrance or flavor material, preferably selected from alcohols, aldehydes, ketones, esters, ethers, acetates, alkenes, nitriles, nitrogen heterocycles, sulfur heterocycles, and Schiff bases.

[0125] The fragrance and flavor materials used in the present invention may be of natural origin (i.e., they are extracted from natural sources and have not been synthetically modified in any way). Preferred fragrance or flavor materials of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, mandarin oil, orange flower extract, cedarwood, vetiver, lavandin, 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, daffodil extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil, or combinations thereof. Preferably, the naturally occurring fragrance or flavor materials are derived from plants. Naturally occurring fragrance or flavor materials may be used alone or in combination, or in combination with synthetic fragrance materials.

[0126] In a preferred method of the present invention, at least one fragrance or flavor material has a vapor pressure of 0.00001 Torr or greater at 25°C.

[0127] In a preferred method of the present invention, at least one fragrance or flavor material has a logP of 3.0 or greater, preferably 3.5 or greater, more preferably 4.0 or greater.

[0128] In a preferred method of the present invention, the at least one fragrance or flavor material has at least two Hansen Solubility Parameters selected from an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bonding (δH) of 2.5 to 11.

[0129] In a preferred method of the present invention, the at least one fragrance or flavor material is part of a fragrance or flavor.

[0130] Preferably, the fragrance or flavor contains at least 20% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of 3.0 or greater, more preferably 3.5 or greater, more preferably 4.0 or greater.

[0131] Preferably, the fragrance or flavor contains at least 40% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0132] Preferably, the fragrance or flavor contains at least 50% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0133] Preferably, the fragrance or flavor contains at least 60% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0134] Preferably, the fragrance or flavor contains at least 10% by weight, preferably at least 30% by weight, more preferably at least 50% by weight, more preferably at least 70% by weight of fragrance or flavor materials of natural origin, based on the total weight of the fragrance or flavor.

[0135] Preferably, the fragrance or flavor contains at least 10% by weight of a fragrance or flavor material that exhibits a biodegradation percentage of 60-100%, more preferably 65%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100% after 28 days based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, as measured according to ISO-14851 version 2019. Because the fragrance or flavor has low water solubility, the fragrance or flavor can be subjected to a biodegradation test on an inert support according to ISO 10634 version 2018: Water quality - Preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium. Additionally, the fragrance or flavor can be checked for its inhibitory effect on microorganisms as detailed in that method.

[0136] Preferably, the fragrance or flavor contains at least 80% by weight, preferably at least 90% by weight, more preferably at least 95% by weight, of fragrance or flavor materials having at least two Hansen solubility parameters selected from an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bonding (δH) of 2.5 to 11, based on the total weight of the fragrance or flavor.

[0137] Preferably, the fragrance or flavor contains only low levels of materials with alcohol functionality (e.g., primary alcohol functionality). In a preferred method of the present invention, the fragrance or flavor contains less than 40% by weight, more preferably less than 20% by weight, of alcohol-containing materials, based on the total weight of the fragrance or flavor. In a particularly preferred method of the present invention, the fragrance or flavor does not contain any alcohol-containing materials. Without being bound by theory, it is believed that alcohols, especially primary alcohols with straight-chain alkyl groups, can easily diffuse through the shell of microcapsules due to their structure, meaning that they can be difficult materials to encapsulate.

[0138] Preferably, the fragrance or flavor material has a high odor impact, which is advantageous as it ensures that even low levels of fragrance are perceived when released from the microcapsules.

[0139] In an alternative, particularly preferred method of the present invention, the active ingredient is a vitamin or mineral. Preferably, the active ingredient is a vitamin or mineral 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, flaxseed oil, omega-3 fatty acids, folic acid, thiamine, riboflavin, niacin, and phosphorus, or a mixture thereof. More preferably, the active ingredient is vitamin D.

[0140] In an alternative, particularly preferred method of the present invention, an additive can be included in the microcapsule core to act as an active protecting agent to reduce any potential degradation of the active ingredient during processing and storage. Preferably, the core additive is an antioxidant or free radical scavenger, such as vitamin E and curcumin. Preferably, the core additive is a pigment, such as a carotenoid, preferably β-carotene. Preferably, the core additive is a natural product containing a mixture of antioxidants, such as polyphenols, and other compounds, such as olive oil and turmeric.

[0141] The vitamins and minerals used in the present invention may be supplied from vitamin- and / or mineral-containing materials, an example of such a material being seaweed.

[0142] In an alternatively preferred method of the present invention, the active ingredient is an agrochemical, such as a pesticide, herbicide, fertilizer, fungicide, insecticide, animal repellent, or combinations thereof.

[0143] Preferably, the pesticide is a natural alternative to synthetic materials and is based on plant extracts and / or plant essential oils (EOs), or components of essential oils, such as thymol. Preferably, the pesticide is a biologically derived agricultural insecticide (biopesticide). Preferably, the pesticide is suitable for use in formulations for the management and production of plants that can be certified organic by organizations such as the USDA (US Department of Agriculture) or European Ecocert.

[0144] 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 a solvent, fat or wax.

[0145] In a preferred method of the invention, the active carrier phase is a solvent. Preferably, the solvent is a low volatility solvent (e.g., having a vapor pressure of less than 0.1 Torr at 25°C, preferably less than 0.01 Torr at 25°C, preferably less than 0.001 Torr at 25°C).

[0146] Preferably, the solvent has low or no odor.

[0147] Preferably, the solvent has at least two Hansen solubility parameters selected from 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 atomic dispersion forces (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0148] Preferably, the solvent has a viscosity of 1.07 g / cm 3 A solvent with this property can advantageously prevent creaming of the encapsulate (e.g., in the final product formulation).

[0149] Preferably, the solvent contains only low levels of materials with alcohol functionality (e.g., primary alcohol functionality). In preferred methods of the invention, the solvent contains less than 40 wt. %, more preferably less than 20 wt. %, of alcohol-containing materials, based on the total weight of the solvent. In particularly preferred methods of the invention, the solvent is free of alcohol-containing materials.

[0150] In a preferred method of the present invention, the active carrier phase is a solvent selected from carboxylic acid esters, fatty acid esters, phthalate esters, triols, diols, rosin resins, isoparaffins, terpenes, and vegetable oils, or combinations thereof.

[0151] Preferably the solvent is Miglyol® 840, Miglyol® 812 N, Miglyol® 829, Miglyol® 829 ECO, Miglyol® Coco 810, Miglyol® 810 N, Miglyol® 128, Miglyol® 808, Miglyol® T-C7, Miglyol® 8810, Miglyol® PPG 810, Miglyol® OE, Miglyol® DO, and Miglyol® 818, Abalyn®, limonene, benzyl benzoate, diethyl phthalate, isopropyl myristate, triethyl citrate, dipropylene glycol, and propylene glycol, triacetin, glycerin, 1,3 propanediol, or combinations thereof, preferably Miglyol® 812 N.

[0152] 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 listed in CTFA Cosmetic Ingredient Handbook, JM Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Vegetable oils are oils derived from plant sources. Alternatively, the solvent is derived from vegetable oils.

[0153] In a preferred method 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.

[0154] Preferably, the wax is selected from Softisan® 100, Softisan® 142, and Softisan® 154, or a combination thereof, preferably Softisan® 100.

[0155] Preferably, the vegetable oil, fat or wax is present in a weight ratio of vegetable protein to active carrier phase greater than 1:1, more preferably greater than 1.5:1, even more preferably 2:1 or greater.

[0156] In the method of the present invention, the method used to form the composition in step (c) is not particularly limited. For example, step (c) 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.

[0157] In a preferred method of the present invention, step (c) is carried out at a temperature in the range of 30°C to 50°C, preferably 35°C to 45°C.

[0158] In a preferred method of the invention, the composition formed in step (c) comprises droplets of the active ingredient or the composition comprises droplets of the active ingredient and a diameter of 0.5 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. 50 and an active carrier phase having

[0159] In a preferred method of the present invention, an additive, preferably a water-soluble additive, is added to the composition in step (b) or between steps (b) and (c), or in step (c).

[0160] Preferably, the additive is selected from plasticizers, surfactants, rheology modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers, and nanoparticles, or mixtures thereof.

[0161] In a preferred method of the invention, the additive is a plasticizer, preferably 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.

[0162] In a preferred method of the present invention, the additive is a polysaccharide, preferably maltodextrin, starch or modified starch, such as Capsul® starch or octenyl succinic anhydride (OSA) modified starch. The use of a polysaccharide additive allows for control of the composition of the matrix of the resulting microcapsules. For example, the use of a polysaccharide means that the amount of plant-derived protein can be controlled.

[0163] In a preferred method of the present 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 dry microcapsules are capable of delivering the additive in addition to the encapsulated active ingredient during end use, for example, upon enzymatic degradation of the plant-derived protein hydrogel matrix in the oral cavity or digestive tract of a human or animal.

[0164] In a preferred method of the invention, the weight ratio of plant-derived protein in the composition formed in step (c) to the composition comprising the active ingredient and the active carrier phase is in the range of 20:1 to 1:20.

[0165] In a preferred method of the present invention, the composition formed in step (c) is a homogeneous composition.

[0166] In a preferred method of the invention, the composition formed in step (c) is a two-phase system (eg, an aqueous two-phase system).

[0167] In the method of the present invention, step (d) involves drying the composition formed in step (c) to form microcapsules or agglomerates of microcapsules or larger objects. Those skilled in the art are familiar with drying techniques and know how to select the conditions necessary to achieve dry microcapsules or larger objects. Suitable drying techniques include spray drying, electrostatic spray drying, spray freeze drying, and spray vacuum drying, in which the slurry is sprayed into a chamber under reduced pressure, thereby allowing the solvent to be removed at a lower temperature, thereby avoiding the problem of thermal decomposition. Advantageously, in the method of the present invention, step (d) can be performed at a relatively low temperature (e.g., a temperature below 100°C). This aids in the formation of small-sized microcapsules and also prevents the loss or decomposition of any encapsulated volatile or heat-sensitive materials. Advantageously, in the method of the present invention, step (d) can be performed relatively quickly (e.g., with a residence time of a few seconds), thereby reducing the loss or decomposition of encapsulated volatile or heat-sensitive materials.

[0168] Drying techniques that can be used independently or in combination to evaporate the solvent from the composition of step (c) include spray drying, fluidized bed drying, film drying, drum drying, belt drying, conduction drying, infrared drying, or a combination thereof. The composition of step (c) can be dispersed and at least partially dried by spray drying to form microcapsules, which are then further dried by a secondary process such as fluidized bed drying. The extended drying time typical of fluidized bed drying typically allows for the use of lower drying temperatures. Some spray dryer designs incorporate this two-stage drying process as an integral part of their design. Some fluidized bed drying processes, which typically involve spraying or dispersing the composition of step (c) onto or into a fluidized bed of particles, e.g., encapsulates of already dried composition (c), are preferred when larger encapsulates are required. The composition can be sprayed onto or into the fluidized bed. Depending on the process conditions, the composition spray agglomerates and / or coats (and thus builds up) the passing microcapsules. An example of a fluidized bed coating and drying process is a Wurster type powder coater. Those skilled in the art will be able to easily define the conditions for controlling the size of the encapsulates from the fluidized bed drying process.

[0169] Depending on the desired form of the dried composition from step (d), other drying processes can be selected. Film drying, in which the composition is cast as a thin film onto a heated belt or drum, can be used to form aesthetic shapes containing the dried composition prepared in step (c). Belt drying, which can use conduction or infrared heating, can be used in combination with other drying techniques to complete drying under controlled conditions.

[0170] During drying in step (d), the plant-derived protein hydrogel slurry forms a plant-derived protein carrier that encapsulates the active ingredient in the form of microcapsules. During drying in step (d), at least a portion of both the first and second cosolvents can evaporate to form microcapsules. Because this evaporation does not occur at the same rate for the two cosolvents, during the process, the slurry is typically more concentrated in the least volatile cosolvent. The microcapsules formed during drying can still contain both the first and second cosolvents trapped within the dried microcapsules, and their ratio may be different from their starting ratio in the mixture in step (a).

[0171] In a preferred method of the present invention, the biodegradation percentage based on O2 consumption of the plant-derived protein carrier measured according to ISO-14851 version 2019 after 28 days, based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, is 60-100%, more preferably 65-10%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100%. ISO-14851 version 2019 describes a method for measuring the biodegradability of materials in natural aqueous environments using biological oxygen demand in a closed respirometer. This is achieved by exposing the material under laboratory conditions in an aqueous standard test medium to an inoculum from previously unexposed, unadapted activated sludge. The measurement is calculated as a percentage of the theoretical oxygen demand calculated from the molecular formula. An internal standard of microcrystalline cellulose is also tested and if its % biodegradation is greater than 60% at the end of the test, the test is valid.

[0172] In a preferred method of the present invention, the biodegradation percentage based on O2 consumption of the microcapsules measured according to ISO-14851 version 2019 after 28 days, based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, is 60-100%, more preferably 65-10%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100%. ISO-14851 version 2019 describes a method for measuring the biodegradability of materials in natural aqueous environments using biological oxygen demand in a closed respirometer. This is accomplished by exposing the material to an inoculum from previously unexposed, unadapted activated sludge under laboratory conditions in an aqueous standard test medium. The measurement is calculated as a percentage of the theoretical oxygen demand calculated from the molecular formula. An internal standard of microcrystalline cellulose is also tested and if its % biodegradation is greater than 60% at the end of the test, the test is valid.

[0173] In a preferred method of the invention, step (d) involves electrostatic spray drying.

[0174] In a preferred method of the invention, the plant-derived protein residue formed during step (d) but not incorporated into microcapsules is recycled and preferably added back to step (a).

[0175] In a preferred method of the invention, the microcapsules have a d of 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, 30 μm or less, 10 μm or less, as measured by laser diffraction. 50 It has.

[0176] In preferred methods of the invention, the microcapsules have a diameter of 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, 30 μm or less, 10 μm or less, as measured by optical microscopy.

[0177] A preferred method of the present invention further comprises subjecting the microcapsules to a post-treatment step. Preferably, the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step or a coating formation step.

[0178] 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 sodium tripolyphosphate (NaTPP), sodium hexametaphosphate, and phenolic compounds (e.g., tannic acid, caffeic acid, etc.).

[0179] 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 genipin, epoxy compounds, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and PolyCup® cross-linking resin, or a combination thereof.

[0180] In a preferred method of the present invention, the post-treatment step comprises a coating formation step.

[0181] In a preferred method of the present invention, the coating formation step comprises treating the microcapsules with a metal compound, preferably selected from a silver compound or a gold compound, preferably a silver compound.

[0182] In a preferred method of the present invention, the coating formation step comprises subjecting the microcapsules to a complex coacervation step using a polysaccharide, preferably selected from xanthan gum, gellan gum, and chitosan, or a combination thereof.

[0183] In a preferred method of the present invention, the coating formation step comprises treating the microcapsules with an aqueous mineral solution to form a mineral coating. Preferably, the aqueous mineral solution comprises an iron salt, a calcium salt, a phosphate salt, a carbonate salt, a titanium salt or a zinc salt, or a combination thereof.

[0184] In a preferred method of the present invention, the coating formation step comprises treating the microcapsules with a silicon-containing compound to form a silicon-based coating. Preferably, the silicon-containing compound is selected from sodium silicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, dimethyldiethoxysilane, and tetramethyl orthosilicate, or a combination thereof. More preferably, the sodium silicate is selected from sodium metasilicate, sodium orthosilicate, and sodium pyrosilicate, with sodium metasilicate being most preferred. As will be understood by those skilled in the art, depending on which phase the silicon compound is present in, the silicon-based coating can be formed either on the internal surface of the plant-derived protein hydrogel (i.e., at the oil droplet interface) or on the external surface of the plant-derived protein hydrogel (i.e., to form a shell around the microcapsules). Water-soluble silicates are most preferred when coating the external surface of the plant-derived protein hydrogel. Organic silicates are most preferred when forming on the internal surface of the plant-derived protein hydrogel.

[0185] As will be understood by those skilled in the art, multiple post-treatment steps can be performed. For example, in a preferred method of the present invention, 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 a further non-covalent cross-linking step (e.g., using NaTPP). For example, microcapsules are subjected to an electrostatic / non-covalent cross-linking step using sodium tripolyphosphate (NaTPP), followed by a silica coating using poly-L-lysine as a deposition aid. In a preferred method of the present invention, microcapsules are subjected to a non-covalent cross-linking step using tannic acid, followed by a coacervate coating formation step using xanthan gum.

[0186] A preferred method of the present invention further comprises adding a flow aid to the microcapsules after step (d). Preferably, the flow aid is fumed silica. Addition of the flow aid can prevent agglomeration of the newly formed microcapsules.

[0187] A preferred process of the present invention comprises further drying the microcapsules after step (d). Preferably, the further drying is selected from fluidized bed drying and / or tray drying.

[0188] Preferred methods of the present invention further comprise resuspending the microcapsules in an external phase, preferably an external aqueous phase, more preferably hard water or an acidic buffer solution.

[0189] Preferred methods of the present invention further include sieving the microcapsules, for example for size classification.

[0190] The present invention also provides biodegradable microcapsules obtained or obtainable by the above method.

[0191] The present invention also provides a method for the preparation of a biodegradable microcapsule composition, comprising: (a) preparing biodegradable microcapsules according to the method described above; (b) suspending the biodegradable microcapsules in an external phase.

[0192] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0193] A preferred method of the present invention further comprises adding a suspending agent to the external phase.

