Biodegradable microcapsule and preparation method thereof

Abstract

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

Description

Field of the Invention

[0001] The present invention relates to methods for preparing biodegradable microcapsules and methods for preparing biodegradable microcapsule compositions. The present invention also relates to biodegradable microcapsules and biodegradable microcapsule compositions per se. The present invention also relates to the use of biodegradable microcapsules and methods involving biodegradable microcapsules, including the preparation of formulated products. The present invention also relates to formulated products per se.

[0002] Background

[0003] Encapsulation technology involves embedding active ingredients in an external matrix, which can be used to protect the active ingredients from their external environment (e.g., exposure to chemicals, air, light, etc.), and also to protect the external environment from the active ingredients themselves, e.g., when they are hazardous to handle. For example, when an active ingredient is added to a product formulation (such as a beverage formulation, a fabric conditioner formulation, etc.), this protects the active ingredient, meaning that the active ingredient in the product formulation has an acceptable shelf life and / or does not activate prematurely. This also means that the external phase of the product formulation is not negatively affected by the active ingredient. The active ingredient is then released when needed (e.g., when the encapsulation breaks or when the encapsulation is enzymatically degraded).

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

[0005] Drying techniques allow for the large-scale preparation of microcapsules, and spray drying of plant proteins has been attempted in an attempt to achieve the production of biodegradable microcapsules or microcapsules for food use. However, in order to obtain robust microcapsules of a small enough size through drying techniques, the material needs to have a low viscosity at high shear rates so that the material can be sprayed (e.g., the material needs to shear thin to a low enough viscosity to successfully undergo spray drying), while also having a high enough protein solid content. Conventional methods for preparing plant-based protein materials suitable for spray drying include hydrolyzing the protein to break it down into lower molecular weight fragments (e.g., by treatment with acid or base or enzymes). However, the resulting spray-dried microcapsules are structurally unstable in water or under acidic or alkaline conditions, at elevated temperatures, and / or under high shear forces, meaning that they are not suitable for incorporation into product formulations through conventional manufacturing processes, especially into liquid aqueous product formulations. In addition, since many oils are unstable under alkaline conditions, the processes involved may have a negative impact on the encapsulated material.

[0006] The use of protein-containing hydrogels in microencapsulation processes is known, for example, from WO2022 / 221710, which describes a process for using hydrogels for preparing microcapsules containing an active substance (usually a flavor). As a basic part of this process, the active substance is first emulsified with an emulsifier (such as gum arabic) and a "filler" (such as maltodextrin). Then, the emulsion is mixed with a protein (such as faba bean protein). Then, the mixture is heated to at least 50 °C for an extended period of time (at least 30 minutes) to in-situ form a hydrogel encapsulating the emulsified active substance. Then, the mixture is cooled and dried to form granules.

[0007] However, the process described in WO2022 / 221710 is not always suitable for temperature-sensitive or highly volatile active substances. This is due to the need for a continuously extended heating time of the mixture containing the active substance to form the hydrogel. Examples of active substances that can be particularly temperature-sensitive include volatile flavorings, vitamins, and probiotics, which are prone to thermal degradation. Since the time period involved is very short, the temperature during the spray drying process is usually of less concern. It would be advantageous for the microencapsulation process to avoid the need for the active substance to undergo an extended high-temperature step. A process for preparing microcapsules that can be used in a wide range of products (such as liquid products, especially liquid aqueous products) would also be advantageous.

[0008] The present invention achieves this by forming a protein hydrogel slurry in which the active substance is dispersed rather than in-situ forming a hydrogel around the droplets of the active substance. The protein hydrogel slurry is formed by first treating a plant protein with a solvent and / or adjusting the pH to below the isoelectric point, and subsequently subjecting it to shear. Then, the subsequent incorporation of the active substance into the protein hydrogel slurry can be carried out at room temperature, thus avoiding limitations regarding the thermal sensitivity of the active substance.

[0009] The hydrogels described and claimed in WO2022 / 221710 do contain protein, but in addition to the protein, an emulsifier and a "filler" are also required. The examples describe using gum arabic and glucose at levels almost equal to or greater than that of faba bean protein. The resulting hydrogels and the granules formed therefrom will have very different properties from the hydrogels and granules of the present invention, especially in terms of their solubility. Relevantly, the examples of WO2022 / 221710 are directed to dry products.

[0010] Microcapsules with significant wall solubility are clearly less suitable for liquid products, especially 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.

[0011] It is well known to use more soluble plant protein materials (such as highly hydrolyzed plant proteins) as encapsulating materials for active substances in spray drying. Using a partially or fully water-soluble material as an encapsulating material for immiscible active substances in a drying process (such as spray drying) generally facilitates the preparation of microcapsule walls that can retain a high integrity and low porosity of the active substance. During the drying process, the soluble material is usually dried to prepare a solid with a lower porosity, as the dissolved substances gradually fill and then block the small holes and gaps formed in the microcapsule wall. Using such soluble materials in the encapsulation and spray drying processes can contribute to the formation of microcapsules with high integrity, which can better retain the active substance during processing.

[0012] Such microcapsules are generally suitable for many dried products, such as protein powders and other foods. Examples are described in, for example, WO2021 / 165289 and EP3042571. However, using soluble wall materials usually results in lower stability of the microcapsules in liquid aqueous compositions and a rapid and significant loss of the active substance.

[0013] As described in the present invention, using a pre-formed protein hydrogel slurry as an encapsulating material for dispersing active substances breaks this pattern. The deformable nature of the protein hydrogel fragments allows for the formation of microcapsule walls with high integrity from less soluble materials, resulting in microcapsules suitable for use in liquid products. The present invention also allows for the use of more heat-sensitive materials in these applications.

[0014] Therefore, there is a need for methods for preparing microcapsules containing active ingredients that are small enough to be incorporated into liquid aqueous product formulations, which are both biodegradable and stable in liquid aqueous product formulations (i.e., the microcapsules maintain their structural integrity under acidic or alkaline conditions, such that the active ingredient is protected during manufacture and the product shelf life). Summary of the Invention

[0016] In a first aspect, the present invention provides a method for preparing biodegradable microcapsules, the method comprising:

[0017] (a) forming a mixture comprising one or more plant-based proteins in a solvent system, wherein the solvent system comprises miscible co-solvents; wherein a first co-solvent increases the solubility of the plant-based proteins, and a second co-solvent decreases the solubility of the plant-based proteins; wherein the co-solvents are added to the mixture in a concentrated or diluted form; and wherein the pH of the plant-based protein mixture is at least 0.5 pH units lower than the isoelectric point of the plant-based proteins;

[0018] (b) subjecting the plant-based protein mixture to shear treatment to form a plant-based protein hydrogel slurry;

[0019] (c) Disperse the active ingredient in the plant-based hydrogel slurry to form a composition; and

[0020] (d) Dry the composition to form microcapsules.

[0021] On the other hand, the present invention provides biodegradable microcapsules obtainable or capable of being obtained by the method as described above.

[0022] On the other hand, the present invention provides a method for preparing a biodegradable microcapsule composition, the method comprising:

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

[0024] (b) Suspend the biodegradable microcapsules in an external phase.

[0025] On the other hand, the present invention provides a biodegradable microcapsule composition obtainable or capable of being obtained by the method as described above.

[0026] On the other hand, the present invention provides a spray-dried biodegradable microcapsule comprising an active ingredient and a plant-based protein carrier, the plant-based protein carrier comprising a plant-based protein, wherein the plant-based protein carrier encapsulates the active ingredient, and wherein the plant-based protein carrier has a solubility of less than 50% when measured at 25 °C and in an aqueous solution at pH 7 at a protein concentration of 5% w / w.

[0027] On the other hand, the present invention provides a composition comprising the biodegradable microcapsules as described above and an external phase.

[0028] On the other hand, the present invention provides a formulated product comprising the biodegradable microcapsules as described above.

[0029] On the other hand, the present invention provides a method for preparing a formulated product, which comprises:

[0030] (a) Prepare biodegradable microcapsules according to the method as described above; and

[0031] (b) Mix the biodegradable microcapsules with a product formulation.

[0032] On the other hand, the present invention provides the use of the biodegradable microcapsules as described above in a formulated product.