[0194] Preferably, the suspending agent is selected from acacia gum, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil category 1, aluminum magnesium silicate, maltodextrin, carboxymethylcellulose, polymethacrylates, polyvinylpyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers such as cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylates, and cross-linked polyvinylpyrrolidone, and expanded clays such as bentonite and laponite.

[0195] The present invention also provides a biodegradable microcapsule composition obtained or obtainable by the above method.

[0196] The present invention also provides dry biodegradable microcapsules comprising an active ingredient and a plant-derived protein carrier comprising a plant-derived protein, wherein the plant-derived protein carrier encapsulates the active ingredient, and the plant-derived protein carrier has a solubility of less than 50% when measured at a protein concentration of 5% w / w in aqueous solution at pH 7 and 25°C.

[0197] Protein solubility is measured using the following protocol: a known amount of spray-dried microcapsules is added to an aqueous solution, which is then centrifuged to separate the soluble and insoluble fractions. After centrifugation, the liquid supernatant (i.e., the soluble fraction) is removed without collecting any of the solids that settle to the bottom (i.e., the insoluble fraction). The resulting supernatant is analyzed for nitrogen content. The protein content in the supernatant is then calculated based on the nitrogen content using a coefficient of 6.25. Protein solubility is defined as the amount of protein in the supernatant divided by the amount of protein in the total aqueous solution.

[0198] In preferred biodegradable microcapsules of the present invention, the plant-derived protein carrier has a solubility of less than 30%, more preferably less than 10%, when measured in aqueous solution at pH 7 and 25°C at a protein concentration of 5% w / w.

[0199] In preferred biodegradable microcapsules of the invention, at least 25%, more preferably at least 40%, even more preferably at least 50%, and even more preferably at least 60% of the initially encapsulated active ingredient remains present within the microcapsules after 10 days of incubation in water at 20° C., as measured by high performance liquid chromatography (HPLC). In alternative preferred spray-dried biodegradable microcapsules of the invention, at least 25%, more preferably at least 40%, even more preferably at least 50%, and even more preferably at least 60% of the initially encapsulated active ingredient remains present within the microcapsules after 10 days of incubation in water at 20° C., as measured by gas chromatography (GC).

[0200] More preferably, the water is acidified to have a pH of less than 4.0, more preferably less than 3.0, more preferably less than 2.0, and more preferably less than 1.0. Therefore, the biodegradable microcapsules of the present invention are insoluble in water and stable even under strongly acidic conditions. This has the advantage that the biodegradable microcapsules of the present invention can be used in a variety of applications where such harsh conditions are common. For example, the biodegradable microcapsules can be added to beverage formulations, which are generally acidic, to deliver active ingredients (e.g., vitamins or minerals) to consumers. The acid stability of the biodegradable microcapsules of the present invention also means that once consumed, the microcapsules can withstand the harsh acidic conditions of the stomach, resulting in the active ingredient not being released until it enters the small intestine, where it can be better absorbed. Therefore, the biodegradable microcapsules of the present invention can provide sustained release of the encapsulated active ingredient. This is because the plant-derived protein hydrogel matrix functions as an enteric coating.

[0201] In a preferred biodegradable microcapsule of the present invention, the plant-derived 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-derived protein is pea protein and / or potato protein. Such proteins are considered to be hypoallergenic proteins.

[0202] In a preferred biodegradable microcapsule of the present invention, the plant-derived protein is pretreated with an organic acid. Preferably, the organic acid is acetic acid, formic acid, propionic acid, α-hydroxy acid, and / or β-hydroxy acid. Particularly preferably, the organic acid is acetic acid and / or lactic acid.

[0203] Preferred α-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 β-hydroxy acids include β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxyβ-methylbutyric acid, 2-hydroxybenzoic acid and carnitine.

[0204] In preferred biodegradable microcapsules of the present invention, the active ingredient is selected from vitamins, minerals, flavor materials, fragrance materials, pro-flavors, pro-fragrances, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antibacterial agents, antiviral agents, anti-inflammatory agents, insecticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioning agents, hair conditioning agents, dyes, pigments, and adhesives, or combinations thereof.

[0205] In certain biodegradable microcapsules of the present invention, the active ingredient is at least one fragrance or flavor material, preferably selected from alcohols, aldehydes, ketones, esters, ethers, acetates, alkenes, nitriles, nitrogen heterocycles, sulfur heterocycles, and Schiff bases.

[0206] The fragrance and flavor materials used in the present invention may be of natural origin (i.e., they are extracted from natural sources and have not been synthetically modified in any way). Preferred fragrance or flavor materials of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, mandarin oil, orange flower extract, cedarwood, vetiver, lavandin, 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, daffodil extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil, or combinations thereof. Preferably, the naturally occurring fragrance or flavor materials are derived from plants. Naturally occurring fragrance or flavor materials may be used alone or in combination, or in combination with synthetic fragrance materials.

[0207] In preferred biodegradable microcapsules of the present invention, at least one fragrance or flavor material has a vapor pressure of 0.00001 Torr or greater at 25°C.

[0208] In preferred biodegradable microcapsules of the present invention, at least one fragrance or flavor material has a logP of 3.0 or greater, preferably 3.5 or greater, more preferably 4.0 or greater.

[0209] In preferred biodegradable microcapsules of the present invention, the at least one fragrance or flavor material has at least two Hansen solubility parameters selected from an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bonding (δH) of 2.5 to 11.

[0210] In preferred biodegradable microcapsules of the present invention, at least one fragrance or flavor material is part of a fragrance or flavor.

[0211] Preferably, the fragrance or flavor contains at least 20% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of 3.0 or greater, more preferably 3.5 or greater, more preferably 4.0 or greater.

[0212] Preferably, the fragrance or flavor contains at least 40% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0213] Preferably, the fragrance or flavor contains at least 50% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0214] Preferably, the fragrance or flavor contains at least 60% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0215] Preferably, the fragrance or flavor contains at least 10% by weight, preferably at least 30% by weight, more preferably at least 50% by weight, more preferably at least 70% by weight of fragrance or flavor materials of natural origin, based on the total weight of the fragrance or flavor.

[0216] Preferably, the fragrance or flavor contains at least 10% by weight of a fragrance or flavor material that exhibits a biodegradation percentage of 60-100%, more preferably 65%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100% after 28 days based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, as measured according to ISO-14851 version 2019. Because the fragrance or flavor has low water solubility, the fragrance or flavor can be subjected to a biodegradation test on an inert support according to ISO 10634 version 2018: Water quality - Preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium. Additionally, the fragrance or flavor can be checked for its inhibitory effect on microorganisms as detailed in that method.

[0217] Preferably, the fragrance or flavor contains at least 80% by weight, preferably at least 90% by weight, more preferably at least 95% by weight, of fragrance or flavor materials having at least two Hansen solubility parameters selected from an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bonding (δH) of 2.5 to 11, based on the total weight of the fragrance or flavor.

[0218] Preferably, the fragrance or flavor contains only low levels of materials with alcohol functionality (e.g., primary alcohol functionality). In preferred biodegradable microcapsules of the present invention, the fragrance or flavor contains less than 40% by weight, more preferably less than 20% by weight, of alcohol-containing materials, 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 any alcohol-containing materials.

[0219] Preferably, the fragrance or flavor material has a high odor impact, which is advantageous as it ensures that even low levels of fragrance are perceived when released from the microcapsules.

[0220] In alternative, particularly preferred biodegradable microcapsules of the present invention, the active ingredient is a vitamin or mineral. Preferably, the active ingredient is a vitamin or mineral 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, flaxseed oil, omega-3 fatty acids, folic acid, thiamine, riboflavin, niacin, and phosphorus, or mixtures thereof. More preferably, the active ingredient is vitamin D.

[0221] In an alternative, particularly preferred biodegradable microcapsule of the present invention, an additive can be included in the microcapsule core to act as an active protectant to reduce any potential degradation of the active ingredient during processing and storage. Preferably, the core additive is an antioxidant or free radical scavenger, such as vitamin E and curcumin. Preferably, the core additive is a pigment, such as a carotenoid, preferably beta-carotene. Preferably, the core additive is a natural product comprising a mixture of antioxidants, such as polyphenols, with other compounds, such as olive oil and turmeric.

[0222] The vitamins and minerals used in the present invention may be supplied from vitamin- and / or mineral-containing materials, an example of such a material being seaweed.

[0223] In 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, fat or wax.

[0224] In preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent. Preferably, the solvent is a low-volatility solvent (e.g., having a vapor pressure of less than 0.1 Torr at 25°C, preferably less than 0.01 Torr at 25°C, preferably less than 0.001 Torr at 25°C).

[0225] Preferably, the solvent has low or no odor.

[0226] Preferably, the solvent has at least two Hansen solubility parameters selected from 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 atomic dispersion forces (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0227] Preferably, the solvent has a viscosity of 1.07 g / cm 3 A solvent with this property can advantageously prevent creaming of the encapsulate (e.g., in the final product formulation).

[0228] Preferably, the solvent contains only low levels of materials with alcohol functionality (e.g., primary alcohol functionality). In preferred spray-dried biodegradable microcapsules of the invention, the solvent contains less than 40% by weight, more preferably less than 20% by weight, of alcohol-containing materials, based on the total weight of the solvent. In particularly preferred spray-dried biodegradable microcapsules of the invention, the solvent is free of alcohol-containing materials.

[0229] In preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent selected from carboxylic acid esters, fatty acid esters, phthalate esters, triols, diols, rosin resins, isoparaffins, terpenes, and vegetable oils, or combinations thereof.

[0230] Preferably the solvent is Miglyol® 840, Miglyol® 812 N, Miglyol® 829, Miglyol® 829 ECO, Miglyol® Coco 810, Miglyol® 810 N, Miglyol® 128, Miglyol® 808, Miglyol® T-C7, Miglyol® 8810, Miglyol® PPG 810, Miglyol® OE, Miglyol® DO, and Miglyol® 818, Abalyn®, limonene, benzyl benzoate, diethyl phthalate, isopropyl myristate, triethyl citrate, dipropylene glycol, and propylene glycol, triacetin, glycerin, 1,3 propanediol, or combinations thereof, preferably Miglyol® 812 N.

[0231] 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 described in CTFA Cosmetic Ingredient Handbook, JM Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Alternatively, the solvent is derived from a vegetable oil.

[0232] In 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.

[0233] Preferably, the wax is selected from Softisan® 100, Softisan® 142, and Softisan® 154, or a combination thereof, preferably Softisan® 100.

[0234] In preferred biodegradable microcapsules of the present invention, the plant-derived protein carrier further comprises an additive, preferably a water-soluble additive.

[0235] Preferably, the additive is selected from plasticizers, surfactants, rheology modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers, and nanoparticles, or mixtures thereof.

[0236] In preferred biodegradable microcapsules of the present invention, the additive is a plasticizer, preferably 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.

[0237] In preferred biodegradable microcapsules of the present invention, the additive is a polysaccharide, preferably maltodextrin or OSA starch. The use of a polysaccharide additive allows for control of the composition of the matrix of the resulting microcapsule. For example, the use of a polysaccharide means that the amount of plant-derived protein can be controlled.

[0238] In preferred biodegradable microcapsules of the present 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 microcapsules are capable of delivering the additive in addition to the encapsulated active ingredient during end use, for example, upon enzymatic degradation of the plant-derived protein hydrogel matrix in the oral cavity or digestive tract of a human or animal.

[0239] In preferred biodegradable microcapsules of the present invention, the microcapsules have a d of 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, 30 μm or less, 10 μm or less, as measured by laser diffraction. 50 It has.

[0240] In preferred biodegradable microcapsules of the present invention, the microcapsules have a diameter of 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, 30 μm or less, 10 μm or less, as measured by optical microscopy.

[0241] In preferred biodegradable microcapsules of the present invention, the plant-derived protein has a protein secondary structure having at least 40% intermolecular β-sheet, at least 50% intermolecular β-sheet, at least 60% intermolecular β-sheet, at least 70% intermolecular β-sheet, at least 80% intermolecular β-sheet, or at least 90% intermolecular β-sheet, wherein the % intermolecular β-sheet content is measured by FTIR.

[0242] Preferred biodegradable microcapsules of the present invention are free or substantially free of crosslinking agents.

[0243] In an alternative preferred biodegradable microcapsule of the present invention, the plant-derived protein carrier is non-covalently modified with a non-covalent cross-linking agent, preferably selected from sodium tripolyphosphate, sodium hexametaphosphate, and phenolic compounds (e.g., tannic acid, caffeic acid, etc.).

[0244] In an alternative preferred biodegradable microcapsule of the present invention, the plant-derived protein carrier is covalently modified with a covalent cross-linking agent, preferably selected from genipin, epoxy compounds, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and PolyCup® cross-linking resin, or a combination thereof.

[0245] In preferred biodegradable microcapsules of the present invention, the plant-derived protein carrier has a coating deposited thereon. Preferably, the coating is a metal coating, a coacervate coating, or a mineral coating.

[0246] In preferred biodegradable microcapsules of the present invention, the metal coating is a silver coating or a gold coating, preferably a silver coating.

[0247] In preferred biodegradable microcapsules of the present invention, the coating is a silicon-based coating formed from a silicon-containing compound, preferably selected from sodium metasilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, dimethyldiethoxysilane, and tetramethyl orthosilicate, or a combination thereof, preferably sodium metasilicate.

[0248] In preferred biodegradable microcapsules of the present invention, the coacervate coating is formed from a polysaccharide, preferably selected from xanthan gum, gellan gum, and chitosan, or combinations thereof.

[0249] In preferred biodegradable microcapsules of the present invention, the mineral coating is formed from an aqueous mineral solution, preferably comprising an iron salt, a calcium salt, a phosphate salt, a carbonate salt, a titanium salt or a zinc salt, or a combination thereof.

[0250] In preferred biodegradable microcapsules of the present invention, the coating is a polymeric coating, preferably formed from shellac.

[0251] Preferred biodegradable microcapsules of the present invention have a coating deposited on a covalently or non-covalently modified plant-derived protein matrix, the preferred characteristics of which are as described above.

[0252] In particularly preferred biodegradable microcapsules of the present invention, the plant-derived protein is pea protein and the active ingredient is vitamin D.

[0253] For preferred biodegradable microcapsules of the present invention, the percentage biodegradation based on O2 consumption of the plant-derived protein carrier measured according to ISO-14851 version 2019 after 28 days, based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, is 60-100%, more preferably 65-100%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100%. ISO-14851 version 2019 describes a method for measuring the biodegradability of materials in natural aqueous environments using biological oxygen demand in a closed respirometer. This is achieved by exposing the material under laboratory conditions in an aqueous standard test medium to an inoculum from previously unexposed, unadapted activated sludge. The measured value is calculated as a percentage of the theoretical oxygen demand calculated from the molecular formula. An internal standard of microcrystalline cellulose is also tested and if its % biodegradation is greater than 60% at the end of the test, the test is valid.

[0254] For preferred biodegradable microcapsules of the present invention, the biodegradation percentage based on the microcapsule's O2 consumption measured according to ISO-14851 version 2019 after 28 days is 60-100%, more preferably 65-100%, even more preferably 70-100%, even more preferably 75-100%, even more preferably 80-100%, even more preferably 85-100%, and most preferably 90-100%, based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand. ISO-14851 version 2019 describes a method for measuring the biodegradability of materials in natural aqueous environments using biological oxygen demand in a closed respirometer. This is accomplished by exposing the material under laboratory conditions in an aqueous standard test medium to an inoculum from previously unexposed, unadapted activated sludge. The measured value is calculated as a percentage of the theoretical oxygen demand calculated from the molecular formula. An internal standard of microcrystalline cellulose is also tested and if its % biodegradation is greater than 60% at the end of the test, the test is valid.

[0255] The present invention also provides a composition comprising the above-described biodegradable microcapsules and an external phase.

[0256] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0257] Preferred compositions of the present invention further comprise a suspending agent in the external phase.

[0258] Preferably, the suspending agent is selected from acacia gum, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil category 1, magnesium aluminum silicate, maltodextrin, carboxymethylcellulose, polymethacrylates, polyvinylpyrrolidone, sodium alginate, starch, zein, water-insoluble cross-linked polymers such as cross-linked cellulose, cross-linked starch, cross-linked CMC, cross-linked carboxymethyl starch, cross-linked polyacrylates, and cross-linked polyvinylpyrrolidone, and expanded clays such as bentonite and laponite.

[0259] The present invention also provides a formulated product comprising the biodegradable microcapsules described above.

[0260] The present invention also provides a method of making a formulated product, comprising: (a) preparing biodegradable microcapsules according to the method described above; (b) mixing the biodegradable microcapsules with a product formulation.

[0261] Preferably, the formulated product is a food, beverage, cosmetic, home care product, personal care product, pharmaceutical, industrial product (e.g., paint, adhesive, sandpaper, tape, etc.), medical device, biomaterial, or pesticide.

[0262] The present invention also provides the use of the biodegradable microcapsules described above in formulated products.