[0033] Definitions

[0034] As used herein, the term "microcapsule" refers to microparticles in any form. For example, the term encompasses core-shell microcapsules (i.e., microcapsules having a central core containing an active ingredient, wherein the core is surrounded by a plant-based protein hydrogel in the form of a shell). Those skilled in the art will understand that core-shell microcapsules can have a multi-core morphology (i.e., wherein the core phase is in the form of multiple droplets) or a single-core morphology (wherein the core phase is in the form of a single droplet). The term also encompasses matrix microcapsules (i.e., microcapsules containing a plant-based protein hydrogel matrix, wherein the active ingredient is dispersed throughout the matrix). As determined by laser diffraction, the microcapsules can have a d of 500 nm to 2 mm 50 .

[0035] As used herein, the term "lower shear step" can refer to a processing step of applying a low level of mechanical energy to a material (preferably by a cutting action) to cause it to predominantly split or break into large discrete fragments. "Lower shear" generally does not include any grinding step that crushes or breaks a material by high-speed impact (e.g., an impact with a differential greater than 2 m / s -1 . In a particular embodiment, during the lower shear step, the hydrogel is broken to provide fragments such that at least 80% by weight of the hydrogel fragments have a maximum size of 1 mm to 100 mm, as determined by optical microscopy.

[0036] As used herein, the term "higher shear step" can refer to a processing step of applying energy to reduce a hydrogel into small fragments (e.g., to form a colloidal dispersion). In a particular embodiment, during the higher shear step, the hydrogel is broken to provide fragments having a d 50 particle size of 0.2 to 50 microns, as determined by laser diffraction. Laser diffraction can be performed according to the methods defined herein.

[0037] To avoid doubt, the higher shear step subjects the hydrogel to a higher level of shear than the lower shear step. In cases where the method involves both a lower shear step and a higher shear step, the higher shear step must occur after the lower shear step (i.e., they are discrete steps that occur in this particular order).

[0038] As used herein, the term "sol-gel transition temperature" refers to the temperature at which a plant-based protein transitions from a liquid state to a hydrogel state. Thus, at temperatures above the sol-gel transition temperature, the plant-based protein will be in a liquid state, while at temperatures below the sol-gel transition temperature, the plant-based protein will be in a hydrogel state. As used herein, the term "fragrance" (which may be used interchangeably with the term "perfume") refers to a component in a formulation that is capable of imparting or altering the odor of a product (such as a fabric conditioner or hair conditioner or 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 (such as a fine perfume), or to provide a sensory cue regarding the benefits and functions of the product (such as the calming effect of a lavender sleep aid, the cleaning concept of a laundry product), or to mask an unpleasant odor (such as in an insect repellent product). A "fragrance" can comprise one or more components, which can be a single chemical entity, referred to herein as an "odorant" (which may be used interchangeably with the term "perfume material"), or a mixture of different "odorants". Odorants can be produced by synthetic processes or extracted from nature (especially from plants) to obtain naturally occurring plant essential oils and plant extracts, such as orange oil. Odorants produced by synthetic processes can be newly introduced chemical substances or odorants that are identical to those found in nature. Then, a skilled perfumer (also known as a nose) can blend odorants from synthetic and natural sources into a fragrance for use in consumer products. Odorants can be obtained from professional fragrance suppliers (referred to as fragrance houses) as individual chemical substances, natural blends, or as proprietary special blends with an undisclosed complete composition. The individual odorants that make up a known natural blend can be found by reference to journals commonly used by those skilled in the art (such as "Perfume and Flavourist" or "Journal of Essential Oil Research"), or are listed in reference texts such as the books by S. Arctander (Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA; and recently reprinted by Allured Publishing Corporation, Illinois (1994)) and "Perfume and Flavour Materials of Natural Origin", S. Arctander, Ed., Elizabeth, N.J., 1960. It should be understood that for the purposes of the present invention, "odorant" includes fragrance precursors, such as acetal fragrance precursors, ketal fragrance precursors, ester fragrance precursors, hydrolysable inorganic-organic fragrance precursors, and combinations thereof.A fragrance substance can be released from a fragrance precursor in a variety of ways, for example, by hydrolysis release, or by a shift in an equilibrium reaction, or by pH change, or by enzymatic release, or by UV radiation release.

[0039] A fragrance substance can be described by its odor intensity, detection threshold, odor saturation, and its characteristics. In fragrance encapsulation, it is preferred to use fragrance substances with a low odor detection threshold and high intensity to maximize the detectability of the encapsulated and released fragrance (even in small amounts).