[0263] The formulated products described in various aspects of the present invention can be in any form suitable for use. Preferably, the formulated products are in the form of vapor sprays, aerosols, emulsions, lotions, liquids, creams, gels, sticks, ointments, pastes, mousses, powders, granular products, substrates, or semi-solids. More preferably, the formulated products are liquids, and even more preferably, aqueous liquids. [Brief explanation of the drawings]

[0264] [Figure 1a] 10X optical micrographs of spray-dried microcapsules of Examples 2B and 2E, respectively. [Figure 1b] 10X optical micrographs of spray-dried microcapsules of Examples 2B and 2E, respectively. [Figure 2a] 10X optical micrographs of spray-dried microcapsules of Examples 4A and 4B, respectively. [Figure 2b] 10X optical micrographs of spray-dried microcapsules of Examples 4A and 4B, respectively. [Figure 3] 10X optical microscope image of the Example 5 control in water. [Figure 4a] 6A and 6B are SEM images of the microcapsules of Example 6A at magnifications of ×80 and ×300, respectively. [Figure 4b] 6A and 6B are SEM images of the microcapsules of Example 6A at magnifications of ×80 and ×300, respectively. [Figure 5a] 6A-6B are SEM images of the spray-dried microcapsules of Example 6B at magnifications of ×60, ×500, and ×1250, respectively. [Figure 5b] 6A-6B are SEM images of the spray-dried microcapsules of Example 6B at magnifications of ×60, ×500, and ×1250, respectively. [Figure 5c] 6A-6B are SEM images of the spray-dried microcapsules of Example 6B at magnifications of ×60, ×500, and ×1250, respectively. [Figure 6] 1 is an SEM image of spray-dried microcapsules of Example 7 at x1200 magnification. [Figure 7a] 7A and 7B are 10x optical microscope images of the microcapsules of Example 7 before (FIG. 7a) and after (FIG. 7b) they were pressed to release the oil. [Figure 7b] 7A and 7B are 10x optical microscope images of the microcapsules of Example 7 before (FIG. 7a) and after (FIG. 7b) they were pressed to release the oil. [Figure 8a] 10X microscope images of concentrated slurries of Examples 15A, 15B, and Comparative Example 15C. [Figure 8b] 10X microscope images of concentrated slurries of Examples 15A, 15B, and Comparative Example 15C. [Figure 8c] 10X microscope images of concentrated slurries of Examples 15A, 15B, and Comparative Example 15C. [Figure 9] 10x optical microscope of microcapsules of Example 20 in 1x strength citrate buffer. [Figure 10a] 1 is a photograph of an inverted tube of Example 21. [Figure 10b] 1 is a photograph of an inverted tube of Example 21. [Example]

[0265] material Pea Protein Isolate (PPI) (80% protein, 4% carbohydrates by weight - ProEarth P16109) was purchased from Cambridge Commodities Ltd.

[0266] Lactic acid (85%, food grade) was purchased from Fisher Scientific.

[0267] Acetic acid (glacial acetic acid 99%) was purchased from Fisher Scientific.

[0268] Sodium benzoate was purchased from Fisher Scientific.

[0269] Vitamin D2 oil (1 MIU / g) was purchased from Prinova Europe, UK.

[0270] Miglyol® 812N was purchased from IOI Oleochemical.

[0271] Softisan® 100 was purchased from IOI Oleochemical.

[0272] Vitamin D2 powder, Vitamin D2 100 CWS (100,000 IU / g), was supplied by Prinova Europe, UK.

[0273] Prinova GA (acacia gum) Vitamin D2 capsule samples were supplied by Prinova Europe, UK.

[0274] Robinsons Double Strength No Added Sugar Squash Apple & Blackcurrant (manufactured by Britvic Plc and commercially available from UK supermarkets).

[0275] MiWadi Single Concentrate Blackcurrant, manufactured by Britvic Plc and available commercially from supermarkets in the Republic of Ireland.

[0276] Thymol was purchased from Fisher Scientific, UK. Calcium sulfate dihydrate (CaSO4.2H2O) was purchased from Fisher Scientific.

[0277] Dry magnesium sulfate (MgSO4) was purchased from Fisher Scientific.

[0278] Potassium chloride Extra Pure, SLR, Eur. Ph., (KCl) was purchased from Fisher Scientific.

[0279] Sodium bicarbonate (NaHCO3), certified by AR for analytical use, was purchased from Fisher Scientific.

[0280] Potassium hydroxide (KOH) (>85%) was purchased from Sigma Aldrich.

[0281] Absolute ethanol 99.8+% (EtOH), certified by AR for analytical use, was purchased from Fisher Scientific.

[0282] Maltodextrin was purchased from Sigma-Aldrich Gillingham, UK. Capsul® starch was supplied by Ingredion, Manchester, UK. Polysorbate 80 was purchased from Sigma-Aldrich Gillingham, UK. All components of phosphate buffered saline (PBS) (pH 7.4) were purchased from Fisher Scientific, UK and prepared in the following ratios: NaCl 138 mM, KCl 2.7 mM, Na2HPO4 10 mM, NaH2PO4 2 mM. Sodium acetate trihydrate was purchased from Alfar Aesar, UK. Calcium chloride dihydrate was purchased from Fisher Scientific, UK. Methylparaben was purchased from Alfar Aesar, UK. Gellan gum (high acyl) was purchased from Special Ingredients Ltd, UK. Linseed oil was purchased from Naissance, UK. Shellac was purchased from AF Suter & Company Ltd, UK.

[0283] Tannic acid was purchased from Fisher Scientific, UK. Xanthan gum was purchased from CP Kelco, UK. Tetraethyl orthosilicate (TEOS) was purchased from Acros Organics, UK. Olive oil was purchased from a UK supermarket. Vitamin E was purchased as DL α-tocopherol 97+% from Fischer Scientific, UK. Curcumin was purchased from Fisher Scientific as Curcumin 95%. β-Carotene was purchased from Sigma Aldrich, USA as synthetic β-carotene ≥ 93% (UV).

[0284] Fragrance Ingredients: [Table 1]

[0285] Standard hard water and buffer composition: 1) Hard water It 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.

[0286] 2) 1x strength buffer 100 mL of 1x strength buffer solution was prepared as follows: i. Weighed out 1.904 g of citric acid monohydrate and 0.276 g of trisodium citrate dihydrate. ii. The mixture was made up to 80 g using deionized water. iii. The pH of the solution was adjusted to 3 using 1 M HCl and 1 M NaOH solutions and the volume of the solution was brought to 100 g with DI water. iv. 0.38 g of CaCl2 dihydrate was added. v. 0.2 g of sodium benzoate was added. v. The solution was placed in an ultrasonic bath at 80°C for 5-10 minutes (i.e., until complete dissolution was observed). vi. The solution was allowed to cool to 20°C.

[0287] 3) 2x strength buffer A 100 mL double strength buffer solution was prepared in the same manner as for the 1x strength buffer, except the levels of all ingredients were doubled and the mixture still finished at 80 g.

[0288] Measurement method Vitamin D2 analysis of dry powder samples in microcapsules: Version A Vitamin D2 is extracted from the sample into dimethyl sulfoxide and then partitioned into hexane. Vitamin D2 is quantified using normal-phase HPLC with UV detection. This method is suitable for measuring high levels of vitamin D2, typically between 35 IU / g and 500,000 IU / g.

[0289] Vitamin D2 analysis of dry powder samples in microcapsules: Version B A powdered sample (approximately 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 without heating for 1-1.5 hours, cooled on ice, and then centrifuged at 14,500 rpm for 10 minutes before analysis by HPLC on a reversed-phase column using 100% methanol as the eluent and UV detection at 265 nm.

[0290] Vitamin D2 assay for dilute liquid systems containing microcapsules: Version A The sample is cold saponified overnight in alcoholic potassium hydroxide (containing pyrogallol). The unsaponified fraction is isolated by extraction into hexane. The extracted sample is then concentrated and injected onto a semi-preparative HPLC column. The fraction of the eluent containing vitamin D2 is collected, evaporated to dryness, dissolved in methanol, and vitamin D2 is isolated using reverse-phase HPLC with UV detection. This method is suitable for measuring low levels of vitamin D2, typically 0.5 IU / g to 100 IU / g. Higher levels can be quantified with further dilution.

[0291] Vitamin D2 assay for dilute liquid systems containing microcapsules: Version B A liquid sample (50 g) was centrifuged at 4,500 rpm for 15 minutes to obtain a sediment. To measure the vitamin D2 concentration in the supernatant, approximately 5 g of the supernatant was taken and 2 g of 50% w / v aqueous potassium hydroxide, 3 g of absolute ethanol, and 0.05 g of sodium ascorbate were added. The sample was then mixed thoroughly. The sample was placed in an ultrasonic bath for 1–1.5 hours without heating; however, the temperature may have risen to 50–60°C by the end. If the mixture was not sonicated for an additional hour, a microscope was used to confirm complete destruction of the microcapsules. The sample was then cooled to room temperature using ice, and approximately 1 mL was then taken and centrifuged at 14,500 rpm for 15 minutes. The supernatant was transferred to an amber vial for HPLC analysis on a reverse-phase column using methanol / water (95:5) as the eluent and UV detection at 265 nm. The vitamin D2 content per 100 g of liquid was reported.

[0292] To measure the vitamin D2 concentration in the sediment, carefully remove the supernatant with a pipette, leaving a small amount of supernatant behind to avoid losing any sediment. This yields 1-2 g of wet sediment, which is then subjected to the same steps as for the 5 g supernatant, as described herein. This method is suitable for measuring low levels of encapsulated vitamin D2, typically 0.5 IU / g to 100 IU / g. For shelf-life testing of encapsulated vitamin D2, only the sediment value is reported, as all vitamin D2 was found to remain within the microcapsules. Any vitamin D2 that leaks from the microcapsules is degraded.

[0293] Hydrogel slurry particle size The particle size of the hydrogel slurries was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. Measurements were performed by diluting the hydrogel within one hour of completion of preparation in an aqueous solution containing acetic or lactic acid adjusted to the same pH. The test material was diluted to the required concentration to ensure the absence of aggregates and the desired optical density for measurement (typically 5-15% obscuration). 50is for the volume distribution calculated by a general analysis using Mie theory.

[0294] Droplet size of the composition for spray drying The droplet size of the oil or wax containing the active ingredient in the composition used for spray drying was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. Measurements were performed by diluting the composition within one hour of completion of production in an aqueous solution containing acetic or lactic acid adjusted to the same pH. The test material was diluted to the required concentration to have the desired optical density for measurement (usually 5-15% obscuration). The quoted d 50 is for the volume distribution calculated by a general analysis using Mie theory.

[0295] Protein Solids The solids content of the hydrogel slurry was determined by the mass remaining upon drying. Approximately 5 g of the hydrogel slurry was pipetted into a small polypropylene dish, and the mass was accurately recorded. The dish was placed in a 40°C oven overnight to dry. The dry mass was measured immediately after removing the dish from the oven, and the solids content of the protein hydrogel slurry was calculated as a percentage of the initial wet mass.

[0296] Viscosity of the composition for spray drying The viscosity of the compositions for spray drying was measured within 1 hour of the end of the composition preparation using an Anton Paar MCR 92 rheometer. The rheometer was set up using a cone (1 degree, 50 mm diameter) and plate geometry. The viscosity was measured at a temperature of 20°C and a shear rate of 50 / s.

[0297] Particle size of spray-dried microcapsules The particle size of the final microcapsule powder was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. The powder was added to reverse osmosis water diluted to the required concentration to obtain the desired optical density for measurement (typically 5-15% obscuration). The dispersion should be checked under an optical microscope for any larger agglomerates of microcapsules. If these are visible, 0.5% by weight of acetic acid can be added to ensure the primary particles are well dispersed. 50 is for the volume distribution calculated by a general analysis using Mie theory.

[0298] Alternatively, particle size can be measured using an optical microscope (e.g., an open-frame microscope equipped with a CellCam 200CR camera, an Aura Pro phase-contrast illuminator, and 4x, 10x, and 20x Universal Plan fluor objectives). Microcapsule powder is added to either reverse osmosis water or 1x strength buffer, as appropriate. In this method, particle size is obtained from the average size measurement of 50 microcapsules for each sample. The optical microscope is then calibrated using a grid from a Hirschmann counting chamber (Fuchs Rosenthal). Using the line tool in ImageJ 1.53, particle diameters are measured from two center-edge points with the overlay text function enabled to avoid capsule overlap.

[0299] Scanning electron microscope (SEM) SEM images were obtained using a Hitachi TM3030.

[0300] optical microscopy Optical microscopy images were obtained using an open-frame microscope equipped with a CellCam 200CR camera, an Aura Pro phase-contrast illuminator, and 4x, 10x, and 20x Universal Plan fluorite objectives. Samples for optical microscopy were prepared by first rehydrating the dried microcapsules in hard water with the following composition: [Table 2]

[0301] Prior to microscopic imaging, the microcapsules were left in hard water or 1x buffer for 5 minutes. A cover slip was then placed on top of the rehydrated sample and images were taken. The slide was then removed from the microscope stage, and firm pressure was applied to the cover slip by hand before the sample was returned to the stage and images of the ruptured capsules were taken.

[0302] Total Fragrance and Fragrance Ingredient Load Fragrance and fragrance material levels in microcapsules can be measured by extracting the fragrance and injecting it onto a GC column. Quantification is achieved by using a calibration curve for a given fragrance material diluted in ethanol.

[0303] To measure the total level of fragrance material in an encapsulated sample, the capsules should first be disrupted by sonication. For example, 10–50 mg of dry encapsulates or 0.3–0.5 g of wet encapsulates collected on a 40 μm cell strainer in 3.0 g of deionized (DI) water and 10% KOH are vortexed to thoroughly mix. The solution is then sonicated for 1–2 minutes at 30% amplitude (approximately 1.5–3.0 kJ total) using a Bandelin ultrasonicator with a small probe, TS104. During sonication, ice is used to maintain the temperature below 20°C and avoid fragrance loss due to evaporation. Visually confirm whether the capsules are completely disrupted using the optical microscope detailed above. If not, repeat the sonication process. Ethanol is added to the mixture, which is then mixed and centrifuged. The supernatant is collected and retained as the first extract. The residue is mixed with ethanol and centrifuged. The supernatant is collected and added to the first extract. This mixture is then diluted appropriately and injected into a GC for analysis.

[0304] For samples containing benzyl salicylate, this is quantified by measuring the benzyl alcohol formed from its hydrolysis.

[0305] Total thymol load Thymol levels in the dried microcapsules were determined by extracting the thymol and injecting it onto a GC column according to the total fragrance loading method herein.

[0306] Olfactory evaluation of microcapsule slurries For any given encapsulation, the total fragrance level is quantified by GC as described herein. Based on the encapsulation load, the material is diluted to deliver the appropriate level of fragrance for olfactory evaluation. Optionally, preservatives and suspending agents are added to the diluted encapsulation sample.

[0307] Using a low-shear micropipette tip, pipette 50 µL of diluted encapsulates onto a standard microscope glass slide, ensuring that most of the surface area is covered, leaving the edges clean for handling the slide. Repeat this at least four times.

[0308] 50 μL of the diluted encapsulate slurry is also pipetted onto a rectangular fragrance evaluation card blotter (approximately 9 cm x 5 cm). This is repeated at least twice.

[0309] The slides and blotters were evaluated by trained evaluators at different time points. 1) Immediately after application, 2) Leave it overnight at 20-25°C to dry, then 3) Leave to dry overnight at 20-25°C, then rub the slide glass with a gloved index finger for 10 strokes (in either direction), 4) Leave to dry overnight at 20-25°C, then press the slide against another slide for 10 seconds and remove. 5) Leave to dry overnight at 20-25°C, then rub with another clean piece of blotting paper for 10 strokes (in either direction).

[0310] The evaluators rated the intensity according to the following scale and commented on the fragrance characteristics: [Table 3]

[0311] moisture The moisture content of the microcapsules is measured by weighing a suitable mass of dry microcapsules into an Ohaus MB23 Moisture Analyzer (Ohaus Europe GmbH, Switzerland) and dispersing the sample evenly throughout the pan. The sample is heated to 120°C and the moisture content is reported as % by weight.

[0312] Microcapsule Integrity The physical integrity of the microcapsules is assessed according to the following method: A sample of microcapsules (0.1 g) is weighed into a 15 mL Falcon tube. Hot deionized water (>70°C) is added to the capsules to a total of 10 g and manually shaken to redisperse the powder or pellet. The sample is then vortexed at 2000 rpm for 90 seconds to completely resuspend the microcapsules. The sample is checked for uniform distribution by light microscopy and can be vortexed for an additional 30 seconds if necessary. The Vitamin D2 Analysis for Dilute Liquid Systems Containing Microcapsules: Version B is then performed for analysis of the supernatant and sediment. Microcapsule integrity is reported as the % of vitamin D2 leaking into the supernatant, which is 0 for full microcapsule integrity.

[0313] Example 1 - Preparation of Protein Hydrogel Slurry with Acetic Acid Preparation of protein hydrogels An 800 mL mixture consisting of 12.5% ​​(w / v) pea protein isolate (PPI) in 30% (v / v) acetic acid solution was prepared.

[0314] The mixture was then heated in a water bath at 85°C, followed by an ultrasonication step to break up large colloidal aggregates (Hielscher UIP), after which a clear solution was obtained. The applied energy was 200 kJ.

[0315] The solution was then poured into a container to a depth of 1 cm and allowed to cool at 5°C for 28 hours to obtain a free-standing protein hydrogel.

[0316] Application of shear to protein hydrogels Shear was then applied to the hydrogel as follows: The protein hydrogel was cut into approximately 1 cm cubes using a low-shear cutting process. The cubes were placed inside a 75 micron filter bag, which was then submerged inside a bucket containing 10 L of reverse osmosis water. This created a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were immersed with occasional gentle agitation. This process was performed to reduce the concentration of acetic acid in the hydrogel by diffusion into the continuous aqueous phase.

[0317] The strained gel cubes were transferred to a bottle containing 4 wt% sodium benzoate and exposed to high shear followed by ultra-high shear (probe sonication up to 200 kJ using a Hielscher sonicator) on ice to form a homogenous, low-viscosity protein hydrogel slurry.