[0040] To impart an odor, a fragrance substance must be volatile, even to a small extent, because the molecules must travel through the air into the nose, where it attaches to specific nerve receptors and triggers a signal within the olfactory system. Fragrance substances can be classified according to their volatility. Preferably, fragrance substances are liquids at 20 °C and atmospheric pressure, but occasionally they can be solids and can be blended with other liquid fragrance substances or solvents. Generally, the fragrance industry refers to volatility and substantivity by roughly classifying substances into one of the following three categories: substances with the lowest volatility and the highest substantivity are classified as base notes, substances with medium volatility and substantivity are classified as middle notes, and substances with the highest volatility and the lowest substantivity are classified as top notes. This is based on the odor perception of the substances and is very subjective. One way to objectively classify the volatility of fragrance substances is by their vapor pressure.

[0041] As used herein, the term "vapor pressure" means the partial pressure of a given chemical substance in air at a defined temperature (e.g., 25 °C) and standard atmospheric pressure (760 mmHg). It defines the affinity of the chemical substance for the gas phase rather than the liquid or solid phase. The higher the vapor pressure, the higher the proportion of the substance in the closed headspace at equilibrium. It is also related to the evaporation rate of the fragrance substance, which is defined in the open environment where the substance leaves the system. The vapor pressure can be easily determined according to the reference procedure ACD / Percepta Desktop Software, version 14.0 (build date: August 26, 2021), Advanced Chemistry Development, Inc (ACD / Labs), Toronto, Canada, www.acdlabs.com.

[0042] A physical parameter related to the encapsulation of fragrance substances is their hydrophobicity, which can be defined according to their partition coefficient P. As used herein, the term "partition coefficient" refers to the ratio of the equilibrium concentrations of a substance in n-octanol and water, and is a measure of the differential solubility of the 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 date: August 26, 2021), Advanced Chemistry Development, Inc (ACD / Labs), Toronto, Canada, www.acdlabs.com. The logP value is predicted from the SMILES string of the fragrance substance 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 separating carbons. logP GALAS is based on an algorithm that takes into account a database of compound training sets and adjusts the values using data of structurally similar compounds. Consensus logP is a model based on the first two algorithms and can be expressed as: Consensus logP = a x logP Classic + b x logP GALAS, where a and b are the coefficients of the model. The latter value (Consensus logP) is the logP value referred to herein.

[0043] Another aspect related to the encapsulation of fragrance substances is their Hansen solubility parameter (HSP). The term HSP refers to the solubility parameter method proposed by Charles Hansen, which was initially used to predict the solubility of polymers in a given solvent, as described in Charles Hansen's The Three Dimensional Solubility Parameter and Solvent Diffusion Coefficient, Danish Technical Press (Copenhagen, 1967). This method has later been reapplied to many other molecules. A fragrance substance (or flavor substance or solvent) and its interaction with its environment are defined by three forces: atomic dispersion force, molecular permanent dipole force, and molecular hydrogen bond force. Substances with similar HSP parameters are more likely to be miscible. These forces can be quantified by three values: δ D , the Hansen dispersion value related to van der Waals interaction (intermolecular force); δ P , the Hansen polarity value related to the dipole moment (charge); and δ Η , the Hansen hydrogen bond ("h-bond") value. The solubility parameter δ (MPa1 / 2 ) is defined as δ 2 = δ D 2 + δ P 2 + δ H 2 = E / V, where E is the cohesive energy of the solvent and V is the molar volume. The HSP values of a given substance can be obtained in two main different ways from the HSPiP (Hansen Solubility Parameters in Practice) software, which is available from www.hansen-solubility.com. These values can be retrieved from the main dataset containing over 20,000 substances by searching by name or CAS number; or predicted using the Y-MB (Yamamoto-Molecular Breaking) method by entering the SMILE string of the given molecule in the DIY section of the software. Additionally, determining the HSP sphere relative to a given fragrance substance is a good way to predict solubility preferences in fragrance blends. The radius Ro of the HSP sphere is defined as Ro = Ra / RED, where Ra is the HSP distance between two molecules (1 and 2), expressed as: Ra 2 = 4(δ D1 - δ D2 ) 2 +(δ P1 - δ P2 ) 2 +(δ H1 - δ H2 ) 2 , and RED is the relative energy difference. This RED value can also be extracted from or predicted by the HSPiP software, and a good solvent for a given substance should show a RED value less than or equal to 1, while a solvent showing a RED value greater than 1 should be considered a poor solvent for the given substance.