[0318] Example 2 - Preparation of spray-dried microcapsules containing vitamin D2 Preparation of wax / vitamin D2 mixture Mixtures of 22% (220,000 IU / g) and 11% (110,000 IU / g) by weight were made from vitamin D2 oil (1 MIU / g) in Softisan® 100 wax. The wax was melted above 40°C, the vitamin oil was added, and the mixture was homogenized by manual shaking. The mixture was kept above 40°C until needed.

[0319] Example 2A: Acid-treated hydrogel slurry solids to wax at a 5 to 1 ratio with vitamin D2 added by sonication 1500 g of protein hydrogel slurry was made according to Example 1, where a mixture of PPI and acetic acid was heated for 20 minutes, the gel cubes were soaked for 2 hours, and then high sheared at 15,000 rpm for 15 minutes, followed by ultra-high shear at 0.25 kJ / mL to form a hydrogel slurry.

[0320] Preparation of the Composition The protein hydrogel slurry was heated to 35°C and 22% by weight of the wax / vitamin D2 molten mixture was added, resulting in a protein hydrogel slurry to wax / vitamin D2 weight ratio of 98.3 to 1.7. The resulting mixture was mechanically high sheared using an Ultra-Turrax disperser at 15,000 rpm for 10 minutes, followed by ultra-high shearing on ice using a Hielscher sonicator applying an energy of 0.1 kJ / mL. This resulted in a viscosity of 27.3 cP and an average droplet size d of 0.67 microns. 50 A composition having the formula:

[0321] Spray drying of the composition The composition was spray dried according to the process settings in Table 1 to produce a mean particle size d of 23.8 microns. 50 The vitamin D2 concentration in the microcapsule powder was measured using method version A described herein and found to be 100,250 ug / 100g.

[0322] Example 2B: Acid-treated hydrogel slurry solids to wax at a 2 to 1 ratio with vitamin D2 added by mechanical mixing 1500 g of protein hydrogel was made according to Example 1, where a mixture of PPI and acetic acid was heated for 20 minutes, the gel cubes were soaked for 2 hours, and then high sheared at 15,000 rpm for 15 minutes, followed by ultra-high shear at 0.25 kJ / mL to form a hydrogel slurry.

[0323] Preparation of the Composition The protein hydrogel was heated to 35°C and 11% by weight of the wax / vitamin D2 molten mixture was added, resulting in a protein hydrogel slurry to wax / vitamin D2 ratio of 96 to 4 by weight. The resulting mixture was mechanically high sheared using an Ultra-Turrax disperser at 15,000 rpm for 10 minutes, resulting in a viscosity of 31.4 cP and an average droplet size d of 4.6 microns. 50 A composition having the formula:

[0324] Spray drying of the composition The slurry was spray dried according to the process settings in Table 1 to produce a mean particle size d of 35.9 microns. 50 The vitamin D2 concentration in the microcapsule powder was measured using method version A described herein and found to be 94,750 ug / 100g.

[0325] Example 2C: Acid-Treated Hydrogel Slurry Solids to Wax at a 5 to 1 Ratio with Vitamin D2 Added by Mechanical Mixing 1500 g of protein hydrogel slurry was made according to Example 1, where a mixture of PPI and acetic acid was heated for 20 minutes, the gel cubes were soaked for 2 hours, and then high sheared at 15,000 rpm for 15 minutes, followed by ultra-high shear at 0.25 kJ / mL to form a hydrogel slurry.

[0326] Preparation of the Composition The protein hydrogel slurry was heated to 35°C and 22% by weight of the wax / vitamin D2 molten mixture was added, resulting in a protein hydrogel slurry to wax / vitamin D2 weight ratio of 96 to 4. The mixture was mechanically high sheared using an Ultra-Turrax disperser at 15,000 rpm for 10 minutes, resulting in a viscosity of 35.8 cP and an average droplet size d of 6.2 microns. 50 A composition having the formula:

[0327] Spray drying of the composition The composition was spray dried according to the process settings in Table 1. Two different settings were used to obtain samples Ci and Cii. The Ci and Cii microcapsules each had an average particle size d of 31.3 microns. 50 The Vitamin D2 concentration in the Cii microcapsule powder was measured using method version A described herein and found to be 95,500 ug / 100 g.

[0328] Example 2D: 5 to 1 ratio of protein solids to wax in sonicated PPI (in 5 wt% acetic acid) with vitamin D2 added by mechanical mixing. Preparation of PPI mixture A mixture of 8 wt% pea protein isolate in 5 wt% (v / v) acetic acid solution was prepared to have a solids concentration comparable to the protein hydrogel slurry prepared in Example 1. The mixture was ultra-high sheared using a Hielscher probe sonicator at an energy input of 1 kJ / mL, resulting in a mean particle size d of 2.9 microns. 50 The pH of the protein mixture was measured at 3.4.

[0329] Preparation of the Composition The protein mixture was heated to 35°C and 22% by weight of the wax / vitamin D2 molten mixture was added, resulting in a weight ratio of 98.3 to 1.7 protein mixture to wax / vitamin D2. The mixture was mechanically high sheared using an Ultra-Turrax disperser at 15,000 rpm for 10 minutes, resulting in a viscosity of 5.0 cP and an average droplet size d of 4.4 microns. 50 A composition having the formula:

[0330] Spray drying of the composition The composition was spray dried according to the process settings in Table 1 to produce a mean particle size d of 20.2 microns. 50 The vitamin D2 concentration in the microcapsule powder was measured using method version A described herein and found to be 86,500 ug / 100 g.

[0331] Comparative Example 2E: 5:1 ratio of protein solids to wax in sonicated PPI with vitamin D added by mechanical mixing. Preparation of PPI mixture A slurry of 8 wt% pea protein isolate in reverse osmosis water was prepared with a solids concentration comparable to the protein hydrogel slurry prepared in Example 1. The mixture was ultra-high sheared using a Hielscher probe sonicator at an energy input of 1 kJ / mL. This resulted in a mean particle size d of 1.4 microns. 50 The pH of the protein mixture was adjusted to 6.85.

[0332] Preparation of the Composition The protein mixture was heated to 35°C and 22% by weight of the wax / vitamin D2 molten mixture was added, resulting in a weight ratio of 98.3 to 1.7 protein mixture to wax / vitamin D2. The mixture was mechanically high sheared using an Ultra-Turrax disperser at 15,000 rpm for 10 minutes, resulting in a viscosity of 4.9 cP and an average droplet size d of 4.6 microns. 50 A composition having the formula:

[0333] Spray drying of the composition The composition was spray dried according to the process settings in Table 1 to produce a mean particle size d of 40.0 microns. 50 The vitamin D2 concentration in the microcapsule powder was measured using method version A described herein and found to be 93,000 ug / 100 g.

[0334] Spray drying conditions for Examples 2A to 2E All compositions prepared in Examples 2A-2E were spray dried in a co-current spray dryer equipped with a 1 m diameter chamber and a Spraying Systems FloMax nozzle. The inlet air flow was maintained at 1.5 kg / min, and the slurry feed addition rate was adjusted within a specified range to maintain the outlet temperature. The feed characteristics and spray drying conditions are summarized in Table 1 below. [Table 4]

[0335] The conditions in Table 1 demonstrate that despite the higher feed viscosities of Examples 2A, 2B, and 2C compared to Examples 2D and 2E, the compositions were successfully spray dried under standard equipment and process conditions, which is believed to be due to the shear thinning properties of the compositions of Examples 2A, 2B, and 2C.

[0336] All compositions were successfully spray dried to an average particle size d of 20-40 microns. 50 Figures 1a and 1b are optical micrographs at 10x magnification of the spray-dried microcapsules of Examples 2B and 2E, respectively, showing the visual similarity of the resulting microcapsule powders.

[0337] Example 3 - Preparation of Protein Hydrogel Slurry with Lactic Acid Preparation of protein hydrogels 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 in a Bandelin sonicator until 100 kJ was delivered. The hot protein solution was poured into a Petri dish and allowed to cool overnight at 5°C to yield a free-standing protein hydrogel.

[0338] Application of shear to protein hydrogels 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 approximately 1.5 hours with occasional gentle agitation. The water was then drained from the bag, and the same soaking procedure was repeated three more times until the pH reached above 3.0.

[0339] The hydrogel was then drained and added to a 5 wt% sodium benzoate solution (pH adjusted to 4-5 using lactic acid) at 20 mL per kilogram 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 determined to be 5.1 wt% by the method described herein.

[0340] Example 4 - Preparation of spray-dried microcapsules containing vitamin D2 Preparation of the Composition Vitamin D2 oil (1 MIU / g) was diluted in Miglyol® 812N or Softisan® 100 wax to a concentration of 6000 μg / g. This concentration was chosen to produce spray-dried particles containing a vitamin D2 concentration of 1,000 μg / g. In the case of Softisan® 100 wax, the wax was melted above 40°C, the vitamin oil was added, and the mixture was homogenized by manual shaking. The mixture was kept above 40°C until needed.

[0341] The required mass of oil or wax and vitamin D2 mixture was then added to the protein hydrogel slurry made in Example 3 to give a mass ratio of 5 to 1 hydrogel slurry solids to vitamin D2:wax / oil mixture. The composition was formed by mixing for 15 minutes at 5000 rpm using an IKA Ultra-Turrax, followed by probe sonication to 0.1 kJ / mL using a Bandelin sonicator.

[0342] Spray drying conditions Spray drying was carried out using a ProCept spray dryer in its three column setup. The column height was 1.8 m and the column diameter was 0.15 m. A two-fluid nozzle with a 0.8 mm tip size was used. The atomizing air flow rate was 10 L / min. Fluids were pumped into the spray dryer at 6 mL / min using a syringe pump. Other settings are shown in Table 2 below. An additional air flow rate of 100 L / min was set for the cyclone. [Table 5]

[0343] The results also demonstrate that protein hydrogel slurries formed using lactic acid can be spray dried under standard conditions using standard equipment. The results also demonstrate that a range of different carrier materials (e.g., oils and waxes) for the active ingredient, here vitamin D2, can be used.

[0344] In the spray drying experiments, the mean particle size d measured by laser diffraction 50 However, two free-flowing powders were produced, 15.8 microns for Example 4A and 32.5 microns for Example 4B. The vitamin D2 concentration in the microcapsule powders was measured using Method Version A described herein and found to be 89,000 μg / 100 g and 86,500 μg / 100 g for Samples 4A and 4B, respectively. Figures 2a and 2b are 10x optical micrographs of the spray-dried microcapsules of Examples 4A and 4B, respectively, showing the visual similarity of the powders produced, with the differences in particle size being observable.

[0345] Example 5 - Accelerated Stability Study of Vitamin D2 Microcapsules in a Beverage Formulation Sample preparation The microcapsules of Examples 4A and 4B were each added to Robinsons Double Concentrate No Added Sugar Apple & Blackcurrant squash. More specifically, 45 mg of microcapsules were added to 1 liter of squash to achieve a vitamin D concentration of 4.5 μg / 100 mL in the product, assuming a vitamin D concentration of 100,000 μg / 100 g in the microcapsules. A comparative example was also prepared by adding 9 mg of Prinova Vitamin D2 powder to the same squash sample. Prinova Vitamin D2 powder is a commercially available powder form of vitamin D2. The three mixtures were heated to 95°C on a hot plate with constant stirring and then held at 95°C for 30 seconds to mimic the temperature profile during flash pasteurization. The mixtures were then allowed to cool to 20°C.

[0346] Accelerated Stability Testing Three squash samples were filled into 200 mL clear plastic bottles and placed in a Binder KBF P 240-230V chamber set up for accelerated ambient light stability testing in a standard configuration with visible and UV-A tubes. Nominal UV-A output was 1.1 W / m. 2 The temperature was set at 20°C and laboratory humidity (typically 50% relative humidity (RH)) was used. The initial vitamin D2 loading in the squash was measured using method version A described herein and then measured again after 4 weeks in a binder chamber. The % decrease in vitamin D2 content over the 4-week period was calculated and recorded in Table 3. Vitamin D2 is expected to degrade significantly when exposed to UV over this time frame. [Table 6]

[0347] As can be seen from Table 3, after 4 weeks in squash, the commercially available powdered vitamin D2 was almost completely degraded, with 95% of the initial vitamin D2 being degraded.

[0348] Figure 3 is a 10x microscope image of Prinova Vitamin D2 powder added to cold tap water, showing the presence of a fine dispersion of material; no microcapsules are present. Without being bound by theory, it is believed that the carrier material in this powder is highly soluble in squash, causing it to disintegrate in water and therefore failing to protect the Vitamin D2 in accelerated stability testing.

[0349] However, for Example 4A, after 4 weeks in the squash, the vitamin D2 level was still approximately half of the original level, demonstrating that the microcapsules with a plant-derived protein carrier and an oil-based active carrier phase protected vitamin D2 from UV exposure, thus reducing degradation.The presence of an insoluble plant-derived protein carrier clearly helped protect the encapsulated vitamin D2 compared to the commercially available materials tested in the comparative experiment.Furthermore, for Example 4B, after 4 weeks in the squash, the vitamin D2 level remained substantially unchanged from the original level, demonstrating that the microcapsules with a plant-derived protein carrier and a wax-based active carrier phase were able to completely protect vitamin D2 from degradation.Without being 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, thus providing better protection for the vitamin from UV.

[0350] Example 6 - Preparation of spray-dried fragrance microcapsules Preparation of fragrance / solvent mixtures An 80:20 weight ratio mixture of delta-damascone and Miglyol® 812 was prepared by gentle mixing.

[0351] Preparation of spray-dried microcapsules Protein hydrogel slurries were made according to Example 1, where a mixture of PPI and acetic acid was heated for 30 minutes, the gel cubes were soaked for 1 hour, and then high sheared using an IKA Ultra-Turrax at 25,000 rpm for 15 minutes, followed by ultra-high shearing using sonication (Hielscher sonicator) at 1.0 kJ / mL to form a hydrogel slurry.

[0352] Average particle size of hydrogel slurry d 50 was measured according to the methods described herein and found to be 2.5 microns. The average solids content of the hydrogel slurry was measured according to the methods described herein and found to be 8.6 wt %.

[0353] Two samples were prepared with the compositions shown in Table 4 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 achieve the desired dispersed droplet size of 5 μm. [Table 7]

[0354] The Example 6A phase readily separated to give a clear liquid upper layer and a lower layer of hydrogel fragments, which could be rehomogenized by vigorous mixing before spray drying. The well-mixed slurry had a viscosity of 150 cP at 50 s−1.

[0355] A 1.5 mg droplet of Example 6A was suspended on a thin wire connected to a microbalance and placed in the center of a 2.5 cm inner diameter pipe. 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 a period of 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.

[0356] The resulting particles had an internal matrix structure and an external smooth skin, as can be seen in the SEM images of Figures 4a and 4b.

[0357] Example 6B was a homogeneous, viscous semi-solid with a viscosity of 1000 cP at 50 s.

[0358] A 2.5 mg droplet of Example 6B was suspended on a thin wire connected to a microbalance and placed in the center of a 2.5 cm inner diameter pipe. 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 a period of 3.5 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.

[0359] The resulting particles had a matrix structure and a porous surface, as can be seen in the SEM images of Figures 5a, 5b, and 5c.

[0360] Example 7 - Preparation of spray-dried fragrance microcapsules Preparation of fragrance / solvent mixtures An 80:20 volume mixture of delta-damascone and Miglyol® 812 was prepared by gentle mixing.

[0361] Preparation of spray-dried microcapsules A protein hydrogel slurry was made according to Example 1. The hydrogel slurry was diluted with acetic acid to an average protein solids content of 8.0% by weight. A 221 g batch was mixed at 8000 rpm for 2 minutes using an UltraTurrax lab mixer. 131 g of a fragrance:oil mixture containing 80% δ-damascone and 20% Miglyol® 812 oil was then added. The composition is shown in Table 5 below. [Table 8]

[0362] The batch was subjected to high shear for 5 minutes using an UltraTurrax lab mixer set at 8000 rpm to create a fine emulsion, with an expected droplet size of approximately 5 microns.

[0363] The samples were then spray dried using a ProCepT R&D Spray Dryer with its three column setup. The column height was 1.8 m and the column diameter was 0.15 m. The air inlet temperature was 130° C. with a flow rate of 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 rate of 140 L / min was introduced below the drying chamber to carry the air to the cyclone.

[0364] The dry microcapsule powder formed from Example 7 was collected from the cyclone and also from the interior surfaces of the equipment to which it adhered, for example, in the transfer pipe to the cyclone.

[0365] The microcapsules of Example 7 were examined under a Hitachi TM3030 SEM, and primarily spherical fragrance encapsulates with diameters of approximately 30 μm or less were observed (FIG. 6). The microcapsules appeared to have an uneven surface, indicating that there was very little free fragrance present and that the sample was adequately dried.

[0366] The microcapsules of Example 7 were rehydrated in hard water and photographed under an optical microscope (Figure 7a). The same capsules were then compressed and broken, releasing the oil contained therein (Figure 7b). Oil droplets are clearly visible around the broken microcapsules, with a very distinct dark interface between the oil and water.

[0367] The microcapsules of Example 7 were analyzed for their total fragrance loading by the methods described herein, which was found to be 28.8%.

[0368] Example 8 - Preparation of spray-dried fragrance microcapsules Preparation of fragrance / solvent mixtures An 80:20 volume mixture of delta-damascone and Miglyol® 812 was prepared by gentle mixing.

[0369] Preparation of spray-dried microcapsules A protein hydrogel slurry was made according to Example 1. The hydrogel slurry was diluted with acetic acid to an average protein solids content of 8.0% by weight. A 221 g batch was mixed at 8000 rpm for 2 minutes using an UltraTurrax lab mixer. 33.1 g of a fragrance:oil mixture containing 80% δ-damascone and 20% Miglyol® 812 oil was then added. The composition is shown in Table 6 below. [Table 9]

[0370] A fragrance emulsion was prepared according to Table 6, using manual shaking instead of high shear mixing to form the emulsion. The droplet size was expected to be approximately 20 microns. The samples were then spray dried using a ProCepT R&D Spray Dryer with its three column setup. The column height was 1.8 m and the column diameter was 0.15 m. The air inlet temperature was 120° C. with a flow rate of 400 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. Fluids were pumped into the spray dryer at mL / min using a syringe pump. An additional air flow rate of 1450 L / min was introduced below the drying chamber to carry the air to the cyclone.

[0371] The dry microcapsule powder that formed was collected from the cyclone and also from the interior surfaces of the equipment to which it adhered, for example, in the transfer pipe to the cyclone.

[0372] The microcapsules of Example 8 were analyzed for their total fragrance loading by the methods described herein, which was found to be 18.7%.

[0373] Example 9 - Preparation of spray-dried fragrance microcapsules A fragrance composition was prepared according to Example 7 as per Table 5, except that instead of high shear mixing with an Ultra Turrax, the emulsion was formed by manual shaking. The droplet size was expected to be approximately 20 microns.

[0374] The samples were then spray dried using a ProCepT R&D Spray Dryer with its three column setup. The column height was 1.8 m and the column diameter was 0.15 m. The air inlet temperature was 120° C. with a flow rate of 400 L / min. A two-fluid nozzle with a tip size of 1.0 mm was used. The atomizing air flow rate was 6.5 L / min. The fluid was pumped into the spray dryer at 2 mL / min using a syringe pump. An additional air flow rate of 150 L / min was introduced below the drying chamber to carry the air into the cyclone.

[0375] The dry microcapsule powder that formed was collected from the cyclone and also from the interior surfaces of the equipment to which it adhered, for example, in the transfer pipe to the cyclone.

[0376] The microcapsules of Example 9 were analyzed for their total fragrance loading by the methods described herein, which was found to be 22.9%.

[0377] Example 10 - Coating of spray-dried fragrance microcapsules with shellac 30 g of 25 wt% shellac solution was diluted in 120 mL of deionized water in a tall 250 mL glass beaker. Mechanical stirring was initiated at 350 rpm with an anchor stirrer at room temperature.

[0378] Separately, to obtain a slurry paste, 1.5 g of dry powder from Example 8 in a 50 mL Falcon tube was added pH 3 hard water dropwise, vortexing between additions. Once the paste was homogenous, more pH 3 hard water was added, still vortexing, to ensure homogenous dispersion of the particles. The microencapsulate slurry was further adjusted to a 50% (v / v) slurry in pH 3 hard water.

[0379] The diluted microencapsulates were added dropwise to the stirring shellac solution using a 3 mL transfer pipette. The mixture was stirred for 30 minutes and then stored in a refrigerator for 3 days to form a coating. The crosslinked microcapsules were then subjected to a washing process, whereby they were suspended in a separatory funnel and sieved through a 38 μm sieve and a 250 μm sieve stacked together to eliminate any agglomerates that may have resulted from the crosslinking process. The capsules collected on the 38 μm sieve were resuspended in hard water in a clean separatory funnel for an additional washing step. The decanted capsules were strained through a 40 μm cell strainer for 5 minutes. Any excess water was absorbed with tissue paper. The strained capsules were diluted with 1x strength buffer to make a 50% by weight slurry.

[0380] The coated microcapsules of Example 10 were analyzed for their total fragrance loading by the method described herein, which was found to be 0.8%.

[0381] Example 11 - Crosslinking of spray-dried fragrance microcapsules with tannic acid and complex coacervation with xanthan gum A 0.1 wt% xanthan gum solution was prepared in a water bath set at 60°C under magnetic stirring until complete dissolution was observed. The solution was stored in a refrigerator overnight to allow the gum to fully hydrate. The next day, the xanthan gum solution was adjusted to pH 3 with 1 M HCl. A 10 wt% tannic acid solution was prepared by manually shaking and vortexing for a few seconds using a Fisherbrand™ ZX4 IR Vortex Mixer, followed by immersion in an ultrasonic bath set at 50°C for 5-10 minutes until completely dissolved.

[0382] Separately, to obtain a slurry paste, 1.5 g of dry powder from Example 8 was added dropwise to pH 3 hard water in a 50 mL Falcon tube, vortexing between additions. Once the paste was homogenous, more pH 3 hard water was added, still vortexing, to ensure homogenous particle dispersion. The microencapsulate slurry was further diluted in a total of 380 mL of hard water and adjusted to pH 3 with 1 M HCl.

[0383] 20.2 g of tannic acid solution was added to the microencapsulated suspension under mechanical stirring at 170 rpm at 50°C, and stirring was continued for 5.5 hours to allow crosslinking to occur. After this, the suspension was stored in a refrigerator overnight. The crosslinked microcapsules were then subjected to the washing process as in Example 10, resulting in brown encapsulated material, indicating that crosslinking had occurred.

[0384] The cross-linked microencapsulated material was then filtered, suspended in 400 mL of hard water, and adjusted to pH 3 with 1 M HCl. The suspension was placed under mechanical stirring at 350 rpm at room temperature. 545.7 g of xanthan gum solution was slowly added with mixing and allowed to stand for 1.5 hours to allow coacervation. The final microcapsules were then subjected to the washing process outlined in Example 10.

[0385] The microcapsules of Example 11 were analyzed for their total fragrance loading by the methods described herein, which was found to be 1.5%.

[0386] Example 12 - Coating of spray-dried fragrance microcapsules with silica by direct immersion in TEOS 300 mg of the dry powder from Example 9 was added directly to 2 g of TEOS and mixed manually to form a suspension. The microencapsulated suspension was mixed at 200 rpm for 6 hours to ensure that the capsules did not settle during the reaction. The microcapsules were then centrifuged at 4500 rpm for 5 minutes to settle, and most of the supernatant was removed. The settled microcapsules were resuspended in the remaining TEOS, pipetted onto filter paper, and air-dried overnight to remove excess TEOS.

[0387] The microcapsules of Example 12 were analyzed for their total fragrance loading by the methods described herein, which was found to be 6.3%.

[0388] Example 13 - Preparation of Protein Hydrogel Slurry with Acetic Acid Preparation of protein hydrogels 1120 g of reverse osmosis (RO) water was added to a 2 liter stainless steel vessel, and 216 g of pea protein isolate was added. The vessel was placed in a 92°C water bath and mixed at 1500 rpm using an overhead stirrer. After 3 minutes of stirring, 480 g of glacial acetic acid was added. The mixture was stirred at 1500 rpm for 15 minutes, then at 1200 rpm for 30 minutes, allowing the temperature of the mixture to exceed 85°C for at least 10 minutes. The mixture was poured into a tray to a depth of approximately 10 mm and allowed to stand at room temperature overnight.

[0389] Application of shear to protein hydrogels Shear was then applied to the hydrogel as follows: The protein hydrogel was cut into approximately 1 cm cubes using a low-shear cutting process. The cubes were divided between two 75-micron filter bags, and each was then submerged in a bucket containing 16 L of RO water. This formed a coarse protein hydrogel slurry within the filter bag. The hydrogel cubes were soaked for 90 to 150 minutes while stirring with an overhead stirrer at 600 to 800 rpm. This step was performed to reduce the concentration of acetic acid in the hydrogel by diffusion into the continuous aqueous phase. The pH of the wash water was then measured, and if it exceeded 3.2, soaking was continued for an additional 30 minutes. If it was below 2.9, half of the water was drained and replaced with fresh RO water, and soaking was continued for an additional 30 minutes. The filter bags were then hung over the bucket and allowed to drain for 5 minutes. The washed gel from both filter bags was transferred to a 5-liter beaker and homogenized using a Silverson mixer at 5000 rpm for 5 minutes, 6000 rpm for 5 minutes, and 7000 rpm for 5 minutes. The smooth slurry was then transferred to 1-liter Nalgene bottles (800 g each) and exposed to high-shear sonication (Hielscher UP500Hdt) while chilled on ice, shaking every 75 kJ until 250 kJ was applied. The hydrogel slurry was then passed through a 200-micron sieve before use.

[0390] Comparative Example 14 - Preparation of Protein Slurry Without Acid 450 g of reverse osmosis water was added to a 1 L stainless steel container, and 50 g of pea protein isolate was added. The container was placed in a 92°C water bath and mixed at 1500 rpm using an overhead stirrer. The mixture was stirred at 1500 rpm for 15 minutes, then at 1200 rpm for 30 minutes, allowing the mixture temperature to exceed 85°C for at least 10 minutes. The mixture was cooled to ambient temperature, and in contrast to the experiment using acetic acid, no gel formed. Shear was then applied using a Silverson mixer at 8000 rpm for 10 minutes, followed by a sonicator (Hielscher UP500Hdt) until 156 kJ was applied. For comparison, 0.3125 kJ / g was used for the protein slurry prepared with organic acids. The mixture was then passed through a 200 micron sieve before use.

[0391] Example 15 - Preparation of spray-dried microcapsules containing vitamin D2 Preparation of the Composition Vitamin D2 oil (1 MIU / g) was diluted in Softisan® 100 wax to a concentration of 6000 μg / g. This concentration was chosen to produce spray-dried particles containing a vitamin D2 concentration of 1,000 μg / g. The wax was melted above 40°C, the vitamin oil was added, and the mixture was homogenized by manual shaking. The mixture was kept above 40°C until needed.

[0392] The required mass of the wax and vitamin D2 mixture was then added to the protein hydrogel slurry of Example 13 and mixed for 10 minutes at 7000 rpm using a Silverson L5M-A homogenizer to form a composition containing vitamin D2 and wax with 11.9% total solids by weight. The composition was then further diluted with 3.0% aqueous acetic acid to produce two samples: Example 15A containing vitamin D2 and wax with 10.0% total solids (8.9% protein solids by weight) and Example 15B containing vitamin D2 and wax with 5.0% total solids (4.4% protein solids by weight).

[0393] The required mass of wax and vitamin D2 mixture was also added to the protein slurry of Comparative Example 14 and mixed for 10 minutes at 7000 rpm using a Silverson L5M-A homogenizer to form a composition, which was then further diluted with 3.0% aqueous acetic acid to produce Comparative Example 15C, which had 11.7% total solids by weight (10% protein solids by weight) including vitamin D2 and wax.

[0394] Spray drying conditions In Examples 15A and 15B, spray drying was carried out using a ProCept spray dryer in its three column setup, with a column height of 1.8 m and a column diameter of 0.15 m.

[0395] In Example 15A, a two-fluid nozzle with a 1 mm tip size was used. The atomizing air flow rate was 10 L / min. The fluids were pumped into the spray dryer at 6 mL / min using a syringe pump. An additional air flow rate of 150 L / min was set for the cyclone.

[0396] In Example 15B, 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. The cyclone was set with an additional air flow rate of 150 L / min.

[0397] In Comparative Example 15C, spray drying was carried out using a Buchi B290 Spray Dryer equipped with a two-fluid nozzle with a tip size of 1.4 mm. The aspirator flow rate was set at 100% and the Q-flow setting was set at 40. Fluid was pumped into the spray dryer using a peristaltic pump at a speed setting of 35% (approximately 10 mL / min). The formed dry microcapsule powder was collected from a collection pot.

[0398] Other process settings are shown in Table 7 below. [Table 10]

[0399] The spray drying experiments showed that the average particle size d was measured by laser diffraction according to the method therein for each of Examples 15A, 15B, and Comparative Example 15C. 50 Three free-flowing powders were produced, with particle sizes of 28.9 microns, 40.8 microns, and 9.1 microns. Note that in the case of the acid-treated vegetable protein encapsulates, the more dilute compositions dried with an ultrasonic nozzle produced larger microcapsules. For non-acid-treated vegetable proteins with similar solids content, the powders had a much smaller average particle size.

[0400] The vitamin D2 concentrations in the microcapsule powders were measured using Method Version B described herein and were found to be 963 μg / g, 919 μg / g, and 1149 μg / g for Examples 15A, 15B, and Comparative Example 15C, respectively, which is close to the expected microcapsule target of 1000 μg / g and suggests very little vitamin D2 loss during the spray drying process.

[0401] Example 16 - Accelerated Stability Study of Vitamin D2 Microcapsules in a Beverage Formulation Beverage sample preparation The microcapsules of Examples 15A, 15B and Comparative Example 15C were each prepared into a concentrated slurry which could then be added to Miwadi Blackcurrant single concentrate squash.

[0402] 0.2 g of each microcapsule was first suspended in 30 g of boiling water with vigorous shaking, followed by vortexing at 2000 rpm for 1 minute to mimic the temperature profile during flash pasteurization. It was noted that the concentrated slurry prepared in Comparative Example 15C appeared cloudier and finer than those in Examples 15A and 15B. Figures 8a, 8b, and 8c are 10x microscope images of the concentrated slurries of Examples 15A, 15B, and Comparative Example 15C. The microcapsules of Examples 15A and 15B were visible and clearly survived the treatment. It is noteworthy that no intact microcapsules are visible in the image of Comparative Example 15C, demonstrating that the microcapsules rapidly disintegrate when subjected to heat and shear. Instead, smaller aggregates of protein and very small particles of wax and vitamin D2 can be seen.

[0403] Accelerated Stability Testing The calculated mass of each concentrated slurry was added to 2 liters of squash and mixed with an overhead mixer at 800 rpm for 5 minutes to ensure homogeneity before transferring to bottles.

[0404] Each of the three squash samples was filled into a 200 mL clear plastic PET bottle, and the initial vitamin D2 loading was measured using Method A described herein. The initial beverage vitamin D loadings for Examples 15A and 15B are shown in Table 7. However, in Comparative Example 15C, the initial measured beverage loading of vitamin D2 was undetectable. Without being bound by theory, this is believed to be due to the microcapsules being suspended in the concentrated slurry preparation and heated, resulting in the release of vitamin D2 due to rupture of the microcapsules. The vitamin D2 oil floated to the top of the bottle and was not sampled.

[0405] The filled PET bottles of Examples 15A and 15B were then placed in a Binder KBF P 240-230 V chamber set up for accelerated ambient light stability testing in a standard configuration equipped with visible light and UV-A tubes. The nominal UV-A output was 1.1 W / m2 The temperature was set at 20°C and laboratory humidity (typically 50% relative humidity (RH)) was used. Vitamin D loading was measured again after 4 weeks in a Binder chamber using Method A described herein. The % decrease in vitamin D2 content was calculated and recorded in Table 8. [Table 11]

[0406] After 4 weeks in the squash, the vitamin D2 in the encapsulated products of the present invention was protected from degradation, resulting in a loss of only 22.5% of the initial vitamin D2 in Example 15A and only 5.5% of the initial vitamin D2 in Example 15B. While unencapsulated vitamin D2 would be expected to degrade significantly upon exposure to UV light within this time frame, this low level of loss demonstrated the protection provided by the microcapsules of the present invention. It can also be seen that the lowest loss was achieved with larger average encapsulate sizes. Without being bound by theory, it is believed that a larger average particle size of the encapsulate results in less degradation due to a smaller surface area available per mass of vitamin D2 for exposure to UV light.

[0407] Example 17 - Encapsulation of natural pesticide actives Example 17A: Thymol Protein Microcapsule Preparation Preparation of protein hydrogels and application of shear to protein hydrogels Protein hydrogel shells were prepared and sheared as described in Example 13.

[0408] The protein solids were measured to be 9.3 wt%. Dilute acetic acid (3 wt% in DI water) was added to reduce the protein solids to 8.0%.

[0409] Preparation of spray-dried microcapsules The diluted dispersion was homogenized in a Silverson L5M-A high shear mixer (8000 rpm for 2 minutes). 16.81 g of thymol diluted to 40% by weight with Miglyol 812N was added to 103.25 g of the diluted dispersion. The mixture was further homogenized with the Silverson at 8000 RPM for 5 minutes. The droplet size was expected to be approximately 5 microns.

[0410] The samples were then spray dried using a Buchi B290 Spray Dryer. The air inlet temperature was 120 °C with a Q flow rate setting of 40. A two-fluid nozzle with a 1.4 mm tip size was used. The aspirator flow rate was set at 100%. Fluid was pumped into the spray dryer using a peristaltic pump at a rate setting of 13% (4-5 mL / min).

[0411] The formed dry microcapsule powder was collected from the collection pot. The microcapsules were analyzed for their total thymol loading, which was found to be 4.9% by weight. The particle size was measured by laser diffraction and found to have a d50 of 23 microns.

[0412] Example 17B: Comparative Maltodextrin Thymol Microcapsule Preparation 45 g of maltodextrin was dissolved in 105 g of deionized water and mixed with a magnetic stir bar until a homogeneous slurry was formed. 15.23 g of thymol diluted to 40% by weight with Miglyol 812N, along with 0.14 g of polysorbate 80, was added to the slurry. The mixture was homogenized in a Silverson L5M-A high shear mixer at 8000 rpm for 5 minutes.

[0413] The samples were then spray dried using a Buchi B290 Spray Dryer. The air inlet temperature was 130 °C with a Q flow rate setting of 40. A two-fluid nozzle with a 1.4 mm tip size was used. The aspirator flow rate was set at 100%. Fluid was pumped into the spray dryer using a peristaltic pump at a rate setting of 13% (4-5 mL / min).