[0044] The fragrance substances can be selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds, and Schiff bases.

[0045] Preferred aldehyde fragrance substances include, but are not limited to, α-amylcinnamaldehyde, anisaldehyde, decanal, dodecanal, methyl-n-nonylacetaldehyde, methyl octyl acetaldehyde, nonanal, benzaldehyde, neral, geranial, 1,1-diethoxy-3,7-dimethylocta-2,6-diene, 4-isopropylbenzaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, α-methyl-p-isopropyl dihydrocinnamaldehyde, 3-(3-isopropylphenyl)butyraldehyde, α-hexylcinnamaldehyde, 7-hydroxy-3,7-dimethyloctanal, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, octanal, phenylacetaldehyde, 2,4-dimethyl-3-cyclohexene-1-carboxaldehyde, hexanal, 3,7-dimethyloctanal, 6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-butanal, nonanal, octanal, 2-nonenal undecenal, 2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-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-methyl hydrocinnamaldehyde, cis-4-decen-1-al, 2,5-dimethyl-2-vinyl-4-hexenal, trans-2-methyl-2-butenal, 3-methylnonanal, α-sinensal, 3-phenylbutyraldehyde, 2,2-dimethyl-3-phenylpropionaldehyde, m-tert-butyl-α-methyl dihydrocinnamaldehyde, geranyloxyacetaldehyde, trans-4-decen-1-al, methoxycitronellal, and mixtures thereof.

[0046] Preferred ester flavor substances include, but are not limited to, allyl cyclohexanepropionate, allyl heptanoate, allyl amyl glycolate, allyl hexanoate, amyl acetate (n-amyl acetate), amyl propionate, benzyl acetate, benzyl propionate, benzyl salicylate, cis-3-hexenyl acetate, citronellyl acetate, citronellyl propionate, cyclohexyl salicylate, dihydroisojasmone, dimethyl benzyl carbinyl acetate, ethyl acetate, ethyl acetoacetate, ethyl butyrate, ethyl 2-methylbutyrate, ethyl 2-methylvalerate, 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, p-tert-butylcyclohexyl acetate, (-)-L-menthyl acetate, o-tert-butylcyclohexyl acetate, methyl benzoate, methyl dihydroisojasmone, α-methylbenzyl acetate, methyl salicylate, 2-phenylethyl acetate, prenyl acetate, cedryl acetate, cyclabute, phenethyl phenylacetate, terpinyl formate, citronellyl anthranilate, tricyclo[5.2.1.Ethyl 0-2,6]decane-2-carboxylate, n-hexyl ethyl acetoacetate, 2-tert-butyl-4-methylcyclohexyl acetate, formic acid, 3,5,5-trimethylhexyl ester, phenethyl crotonate, cyclogeranyl acetate, geranyl crotonate, ethyl citronellate, geranyl isobutyrate, methyl 2-nonynoate-2,6-octadienoate, citronellyl valerate, 2-hexenyl cyclopentanone, cyclohexyl anthranilate, L-tigloyl citronellate, butyl tiglate, pentyl tiglate, geranyl octanoate, 9-decenyl acetate, 2-isopropyl-5-methylhexyl-1 butyrate, n-pentyl benzoate, 2-methylbutyl benzoate (and its mixture with pentyl benzoate), dimethyl benzyl carbinyl propionate, dimethyl benzyl carbinyl acetate, trans-2-hexenyl salicylate, dimethyl benzyl carbinyl isobutyrate, 3,7-dimethyloctyl formate, rose formate, rose isovalerate, rose acetate, rose butyrate, rose propionate, cyclohexylethyl acetate, neryl butyrate, tetrahydrogeranyl butyrate, myrcenyl acetate, methyl 2,5-dimethyl-2-vinylhex-4-enoate, 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, methyl 1-methylcyclohex-3-ene-carboxylate, propyl tiglate, propyl / isobutyl cyclopent-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, isopentenyl hexanoate, furfuryl propionate, diethyl malate, isopropyl 2-methylbutyrate, dimethyl malonate, borneol formate, styralyl acetate, 1-(2-furyl)-1-acetone, l-citronellyl acetate, 3,7-dimethyl-1,6-nonadien-3-yl acetate, neryl crotonate, dihydromyrcenyl acetate, tetrahydromyrcenyl acetate, lavandulyl acetate, 4-cyclooctenyl isobutyrate, cyclopentyl isobutyrate, 3-methyl-3-butenyl acetate, allyl acetate, geranyl formate, cis-3-hexenyl hexanoate and mixtures thereof.

[0047] Preferred alcohol flavoring substances include, but are not limited to, benzyl alcohol, β-γ-hexenol (2-hexen-1-ol), cedrol, citronellol, cinnamyl alcohol, p-cresol, cuminol, dihydromyrcenol, 3,7-dimethyl-1-octanol, dimethylbenzyl carbinol, eucalyptol, eugenol, fenchol, geraniol, hydratopic 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, 1-3,7-dimethyl octane-1-ol, 2-(furfuryl-2)-heptanol, 6,8-dimethyl-2-nonanol, ethyl norbornyl cyclohexanol, β-methyl cyclohexane ethanol, 3,7-dimethyl-(2),6-octadiene (diene)-1-ol, trans-2-undecen-1-ol, 2-ethyl-2-isopentenyl-3-hexenol, isobutyl benzyl carbinol, dimethylbenzyl carbinol, ocimenol, 3,7-dimethyl-1,6-nonadien-3-ol (cis and trans), tetrahydromyrcenol, α-terpineol, 9-decenol-1,2-(2-hexenyl)-cyclopentanol, 2,6-dimethyl-2-heptanol, 3-methyl-1-octen-3-ol, 2,6-dimethyl-5-hepten-2-ol, 3,7,9-trimethyl-1,6-decadien-3-ol, 3,7-dimethyl-6-nonen-1-ol, 3,7-dimethyl-1-octyn-3-ol, 2,6-dimethyl-1,5,7-octatrieneol-3, dihydromyrcenol, 2,6,-trimethyl-5,9-undecadienol, 2,5-dimethyl-2-propylhex-4-enol-1, (Z)-3-hexenol, o-, m-, p-methyl phenethyl alcohol, 2-methyl-5-phenyl-1-pentanol, 3-methyl phenethyl alcohol, p-methyl dimethylbenzyl carbinol, methyl benzyl carbinol, p-methyl phenethyl alcohol, 3,7-dimethyl-2-octen-1-ol, 2-methyl-6-methylene-7-octen-4-ol and mixtures thereof.

[0048] Preferred ketone flavor substances include, but are not limited to, 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 pentyl ketone, α-ionone, β-ionone, acetone, octahydro-2,3,8,8-tetramethyl-2-acetylnaphthalene, α-irone, 1-(5,5-dimethyl-1-cyclohexen-1-yl)-4-penten-1-one, 3-nonanone, ethyl hexyl ketone, menthone, 4-methylacetophenone, γ-methylionone, methyl pentyl ketone, methylheptenone (6-methyl-5-hepten-2-one), methyl heptyl ketone, methyl hexyl ketone, δ-muscone, 2-octanone, 2-pentyl-3-methyl-2-cyclopenten-1-one, 2-heptyl cyclopentanone, α-methylionone, 3-methyl-2-(trans-2-pentenyl)-cyclopentenone, octenyl cyclopentanone, n-pentyl cyclopentenone, 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.

[0049] Preferred ether flavor substances include, but are not limited to, diphenyl ether, p-tolyl methyl ether, 4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydro-cyclopenta(g)-2-benzopyran, β-naphthyl methyl ether, methyl isobutenyl tetrahydropyran, 5-acetyl-1,1,2,3,3,6-hexamethyl indane (tonalid), 7-acetyl-1,1,3,4,4,6-hexamethyl tetralin (galaxolide), 2-phenethyl-3-methylbut-2-enyl ether, ethyl geranyl ether, phenethyl isopropyl ether and mixtures thereof.

[0050] Preferred olefin flavor substances include, but are not limited to, allo-ocimene, camphene, β-caryophyllene, cadinene, diphenylmethane, d-limonene, dihydro myrcenol (lymolene), β-myrcene, p-cymene, 2-α-pinene, β-pinene, α-terpinene, γ-terpinene, terpinolene, 7-methyl-3-methylene-1,6-octadiene and mixtures thereof.