[0414] The dry microcapsule powder that formed was collected from the collection pot. The microcapsules were analyzed for their total thymol loading, which was found to be 3.4% by weight. These microcapsules were so fragile that it was not possible to measure their particle size. When placed on a microscope slide with a cover slip, it could be seen that the weight of the cover was sufficient to break them.

[0415] Example 17C: Aqueous Release Test The thymol microcapsule powder from Example 12A was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben and a solution of sodium acetate and calcium chloride according to Table 9, so that the samples contained 4 mg of thymol. Duplicate samples were prepared so that samples could be analyzed at t=0 hours, 24 hours, 72 hours, 1 week, and 2 weeks of incubation. [Table 12]

[0416] Samples were mixed on day 0 and placed in a 37°C incubator in the dark, except for the t=0 sample, which was analyzed immediately. At each time point, samples were centrifuged at 4900 RPM for 30 minutes, and the supernatant was transferred to a 50 mL Falcon tube. Ethanol was added to dilute the supernatant sample 10-fold. The thymol concentration in the supernatant was measured by gas chromatography and used to calculate the amount of thymol released from the microcapsules.

[0417] To the pelleted material from each sample after centrifugation, 2.9 mL of DI water and 0.1 mL of 10% KOH solution were added. The mixture was sonicated (Bandelin Sonopuls HD4200 with probe TS104) at 30% amplitude for 2 minutes, vortexed, and centrifuged at 4900 rpm for 5 minutes. 100 μL of the supernatant from each sample was diluted 10-fold with ethanol, and the thymol concentration 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. The amount of thymol remaining was considered the amount of thymol originally unreleased in the initial supernatant. The results are shown in Table 10. [Table 13]

[0418] The release studies demonstrated that the majority of the active ingredient, i.e., more than 90%, remained encapsulated over a two-week period, making such slurries suitable for storage prior to field and crop application.

[0419] Example 17D: Comparative Maltodextrin-Thymol Release Study The thymol microcapsule powder of Comparative Example 17B was suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben according to Table 11 so that the sample contained 4 mg of thymol, the same level as Example 17A. There were only minor differences in the composition of the suspension, which did not contain the insignificant low levels of NaOAc and CaCl in this example. [Table 14]

[0420] Samples were prepared, mixed, and incubated for 1 hour. Samples were centrifuged at 4900 RPM for 10 minutes, and the supernatant was transferred to a 50 mL Falcon tube. Ethanol was added to dilute the supernatant sample 10-fold. The thymol concentration in the supernatant was measured by gas chromatography and used to calculate the amount of thymol released from the capsules in buffered water.

[0421] After centrifugation of the samples, 2.9 mL of deionized water and 0.1 mL of 10% KOH solution were added to the pelleted material. The mixture was sonicated (Bandelin Sonopuls HD4200 with probe TS104) at 30% amplitude for 2 minutes, vortexed, and centrifuged at 4900 rpm for 5 minutes. 100 μL of the supernatant was diluted 10-fold with ethanol, and the thymol concentration 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. The amount of thymol remaining was considered to be the amount of thymol originally unreleased in the initial supernatant. The results are shown in Table 12. [Table 15]

[0422] This release test demonstrates that in maltodextrin microcapsules, more than 40% of the active ingredient is released in aqueous buffer solution after 1 hour in Comparative Example 17B.Such slurry is not suitable for storage before application to fields and crops.When prepared into an aqueous composition or in the presence of water in the environment, such as rain, it will release the active ingredient early, and therefore will not show sustained release properties.

[0423] Example 18 - Preparation of spray-dried microcapsules containing fragrance oil Fragrance / Solvent Mixture Two different fragrances (18A and 18B) were prepared according to Table 13. Each fragrance was then blended with one of two solvents, either Miglyol® 812N or isopropyl myristate (IPM), in an 80:20 weight ratio of fragrance to solvent.

[0424] Fragrance 18A was composed exclusively of moderately volatile fragrance materials with similar volatility ranging from very low to very high hydrophobicity. Fragrance 18B was composed of similarly hydrophobic materials with a full range of volatility, including low, medium, and high volatility materials. Both fragrances had fragrance materials with HSP parameters ranging from δD of 15-20, δP of 1-7, and δH of 2.5-8. [Table 16]

[0425] Preparation of spray-dried microcapsules A protein hydrogel slurry was made according to Example 13.

[0426] The first batch of fragrance composition was prepared as follows: 100-120 g of the protein hydrogel slurry batch from Example 13 was diluted to 20% with a 3% acetic acid solution to obtain a slurry with a protein solids content of 8.0% by weight. The diluted slurry was then stirred in a 400 mL Nalgene beaker at 8000 rpm for 2 minutes using a Silverson lab mixer. 31.5% by weight (based on the weight of the slurry) of Fragrance 18B / Miglyol® 812N blend was then added to the slurry and emulsified at 8000 rpm for an additional 5 minutes using a Silverson mixer to form a fine emulsion. This fine emulsion was aged at room temperature for approximately 2 days before being spray dried. This mixture was used in Experiment 2 TTB.

[0427] A second batch of fragrance composition was prepared as above, except that the fragrance blend was emulsified by gentle hand shaking immediately prior to spray drying rather than using a Silverson mixer. This mixture was used in Experiment 3 TTB.

[0428] A third batch of fragrance composition was prepared similarly to the first batch, except that Miglyol® 812N was replaced with isopropyl myristate (IPM) to form the fragrance:solvent blend. The fragrance and slurry mixture was emulsified using a Silverson mixer at 8000 rpm for an additional 5 minutes to form a fine emulsion. This fine emulsion was aged at room temperature for approximately 2 days before being spray dried. This mixture was used in Experiment 6 TTB.

[0429] A fourth batch of fragrance composition was prepared with the same composition and in the same manner as the materials used in Experiment 6 TTB and the materials used in Experiment 7 CPB and Experiment 8 TTB.

[0430] Samples were spray dried using a ProCepT R&D Spray Dryer with a three column setup: column height 1.8 m, column diameter 0.15 m.

[0431] The air inlet temperature was varied to obtain a dry powder. The air inlet temperature was kept as low as possible to reduce the loss of volatile fragrance materials. The dry inlet air flow rate was 0.4 m 3 The spray dryer was maintained at a constant 4 mL / min. A two-fluid nozzle from Spray Systems with a 1.0 mm tip size was used. The atomization air flow rate was varied between experiments as shown in Table 14 below. Fluid was pumped into the spray dryer at 4 mL / min using a syringe pump in all experiments. An additional air flow of 150 L / min was introduced below the drying chamber to carry the air into the cyclone.

[0432] The dry microcapsule powder formed was collected in a collection pot and also from the interior surfaces of the equipment to which it adhered, for example, in the transfer pipe to the cyclone. The particle size of the microcapsules was measured by laser diffraction.

[0433] Fragrance 18B was successfully encapsulated as shown in Table 14. [Table 17]

[0434] When dried at a lower temperature (inlet approximately 69°C), a maximum particle size of 30.1 microns was produced by a coarse fresh emulsion prepared immediately prior to spray drying with Miglyol® 812N as the solvent. Changing the fragrance solvent from Miglyol® 812N to isopropyl myristate in the fine aged emulsion had little effect on particle size.

[0435] Decreasing the atomizing air flow rate resulted in larger particles (compare Experiment 8TT with Experiment 7CP).

[0436] The fragrance material composition of these microcapsules was analyzed using the GC fragrance loading quantification method described herein, and the results are listed in Table 15. [Table 18]

[0437] Samples dried at lower air inlet temperatures typically had higher total fragrance loadings than those dried at higher air inlet temperatures.

[0438] When dried at a lower temperature (inlet approximately 69°C), isopropyl myristate provided higher total loadings for the fine emulsions than Miglyol® 812N. In addition, at these drying conditions, the coarse emulsions provided higher loadings.

[0439] When the atomizing air flow rate was reduced and drying was performed at higher temperatures (at least 80°C at the inlet), higher total loadings were achieved than with higher air atomization.

[0440] Fragrance 18B demonstrated that, for similarly hydrophobic fragrance materials, materials with a range of volatility can be encapsulated. However, as expected, the least volatile fragrance materials were concentrated in the encapsulates, with benzyl salicylate accounting for 48.4-56.3% of the fragrance, compared to 20% of the fragrance before encapsulation. Drying the crude emulsion at lower temperatures improved the encapsulation of more volatile fragrance materials, such as limonene, from 0.6% to 1.5% of the fragrance.

[0441] Fragrance composition 18A was also successfully encapsulated as listed in Table 16.

[0442] The first batch of fragrance composition was prepared as follows: 100g-120g of the protein hydrogel slurry batch of Example 13 was diluted to 20% by weight with a 3% acetic acid solution to obtain a slurry with 8.0% by weight protein solids. The diluted slurry was then stirred in a 400mL Nalgene beaker using a Silverson lab mixer at 8000 rpm for 2 minutes. 28.3% by weight (based on the weight of the slurry) of Composition 18A / Miglyol® 812N blend was then added to the slurry and emulsified using a Silverson mixer at 8000 rpm for an additional 5 minutes to form a fine emulsion. This fine emulsion was aged at room temperature for approximately 2 days before being spray dried. This mixture was used in Experiment 1 TTA.

[0443] 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 under gentle stirring. This mixture was used in Experiment 9 TTA.

[0444] A third batch of fragrance composition was prepared identically to the first batch above, except that the fragrance blend was sheared and emulsified immediately prior to spray drying rather than aging. This mixture was used in Experiment 3 TTA.

[0445] A fourth batch of fragrance composition was prepared as above, except that the fragrance blend was emulsified by gentle hand shaking immediately prior to spray drying rather than using a Silverson mixer. This mixture was used in Experiment 2 TTA.

[0446] A fifth batch of fragrance composition was prepared similar to the first batch, except that Miglyol® 812N was replaced with isopropyl myristate (IPM). This mixture was used in Experiment 4 TTA.

[0447] A sixth batch of fragrance composition was prepared identically to the fifth batch, and this mixture was used in Test 5CPA.

[0448] The same equipment as in the previous experiment was used, with the same dry air flow rate, emulsion flow rate, and transport air flow rate as in Fragrance Composition 18B. The dry microcapsule powder formed was again collected in a collection pot and also collected from the interior surfaces of the equipment where the dry microcapsule powder adhered, for example, in the transport pipe to the cyclone. The particle size of the microcapsules was measured by laser diffraction. [Table 19]

[0449] The largest particle size was produced in the coarse fresh emulsion with Miglyol® 812N as the fragrance solvent, prepared immediately before spray drying.

[0450] The fine aged emulsion with Miglyol® 812N as the fragrance solvent resulted in the smallest microencapsulate particle size. Diluting the fine aged emulsion and operating at a lower drying temperature did not affect particle size. Changing the fragrance solvent from Miglyol® 812N to isopropyl myristate in the fine aged emulsion had little effect on particle size.

[0451] The aged and spray dried fine emulsions had smaller particle size than the same freshly made emulsions.

[0452] The fragrance material composition of these microcapsules was analyzed using the GC fragrance loading quantification method described herein, and the results are listed in Table 17. [Table 20]

[0453] All samples had similar total fragrance loadings, but the fine aged sample with IPM sprayed at the lower air atomization rate had the highest loading of 30%. This is significantly less than Fragrance Composition 18B, which had the highest total fragrance loading of 51%. Without being bound by theory, this is likely due to the presence of very low log P fragrance materials in Fragrance 18A.

[0454] Fragrance Composition 18A contained four fragrance materials of similar medium volatility and varying hydrophobicity, demonstrating the ability to encapsulate materials with a range of hydrophobicity. The encapsulated fragrance compositions contained higher amounts of dodecanenitrile, the most hydrophobic fragrance material, for all examples. This was least noticeable in the coarse emulsion samples.

[0455] δ-damascone was the least concentrated fragrance material, despite having a higher log P than geraniol and undecavertol. These two alcohols were well encapsulated (especially geraniol), demonstrating that it is still possible to encapsulate these relatively hydrophilic fragrance materials within fragrances.

[0456] Olfactory evaluation of spray-dried microcapsules A concentrated slurry of Test 3TTA microcapsules was prepared prior to making diluted formulations for olfactory evaluation. To obtain a slurry paste, some 1x buffer solution containing a preservative was added dropwise to the dried microcapsules in a 50 mL Falcon tube, vortexing at 2000 rpm between additions. Once the paste appeared homogeneous, more buffer solution was added for further dilution, still using the vortex to ensure uniform dispersion of the microcapsules. The suspension was allowed to stand for 1 hour to allow for possible swelling of the microcapsules, and then strained through a 40 μm cell strainer for 5 minutes. Any excess water was absorbed with tissue paper.

[0457] The total fragrance level was analyzed by GC according to the method described herein and was found to be 12.6% by weight. This is lower than the total fragrance level of spray-dried microcapsules. Without being bound by theory, it is believed that the hydrogel shell hydrates and swells, increasing the shell mass. Furthermore, a small amount of surface fragrance may have been lost in the aqueous phase.

[0458] A dilute slurry of gellan gum-containing microcapsules was prepared containing 0.1% total fragrance by weight for olfactory evaluation. The gellan gum helps to keep the microcapsules well suspended. A gellan gum solution was prepared by mixing 9.5 mg of gellan gum with 15 g of deionized water in a 50 mL Falcon tube. The mixture was heated to above 90°C for 20 minutes and stirred at 500 rpm using an Eppendorf Thermomixer C set at 100°C. After cooling to room temperature, 15 g of double-strength buffer solution was added. Using a micropipette equipped with a low-shear tip, the concentrated encapsulate slurry was added to the gellan gum solution, the sample tube was closed, and the mixture was mixed by repeated inversion.

[0459] The pH of the diluted slurry was 3.05. The odor of the diluted slurry was evaluated according to the methods described herein. The results are shown in Table 18. [Table 21]

[0460] Upon initial application, there was a strong, characteristic fragrance odor, indicating that some of the fragrance oil was bound to the surface of the hydrogel shell. After 24 hours of drying, the odor was very weak, indicating that most of the surface fragrance had evaporated. When rubbed or pressed against glass, a strong, characteristic odor was noted as the force ruptured the microcapsules, releasing the fragrance. When rubbed against card, the odor was of medium intensity, and the fragrance profile was somewhat different from that on glass, indicating that only a portion of the encapsulated fragrance had been released.

[0461] Example 19 - Crosslinking of spray-dried fragrance microcapsules Spray-dried microcapsules were prepared as in Example 18, Experiment 3TTA.

[0462] 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. Once the paste appeared homogeneous, more pH 3 hard water was added for further dilution, still using the vortex 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.

[0463] A 10 wt% tannic acid solution was prepared by manually shaking and vortexing for a few seconds using a Fisherbrand™ ZX4 IR Vortex Mixer, followed by immersion in an ultrasonic bath set at 50°C for 5-10 minutes until completely dissolved.

[0464] 10.6 g of tannic acid solution was added to the microcapsule suspension under mechanical stirring at 170 rpm at 50°C and stirred for 5.5 hours. The suspension was then stored in a refrigerator for approximately 36 hours. The cross-linked microcapsule suspension was sieved through a 38 μm sieve, washed with pH 3 hard water, and resuspended in pH 3 hard water in a clean separatory funnel. The decanted capsules were again collected through a 38 μm sieve, washed with a 1x strength buffer solution containing a preservative, and resuspended in this buffer solution in a separatory funnel. The capsules were collected as a concentrated slurry after decantation and appeared as brown microencapsulated material, indicating that cross-linking had occurred.

[0465] The cross-linked capsule slurry was directly strained through a 40 μm cell strainer for 5 minutes, and excess water was absorbed with tissue paper. The total fragrance level was analyzed by GC according to the method described herein and found to be 4.1 wt %. This is lower than the total fragrance level of the microcapsules before cross-linking. Without being bound by theory, this is thought to be due to loosely bound fragrance on the surface that is removed by the washing process during cross-linking. Particle size was measured by laser diffraction and found to be D 50The particle size was 47.9 μm, which was larger than the particle size of the original spray-dried encapsulates, indicating that the hydrogel shell had hydrated and the encapsulates had swelled.

[0466] Olfactory evaluation of spray-dried and cross-linked microcapsules A diluted slurry of microcapsules containing gellan gum was prepared to contain 0.1% by weight of total fragrance. The gellan gum helped to keep the microcapsules well suspended. This was done according to the method of Example 18. The pH of the diluted slurry was 3.04. The odor of the diluted slurry was evaluated according to the method described herein. The results are shown in Table 19. [Table 22]

[0467] Upon initial application, a mild, characteristic odor of the fragrance was present, indicating that a small amount of fragrance was bound to the surface of the hydrogel shell. After 24 hours of drying, the odor was very weak, indicating that most of the surface fragrance had evaporated. When rubbed or pressed on glass, a strong, characteristic odor was noted as the force ruptured the microcapsules, releasing the fragrance. When rubbed on card, a mild odor was noted, and the fragrance profile was similar to that on glass.

[0468] Example 20 - Preparation of spray-dried microcapsules containing linseed oil Preparation of protein hydrogels and application of shear to protein hydrogels A protein hydrogel slurry was prepared as described in Example 13. The protein solids content was measured to be 9.5% by weight.

[0469] Preparation of spray-dried microcapsules Flaxseed oil is a dietary supplement used for its high level of omega-3 fatty acids, which is also a vegan alternative to fish oil.The slurry was homogenized using a Silverson L5M-A high shear mixer at 8000 rpm for 2 minutes.Flaskseed oil was added to the slurry at a 1:1 oil to protein solids weight ratio, and the mixture was further homogenized using a Silverson at 8000 rpm for 5 minutes to produce a fine emulsion.

[0470] The slurries were spray dried within one day of preparation using a Buchi B290 Spray Dryer using a two-fluid nozzle with a 1.4 mm tip size. The aspirator flow rate was set at 100% and the Q-flow setting was 40. The fluids were pumped into the spray dryer using a peristaltic pump at a rate setting of 13% (4-5 mL / min). The air inlet temperature was varied from 183 to 180 °C, and the air outlet temperature was varied from 126 to 122 °C.

[0471] 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 described herein and found to have a d50 of 45.3 microns. The powder had no rancid off-flavor, indicating that the temperature in the dryer did not decompose the linseed oil. It had a weak nutty odor and a very slight vinegar odor, indicating that only low levels of surface oil were present.

[0472] FIG. 9 shows a 10x optical microscope image of Example 20 suspended in 1x strength citrate buffer, where it can be seen that the encapsulates remain intact.

[0473] The microcapsule powder of Example 20 was also suspended in Miwadi blackcurrant single concentrate at a concentration of 12.5 g / L by vortexing at 2000 rpm for 1 minute. The encapsulated material did not impart any negative odor to the characteristic red fruit note of the original product, demonstrating that the encapsulated material is insoluble and the flaxseed oil remains encapsulated, thereby having little effect on the product aroma. In comparison, the same level of flaxseed oil cannot be incorporated into a beverage without an emulsifier, imparting a very pronounced nutty flavor and a distinctive effect on the beverage's taste.

[0474] Example 21 - Demonstration of protein gel formation The reverse osmosis (RO) water and pea protein isolate were added to a 1 liter stainless steel vessel, placed in a 92°C water bath, and mixed at 1300 rpm using an overhead stirrer. After stirring for 3 minutes, glacial acetic acid was added. The mixture was stirred at 1300 rpm for a total of 45 minutes, ensuring the temperature remained above 80°C. The mixture was then subjected to high shear using a Silverson L5M-A homogenizer at 8000 rpm for 10 minutes.

[0475] 20 mL aliquots of the hot liquid were measured into 50 mL Falcon tubes, sealed, and refrigerated overnight in a normal upright position. High levels of organic acids were tested as listed in Table 20a.

[0476] 5g aliquots of the hot liquid were weighed into 14mL glass vials, sealed and refrigerated overnight in a normal upright position. Lower levels of organic acids were tested as listed in Table 20b. [Table 23] [Table 24]

[0477] After a minimum of 12 hours of refrigeration, gel formation was assessed by inverting the vials.

[0478] As can be seen in Figure 10a, the use of higher levels of acid, i.e., 30% and 10% glacial acetic acid, resulted in the formation of solid gels that did not fall under gravity. When no acid was used, no gel formed and fell into the inverted Falcon tube lid.

[0479] As can be seen in Figure 10b, the use of 5%, 3%, and 2% glacial acetic acid resulted in the formation of solid gels that did not fall under gravity (within a 20-minute observation period). The use of 1% glacial acetic acid and no acid resulted in no gels forming and falling into the inverted vial lid.

[0480] Correlating the gel test results with pH measurements of the hot slurries after homogenization, the mixtures that formed gels were more than 0.5 units lower than the isoelectric point of pea protein isolate, i.e., 4.5 according to Guldekin et al., Food Hydrocolloids (2023), 145:109029.

[0481] Example 22 - Protein Slurry Preparation A: Preparation of protein slurry with acetic acid and sonication 336 g of pea protein isolate (PPI) was added to 2731 g of room temperature reverse osmosis water and placed in a 90°C water bath. The mixture was stirred with an overhead stirrer equipped with a propeller impeller at 1700 rpm for approximately 1-2 minutes to ensure uniform wetting of the PPI powder. 269 g of 80% (v / v) acetic acid solution was added to the stirred mixture, a lid was placed on the vessel, and stirring was continued for an additional 45 minutes. The temperature of the mixture was checked. If it was below 85°C, stirring was continued until this temperature was reached.

[0482] The hot mixture was then transferred to a Silverson mixer and homogenized at 8000 rpm for 15 minutes. The vessel was then covered and allowed to cool until the mixture's temperature was below 40° C., which may take several hours, typically overnight.

[0483] Once this temperature was reached, the mixture was gently stirred at 200 rpm for 15 minutes in an overhead mixer fitted with an anchor impeller to make the mixture pourable, which was then divided into smaller 1 litre batches in bottles.

[0484] Each bottle, containing approximately 860-890 g of the mixture, was sonicated in an ice bath using a Hielscher UIP500hdT to ensure the mixture temperature remained below 40°C. This was done at 100% amplitude for 22-25 minutes until the total energy added was 175 kJ. The sonication was stopped at the quarter and midpoint of the process, and the bottle was manually shaken for 1 minute to ensure homogeneous mixing. The mixture was then passed through a 200-micron sieve to produce a homogeneous, low-viscosity liquid. The viscosity was measured to be 81.8 mPas at 50 s-1, and the pH was 3.5.

[0485] B: Preparation of protein slurry with acetic acid and high pressure homogenization The method of Example 22A was followed until the mixture cooled to below 40°C. The sample was processed using a Pressure Cell Homogeniser SPCH-EP Model FPG12805 (Homogenising System Ltd) equipped with a piston gap valve (HPVS-1) with a contact diameter of 1 mm. The slurry was passed twice at 100 MPa. 0.1 wt% methylparaben preservative was added. The viscosity was 50 s. -1 The pressure was measured at 390.8 mPas and the pH was 3.6.

[0486] C: Comparative Example: Preparation of Protein Slurry Without Acid The method of Example 22B was followed with the following modifications: 2744.0 g of reverse osmosis water was used in the formulation, and 256 g of reverse osmosis water was added in place of the acetic acid. The viscosity was 50 s -1 The pressure was measured at 188.3 mPas and the pH was 6.5.

[0487] D: Comparative Example: Preparation of Protein Slurry with HCl The method of Example 22B was followed with the following modifications: 2658.7 g of reverse osmosis water was used in the formulation, and 341.3 g of 1 M HCl was added instead of acetic acid. The viscosity was 50 s -1 The pressure was measured at 204.8 mPas and the pH was 3.1.

[0488] Example 23 - Preparation of spray-dried microcapsules containing vitamin D2 with modified slurry Preparation of protein slurry and application of shear to the protein slurry Protein slurries were prepared as described in Example 22B and Comparative Examples 22C and 22D, and the protein solids were all calculated to be 10.07% by weight.

[0489] Preparation of spray-dried microcapsules Vitamin D2 oil (1 MIU / g) was diluted to a concentration of 6000 ug / g in the carrier Softisan® 100 wax. The wax was melted above 40°C, Vitamin D2 oil was added, and the mixture was homogenized by manually shaking. The mixture was kept above 40°C until needed.

[0490] The required masses of vitamin D2 and wax were then added to each protein slurry 22B, C, and D at a protein:carrier solids weight ratio of 5:1, which was then mixed for 10 minutes at 8000 rpm using a Silverson L5M-A homogenizer to form emulsions of mixtures 23B, C, and D containing vitamin D2 and wax with a total solids content of 11.5% by weight.

[0491] Each emulsion was spray dried within one day of preparation using a Buchi B290 Spray Dryer using a two-fluid nozzle with a 1.4 mm tip size. The aspirator flow rate was set at 100% and the Q-flow setting was 40. The air inlet temperature was maintained at 180°C. Fluids were pumped into the spray dryer using a peristaltic pump at rate settings of 6.7 to 23 g / min to achieve outlet air outlet temperatures of 85 to 120°C. Process conditions and results are shown in Table 21. [Table 25]

[0492] A dry microcapsule powder was formed, indicating encapsulation of the wax and vitamin D. Moisture content, particle size by laser diffraction, and vitamin D2 content (method version B) were all measured using the methods described herein.

[0493] All three examples had very similar vitamin D2 contents. The large differences in run speed and outlet temperature between Examples 23B, 23C, and 23D were due to differences in viscosity, as the emulsion samples without added organic acids had lower viscosities than the emulsion samples made with organic acids. This was caused by the fact that they were prepared using protein slurries with very different viscosities.

[0494] Example 24 - Preparation of spray-dried microcapsules containing vitamin D2 with different carriers Preparation of protein slurry and application of shear to the protein slurry A protein hydrogel slurry was prepared as described in Example 22 A. The protein solids content was measured to be 11.6 wt %.

[0495] Preparation of spray-dried microcapsules Vitamin D2 oil (1 MIU / g) was diluted to a concentration of 6000 ug / g in the carrier Softisan® 100 wax. The wax was melted above 40°C, Vitamin D2 oil was added, and the mixture was homogenized by manually shaking. The mixture was kept above 40°C until needed.

[0496] Vitamin D2 oil (1 MIU / g) was also separately diluted in the carrier olive oil to a concentration of 6000 μg / g, and the mixture was homogenized by manual shaking. The mixture was kept at room temperature until needed.

[0497] The required masses of vitamin D2 and carrier were then added to the protein hydrogel slurry of Example 22A at protein:carrier solids weight ratios of 5:1 and 2:1, which were then mixed at 7000 rpm for 10 minutes using a Silverson L5M-A homogenizer to form an emulsion.

[0498] The slurry was spray dried using a pilot-scale co-current spray dryer using a rotary disk atomizer. The drying chamber was approximately 1 m in diameter and 2.1 m high. The nominal evaporation capacity was 3 kg HO / h at 150 °C and the nominal air flow was 125 m. 3 / hr. The fluid was pumped to the spray dryer using a peristaltic pump with the flow rate adjusted to achieve the target outlet temperature. The air inlet temperature was varied from 179 to 181°C, and the air outlet temperature was varied from 92 to 95°C. The processing conditions and results are shown in Table 22. [Table 26]

[0499] A dry microcapsule powder was formed, indicating encapsulation of vitamin D2 and the carrier. Moisture content, particle size by laser diffraction, and vitamin D2 content (Method Version B) were all measured using the methods described herein.

[0500] The vitamin D2 content of the microcapsule powders was higher than the 1000 μg / g target in all cases. In Examples 24B and C, the increased levels may be due to uneven distribution of oil and protein between the sample obtained from the collection vessel and smaller particles not recovered by the cyclone.

[0501] Example 25 - Preparation of spray-dried microcapsules containing vitamin D2 and a core additive Preparation of protein hydrogels and application of shear to protein hydrogels A protein hydrogel slurry was prepared as described in Example 22 A. The protein solids content was measured to be 11.8 wt %.

[0502] A protein hydrogel slurry was also prepared as described in Example 13. The protein solids content was measured to be 10.2 wt %.

[0503] Preparation of spray-dried microcapsules Vitamin D2 oil (1 MIU / g) was diluted in Softisan® 100 wax to a concentration of 6000 μg / g. The wax was melted above 40°C, vitamin D2 oil was added, and the mixture was homogenized by manual shaking. A 10 mg / g solution of curcumin in absolute ethanol was added to the mixture, with curcumin being 0.46% by weight of the mass of vitamin D2 in the emulsion. This mixture 25A was homogenized by manual shaking and held above 40°C until needed.

[0504] Vitamin D2 oil (1 MIU / g) was diluted in Softisan® 100 wax to a concentration of 6000 μg / g. The wax was melted above 40°C, vitamin D2 oil was added, and the mixture was homogenized by manual shaking. A 10 mg / g solution of curcumin in absolute ethanol was added to the mixture, with curcumin being 0.62% by weight of the mass of vitamin D2 in the emulsion. This mixture 25B was homogenized by manual shaking and held above 40°C until needed.

[0505] Vitamin D2 oil (1 MIU / g) was diluted in Softisan® 100 wax to a concentration of 6000 ug / g. The wax was melted above 40°C and vitamin D2 oil was added, followed by vitamin E at 10% by weight of the mass of vitamin D2 in the emulsion. The mixture was homogenized by manual shaking at 25°C and held above 40°C until needed.

[0506] Vitamin D2 oil (1 MIU / g) was diluted in Softisan® 100 wax to a concentration of 6000 μg / g. The wax was melted above 40°C, vitamin D2 oil was added, and the mixture was homogenized by manual shaking. A 100 mg / g solution of β-carotene in absolute ethanol was added to the mixture, with β-carotene being 6.2% by weight of the mass of vitamin D2 in the emulsion. This mixture 25D was homogenized by manual shaking and held above 40°C until needed.

[0507] The required mass of each of Mixes 25A, C, and D was then added to the protein hydrogel slurry of Example 22A, and Mix 25B was added to the protein hydrogel slurry of Example 13. Wax was added to the slurry at a protein:wax solids weight ratio of 5:1, which was then mixed for 10 minutes at 7000 rpm using a Silverson L5M-A homogenizer to form emulsions with a total solids content of 13.8% by weight for Mixes 25A, C, and D, including vitamin D2, wax, and additives, and a total solids content of 11.9% by weight for Mix 25B, including vitamin D2, wax, and additives.

[0508] The slurry was spray dried within one day of preparation using a Buchi B290 Spray Dryer using a two-fluid nozzle with a 1.4 mm tip size. The slurry was initially heated to 40-45°C. The aspirator flow rate was set at 100%, with a Q-flow setting of 40. The fluid was pumped into the spray dryer using a peristaltic pump at a speed setting of 35% (13-14 mL / min). The air inlet temperature was varied from 181-179°C, and the air outlet temperature was varied from 127-111°C. The processing conditions and results are shown in Table 23. [Table 27]

[0509] A dry microcapsule powder was formed, indicating that the vitamin D and carrier were encapsulated. It was noted that the additives slightly altered the overall cream color of the powder. Examples 25A and B were pale yellow, Example 25C was whiter, and Example 25D was pale orange. Moisture content, particle size by laser diffraction, and vitamin D2 content (Method Version B) were all measured using the methods described herein.

[0510] Example 26 - Integrity of spray-dried microcapsules containing vitamin D2 Microcapsule integrity was measured according to the method described herein, and the results are shown in Table 24. For each encapsulation, 1% of the dry powder was added to water to ensure vitamin D levels were within the method detection limits.

[0511] Comparative testing was performed with a commercially available polysaccharide vitamin D microcapsule, Prinova GA. The vitamin D content in the powder was determined to be 2727.4 μg / g using Method B described herein, and the moisture content was determined to be 7.4 wt % using the method described herein. [Table 28]

[0512] The comparative Prinova GA microcapsules are so soluble in water that they do not form a visible sediment. Because they are highly soluble, they release most of the encapsulated vitamin D2 into the water. Leakage was measured at 75%.

[0513] In contrast, the compositions of the present invention of Examples 23B and 24C were highly insoluble in water and maintained their integrity, with 0% leakage of encapsulated vitamin D2.

[0514] Comparative Examples 23C and 23D had leakage of 15% and 8%, demonstrating the importance of treating the vegetable protein with organic acids. [Table 29]

[0515] The compositions of the present invention of Examples 25A, B, C, and D maintained their integrity and had 0% leakage, as shown in Table 25, demonstrating that the use of active protectants does not adversely affect the integrity of the microcapsules.

[0516] Example 27 - Accelerated Stability Study of Vitamin D2 Microcapsules in a Beverage Formulation Beverage sample preparation The microcapsules of Examples 24A and B and Examples 25A, B, C, and D were each prepared into concentrated slurries that could then be added to MiWadi Blackcurrant single concentrate squash.

[0517] 0.2 g of each microcapsule was first suspended in 30 g of boiling water with vigorous shaking, followed by vortexing at 2000 rpm for 1 min to mimic the temperature profile during flash pasteurization.

[0518] Accelerated Stability Testing The calculated mass of each concentrated slurry was added to 2 liters of squash and mixed with an overhead mixer at 500 rpm for 5 minutes to ensure homogeneity before transferring to bottles.

[0519] Three squash samples were filled into 200 mL clear plastic PET bottles, and the initial vitamin D2 loading was measured using Method B described herein. The filled bottles were then placed in a Binder KBF LQC 720 chamber configured to operate with only a visible light bulb. The total light dose for the test was determined by the expected visible light intensity in a typical supermarket (generally 600 lux) and operated 24 hours a day for 6 or 9 months. This corresponded to 2.6 MLUXh (representing a 6-month shelf life) or 3.9 MLUXh (representing a 9-month shelf life) at 35°C and laboratory humidity (typically 50% relative humidity (RH)). The photometry function on the Binder KBF LQC 720 was used to achieve the required dose. After the equivalent of 6 and 9 months on the shelf, the vitamin D loading was again measured using Method B described herein. The % reduction in vitamin D2 content was calculated and recorded in Table 26 for Examples 24A and B and in Table 27 for Examples 25A, B, C, and D. [Table 30]

[0520] The results in Table 26 show that after a comparable shelf life of 9 months in a beverage, vitamin D2 in the encapsulated products of the present invention was protected from degradation. Unencapsulated vitamin D2 would be expected to degrade below the method detection limit upon exposure to light over this time frame. Examples 24A and 24B, which had a protein to wax ratio greater than 1:1, lost only 48% and 46%, respectively, of the initial vitamin D2. This low level of loss demonstrates the protection afforded by the microcapsules of the present invention during long-term storage. [Table 31]

[0521] The results in Table 27 show that after a comparable shelf life of 9 months in a beverage, vitamin D2 in the encapsulates of the present invention was protected from degradation despite the very small average particle size provided by the spray dryer used. Unencapsulated vitamin D2 is expected to degrade below the method detection limit upon exposure to light over this time frame. Example 25B had the lowest vitamin D2 loss at 41%, which, without being bound by theory, is believed to be due to the combination of the protective curcumin core additive and the larger average particle size. Examples 25A, C, and D all had slightly higher losses than Example 25B, which is believed to be due to the very small average particle size. Nevertheless, because the core additive provided additional protection, their stability performance was consistent with Example 24A, which has a much larger average particle size. This low level of loss demonstrates the protection provided by the microcapsules of the present invention during long-term storage. Without being bound by theory, it is believed that the larger the average particle size of the encapsulates, the less available surface area per mass of vitamin D2 for exposure to light, resulting in less degradation.

[0522] Example 28 - Preparation of spray-dried microcapsules containing fragrance with shell additives Fragrance / Solvent Mixture Fragrance 28A was prepared according to Table 28 and blended with Miglyol® 812N at an 80:20 fragrance:solvent weight ratio. Fragrance 28A was composed of similar hydrophobic materials having a full range of volatility, including low-, medium-, and high-volatility materials. The fragrance had fragrance materials with HSP parameters ranging from δD of 15-20, δP of 1-7, and δH of 2.5-8. [Table 32] Preparation of spray-dried microcapsules

[0523] A first batch of fragrance composition was prepared as follows: The batch of protein hydrogel slurry from Example 13 was homogenized using a Silverson lab mixer at 8000 rpm for 2 minutes. 5.1 wt. % (based on the weight of the slurry) of fragrance 28A / Miglyol® 812N blend was then added to the slurry and emulsified using an overhead stirrer equipped with a 4-blade impeller at 800 rpm for 2 minutes to form a coarse emulsion. This emulsion was used in Example 28B.

[0524] A second batch of fragrance composition was prepared as follows: The batch of protein hydrogel slurry from Example 13 was combined with 2.0 wt.% (based on the weight of the slurry) of glycerol. The mixture was homogenized using a Silverson lab mixer at 8000 rpm for 2 minutes. 6.1 wt.% (based on the weight of the slurry) of fragrance 28A / Miglyol® 812N blend was then added to the slurry, and emulsified using an overhead stirrer equipped with a 4-blade impeller at 800 rpm for 2 minutes to form a coarse emulsion. This emulsion was used in Example 28C.

[0525] A third batch of fragrance composition was prepared as follows: The batch of protein hydrogel slurry from Example 13 was homogenized using a Silverson lab mixer at 8000 rpm for 2 minutes. 4.9 wt. % (based on the weight of the slurry) of fragrance 28A / Miglyol® 812N blend was then added to the slurry and emulsified using a Silverson mixer at 8000 rpm for an additional 5 minutes to form a fine emulsion. This mixture was used in Example 28D.

[0526] A fourth batch of fragrance composition was prepared as follows: The batch of protein hydrogel slurry from Example 13 was heated to 70°C in a water bath and combined with 3.6 wt% (based on the weight of the slurry) Capsul® starch. The mixture was homogenized using a Silverson lab mixer at 8000 rpm for 2 minutes. The mixture was allowed to cool to ambient temperature overnight. 6.4 wt% (based on the weight of the slurry) of fragrance 28A / Miglyol® 812N blend was then added to the slurry and emulsified using a Silverson mixer at 8000 rpm for an additional 5 minutes to form a fine emulsion. This emulsion was used in Example 28E.

[0527] Each emulsion was spray dried using a pilot-scale co-current spray dryer using a rotary disk atomizer. The drying chamber was approximately 1.2 m in diameter and 2.1 m in height. The nominal evaporation capacity was 3 kg HO / hr at 150 °C, and the nominal air flow was 125 m. 3 The flow rate was 1 / hr. The fluid was pumped into the spray dryer using a peristaltic pump with the flow rate adjusted to achieve the target outlet temperature. The air inlet temperature was varied from 129 to 135°C, and the air outlet temperature was varied from 69 to 70°C.

[0528] Fragrance 28A was successfully encapsulated in Examples 28B, C, D, and E, as shown in Table 29. [Table 33]

[0529] In all four examples, dry microcapsule powders were formed, indicating that the fragrance was encapsulated. Particle size and fragrance content by laser diffraction were measured using the methods described herein. The addition of glycerol or Capsul® starch to the encapsulate shell did not significantly affect the fragrance loading or particle size. The use of fine emulsions resulted in larger average encapsulate particle sizes.

[0530] The individual fragrance materials in Oil Example 28A were measured using the methods herein as shown in Table 30. [Table 34]

[0531] All fragrance ingredients were encapsulated, but not equally at 20 wt% each, indicating an acceptable level of selectivity. There were very few differences between the four encapsulations, indicating that the shell modifications did not affect fragrance selectivity during encapsulation.

[0532] δ-damascone and dipentene had the lowest encapsulation levels. δ-damascone is the fragrance material with the lowest log P. Without being bound by theory, it is expected that materials with higher log P are more easily emulsified and therefore preferentially encapsulated. Dipentene is the most volatile material and therefore most likely to be lost by evaporation during the drying process. However, it was still present at about three-quarters of the expected level.

[0533] The encapsulates were also evaluated for their olfactory performance according to the olfactory evaluation method described herein. Example 28C performed similarly to Example 28B, with a strength rating of 3.5 when rubbed and pressed on glass, and 4.5 when rubbed on absorbent paper. Example 28E performed similarly to Example 28D, with a strength rating of 3.5 when rubbed on glass, and 4.0 when pressed on glass and rubbed on absorbent paper. This confirms that the addition of glycerol or Capsul® starch does not prevent the dried encapsulates from breaking when rubbed or pressed, thereby releasing the fragrance so that the scent can be released.

Claims

1. 1. A method for the preparation of biodegradable microcapsules, comprising: (a) forming a mixture comprising one or more plant-derived proteins in a solvent system, the solvent system comprising miscible co-solvents, a first co-solvent increasing the solubility of the plant-derived protein and a second co-solvent decreasing the solubility of the plant-derived protein, the co-solvents being added to the mixture in either concentrated or diluted form, and the pH of the plant-derived protein mixture being at least 0.5 pH units lower than the isoelectric point of the plant-derived protein; (b) subjecting the plant-derived protein mixture to a shear treatment to form a plant-derived protein hydrogel slurry; (c) dispersing an active ingredient in the plant-derived hydrogel slurry to form a composition; (d) drying the composition to form microcapsules.

2. 10. The method of claim 1, further comprising the step of derivatizing at least a portion of the plant-derived protein in the mixture to form a plant-derived protein hydrogel by one or more of cooling, addition of salt, addition of anti-solvent, solvent reduction, addition of a cross-linking agent, electrostatic cross-linking, preferably by cooling and / or solvent reduction, most preferably by cooling.

3. 3. The method of claim 2, wherein the step of inducing at least a portion of the plant-derived protein in the mixture to form a plant-derived protein hydrogel involves a sol-gel transition.

4. The method of any one of claims 1 to 3, wherein the mixture formed in step (a) is a slurry, dispersion, emulsion, or solution.

5. The method according to any one of claims 1 to 4, wherein the drying in step (d) is carried out by spray drying.

6. The method according to any one of claims 1 to 5, wherein the drying in step (d) is carried out by fluidized bed drying.

7. 2. The method of claim 1, wherein the plant-derived protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, faba bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, preferably selected from pea protein and / or potato protein.

8. 2. The method of claim 1, wherein the first co-solvent is an organic acid, preferably the organic acid is acetic acid, lactic acid, formic acid, propionic acid, an α-hydroxy acid, and / or a β-hydroxy acid, more preferably lactic acid or acetic acid.

9. 2. The method of claim 1, wherein the second co-solvent is selected from water, ethanol, and / or ethyl acetate, more preferably selected from water and / or ethanol, even more preferably water.

10. 10. The method of any one of claims 3 to 9, wherein the sol-gel transition is achieved by heating the protein mixture to a first temperature higher than the sol-gel transition temperature of the one or more plant-derived protein mixtures, and then reducing the temperature to a second temperature lower than the sol-gel transition temperature of the one or more plant-derived protein mixtures to form a hydrogel.

11. The shearing treatment comprises a single shearing step which involves fragmenting the plant-derived protein hydrogel into fragments, preferably the fragments having a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. ５０ The method according to any one of claims 1 to 10, comprising:

12. The method according to any one of claims 1 to 10, wherein the shearing treatment comprises a first shearing step followed by a second shearing step.

13. 13. The method of claim 12, wherein the first shearing step involves fragmenting the plant-derived protein hydrogel into fragments, and preferably at least 80% by weight 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, and even more preferably 20 mm to 30 mm, as measured by optical microscopy.

14. The second shearing step involves further fragmenting the plant-derived protein hydrogel, and preferably the fragments produced in the second shearing step have a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. ５０ 14. The method of claim 12 or 13, comprising:

15. 15. The method according to any one of claims 12 to 14, wherein step (b) further comprises subjecting the plant-derived protein hydrogel slurry to a solvent reduction step, preferably a solubilizing solvent reduction step, between the first shearing step and the second shearing step.

16. The solvent reduction step (i) contacting the fragments of the plant-derived hydrogel slurry with a non-solubilizing solvent; (ii) separating the fragments of the plant-derived hydrogel slurry from the non-solubilizing solvent to obtain a washed plant-derived protein hydrogel slurry; (iii) optionally repeating steps (i) and (ii).

17. 17. The method of any one of claims 1 to 16, further comprising altering the pH of the plant-derived protein hydrogel slurry so that it differs by more than 1 pH unit from the isoelectric point of the plant-derived protein.

18. 18. The method of claim 17, wherein the step of altering the pH of the plant-derived protein hydrogel slurry occurs after or consecutively with step (b).

19. 19. The method of any one of claims 1 to 18, wherein the composition formed in step (c) is a shear thinning composition.

20. The composition formed in step (c) is heated at 20° C. and 50 s －１ Viscosity in the range of 1 to 10,000 cP at 20°C and 50 s －１ Viscosity in the range of 10 to 7500 cP at 20°C and 50 s －１ 20. The method of any one of claims 1 to 19, wherein the composition has a viscosity in the range of 15 to 5000 cP at RT.

21. 20. A method according to any one of the preceding claims, wherein the composition formed in step (c) has a protein solids content in the range of from 1% to 25%, preferably from 2% to 20%, more preferably from 4% to 15%, even more preferably from 5% to 12% by weight based on the total weight of the composition.

22. 22. The method of any one of claims 1 to 21, wherein the active ingredient is selected from vitamins, minerals, flavor materials, fragrance materials, pro-flavors, pro-fragrances, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antibacterial agents, antiviral agents, anti-inflammatory agents, insecticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioning agents, hair conditioning agents, dyes, pigments, and adhesives, or combinations thereof.

23. the active ingredient is at least one fragrance or flavor material; the at least one fragrance or flavor material has a vapor pressure of 0.00001 Torr or greater at 25°C; and / or 23. The method of claim 22, wherein said at least one fragrance or flavor material has a log P of 3.0 or greater, preferably 3.5 or greater, more preferably 4.0 or greater.

24. 23. The method of claim 22, wherein the active ingredient is at least one fragrance or flavor material that is part of a fragrance or flavor, preferably the fragrance or flavor containing at least 20% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a log P of 3.0 or greater, more preferably 3.5 or greater, more preferably 4.0 or greater.

25. 23. The method of claim 22, wherein the active ingredient is a vitamin or 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, omega-3, folic acid, thiamine, riboflavin, niacin and phosphorus, or mixtures thereof, more preferably vitamin D.

26. A method according to any preceding claim, wherein the active ingredient is part of a composition comprising the active ingredient and an active carrier phase.

27. 27. The method of claim 26, wherein the active carrier phase is a solvent, fat or wax.

28. The composition formed in step (c) comprises droplets of the active ingredient or the composition comprises droplets of the active ingredient and a diameter of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns, as measured by laser diffraction. ５０ and an active carrier phase having the formula:

29. 29. The method according to any one of the preceding claims, wherein plant-derived protein residues formed during step (d) but not incorporated into the microcapsules are recycled in the method, preferably added back to step (a).

30. The method further comprises subjecting the microcapsules to a post-treatment step, and preferably the post-treatment step comprises a non-covalent cross-linking step, a covalent cross-linking step, or a coating formation step, and the coating formation step comprises: (i) treating the microcapsules with a metal or silicon-containing compound, and / or (ii) subjecting the microcapsules to a complex coacervation process using a polysaccharide, and / or (iii) treating the microcapsules with an aqueous mineral solution; 30. The method according to any one of claims 1 to 29, wherein the post-treatment step preferably comprises a non-covalent cross-linking step using tannic acid followed by (ii) a complex coacervation step using xanthan gum.

31. Biodegradable microcapsules obtained or obtainable by the method according to any one of claims 1 to 30.

32. 1. A method for the preparation of a biodegradable microcapsule composition, comprising: (a) preparing biodegradable microcapsules by the method of any one of claims 1 to 30; (b) suspending the biodegradable microcapsules in an external phase.

33. 33. A biodegradable microcapsule composition obtained or obtainable by the method of claim 32.

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

35. 35. The biodegradable microcapsules of claim 34, wherein at least 25%, more preferably at least 40%, even more preferably at least 50%, and even more preferably at least 60% of the initially encapsulated active ingredient remains present in the microcapsules after incubation in water at 20°C for 10 days, as measured by HPLC.

36. 36. Biodegradable microcapsules according to claim 34 or 35, wherein the plant-derived protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, faba bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, preferably selected from pea protein and / or potato protein.

37. The biodegradable microcapsules according to any one of claims 34 to 36, wherein the plant-derived protein has been pretreated with an organic acid, and preferably the organic acid is acetic acid, lactic acid, formic acid, propionic acid, an α-hydroxy acid and / or a β-hydroxy acid, and preferably lactic acid or acetic acid.

38. 38. The biodegradable microcapsules of any one of claims 34 to 37, wherein the active ingredient is selected from vitamins, minerals, flavor materials, fragrance materials, pro-flavors, pro-fragrances, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antibacterial agents, antiviral agents, anti-inflammatory agents, insecticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioning agents, hair conditioning agents, dyes, pigments, and adhesives, or combinations thereof.

39. the active ingredient is at least one fragrance or flavor material; the at least one fragrance or flavor material has a vapor pressure of 0.00001 Torr or greater at 25°C; and / or 39. Biodegradable microcapsules according to claim 38, wherein the at least one fragrance or flavour material has a log P of 3.0 or greater, preferably 3.5 or greater, more preferably 4.0 or greater.

40. 39. The biodegradable microcapsule of claim 38, wherein the active ingredient is at least one fragrance or flavor material that is part of a fragrance or flavor, and preferably the fragrance or flavor contains at least 20% by weight, based on the total weight of the fragrance or flavor, of fragrance or flavor materials having a log P of 3.0 or greater, more preferably 3.5 or greater, more preferably 4.0 or greater.

41. 39. The biodegradable microcapsules of claim 38, wherein the active ingredient is a vitamin or 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.

42. 42. The biodegradable microcapsules according to any one of claims 34 to 41, wherein the active ingredient is part of a composition comprising the active ingredient and an active carrier phase.

43. 43. The biodegradable microcapsule of claim 42, wherein the active carrier phase is a solvent, a fat or a wax.

44. The biodegradable microcapsules of any one of claims 34 to 43, wherein the microcapsules have a diameter of 250 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 50 μm or less, 30 μm or less, or 10 μm or less, as measured by optical microscopy.

45. 45. The biodegradable microcapsules of any one of claims 34 to 44, wherein the plant-derived protein has a protein secondary structure with at least 40% intermolecular beta-sheet, at least 50% intermolecular beta-sheet, at least 60% intermolecular beta-sheet, at least 70% intermolecular beta-sheet, at least 80% intermolecular beta-sheet, or at least 90% intermolecular beta-sheet, wherein the % content of intermolecular beta-sheet is measured by FTIR.

46. 46. ​​The biodegradable microcapsules of any one of claims 34 to 45, wherein the plant-derived protein carrier is non-covalently modified with a non-covalent crosslinking agent, or the plant-derived protein carrier is covalently modified with a covalent crosslinking agent, or the plant-derived protein carrier has a coating deposited thereon, the coating being a metal coating, a silicon-based coating, a polymer coating, a coacervate coating, or a mineral coating, and the silicon-based coating is formed from a silicon-containing compound, preferably the silicon-containing compound is selected from sodium silicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, dimethyldiethoxysilane, and tetramethyl orthosilicate, or a combination thereof, more preferably the sodium silicate is selected from sodium metasilicate, sodium orthosilicate, and sodium pyrosilicate, with sodium metasilicate being most preferred.

47. The biodegradable microcapsules according to any one of claims 34 to 46, wherein the plant-derived protein is pea protein and the active ingredient is vitamin D.

48. A composition comprising the spray-dried biodegradable microcapsules of any one of claims 34 to 47 and an external phase.

49. A formulated product comprising the spray-dried biodegradable microcapsules of any one of claims 34 to 47.

50. 1. A method of making a formulated product, comprising: (a) preparing biodegradable microcapsules by the method of any one of claims 1 to 30; (b) mixing the biodegradable microcapsules with a product formulation.

51. Use of the biodegradable microcapsules according to any one of claims 34 to 47 in formulated products.

52. 52. The method of claim 50 or the use of claim 51, wherein the formulated product is a food, beverage, cosmetic, home care product, personal care product, pharmaceutical, industrial product (e.g., paint, adhesive, sandpaper, tape, etc.), medical device, biomaterial, or pesticide.

Images