[0051] Preferred nitrile flavor substances 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.

[0052] Preferred Schiff base flavoring substances include but are not limited to citronellyl nitrile, nonanal / methyl anthranilate, N-octylidene-methyl anthranilate, hydroxycitronellal / methyl anthranilate, cyclamen aldehyde / methyl anthranilate, methoxyphenyl propionaldehyde / methyl anthranilate, ethyl p-aminobenzoate / hydroxycitronellal, citral / methyl anthranilate, 2,4-dimethylcyclohex-3-ene-carboxaldehyde methyl anthranilate, hydroxycitronellal indole and mixtures thereof.

[0053] As used herein, the term "flavorant" refers to a component in a formulation that can impart or modify the taste and odor of a product, such as toothpaste or food. Flavorants are typically used to impart an overall pleasant taste and odor, or taste and odor profile, to a product, either simply to provide a pleasant experience (e.g., in food) or to mask an unpleasant taste or odor (e.g., in pharmaceuticals). Flavorants or flavoring substances can be described in terms of their odor intensity, detection threshold, and quality. A "flavorant" can comprise one or more components, which can be a single chemical entity (referred to herein as a "flavoring substance") or a mixture of different "flavoring substances". Flavoring substances can be produced by synthetic processes or can be extracted from natural sources, particularly from plants, to yield naturally occurring plant and animal oils and secretions, such as vanilla extract. A skilled flavorist can then blend flavoring substances from synthetic and natural sources into a flavorant for use in consumer products. Flavoring substances can be obtained from specialty flavorant suppliers (referred to as flavor houses) as individual chemicals, natural blends, or as proprietary special blends with the complete composition not disclosed. The individual flavoring substances that make up known natural blends can be found by reference to journals commonly used by those skilled in the art (e.g., "Perfume and Flavourist" or "Journal of Essential Oil Research"), or in reference texts such as the books by S. Arctander (Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA; and recently reprinted by Allured Publishing Corporation Illinois (1994)); "Perfume and Flavour Materials of Natural Origin", S. Arctander, Ed., Elizabeth, N.J., 1960; and "Flavourings", E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998. It should be understood that flavorants can be volatile or have volatile components that can be detected by the nose in the same manner as fragrances. Thus, flavoring substances can also be classified according to their physical properties (e.g., volatility and hydrophobicity) using the methods described above for fragrance substances. Flavoring substances can also be described according to their Hansen solubility parameters using the methods described above for fragrance substances.

[0054] Sources of flavoring substances include essential oils, concretes, absolutes, resins, resinoids, balsams and tinctures. Preferred flavoring substances include anise oil, ethyl-2-methylbutyrate, vanillin, cis-3-heptenol, cis-3-hexenol, trans-2-heptenal, butyl valerate, 2,3-diethylpyrazine, methylcyclopentenolone, benzaldehyde, valerian oil, 3,4-dimethoxyphenol, amyl acetate, pentyl cinnamate, γ-butyrolactone, trimethylpyrazine, phenylacetic acid, isovaleraldehyde, ethyl maltol, ethyl vanillin, ethyl valerate, ethyl butyrate, cocoa extract, coffee extract, peppermint oil, spearmint oil, clove oil, anethole, cardamom oil, wintergreen oil, cinnamaldehyde, ethyl-2-methylvalerate, γ-hexenolide, 2,4-decadienal, 2,4-heptadienal, methylthiazolol (4-methyl-5-b-hydroxyethylthiazole), 2-methylbutanethiol, 4-mercapto-2-butanone, 3-mercapto-2-pentanone, 1-mercapto-2-propane, benzaldehyde, furfural, furfuryl alcohol, 2-mercaptopropionic acid, alkylpyrazines, methylpyrazine, 2-ethyl-3-methylpyrazine, tetramethylpyrazine, polysulfides, dipropyl disulfide, methylbenzyl disulfide, alkylthiophenes, 2,3-dimethylthiophene, 5-methylfurfural, acetylfuran, 2,4-decadienal, guaiacol, phenylacetaldehyde, β-decalactone, d-limonene, acetoin, amyl acetate, maltol, ethyl butyrate, levulinic acid, piperonal, ethyl acetate, n-octanal, n-valeraldehyde, n-hexanal, diacetyl, monosodium glutamate, monopotassium glutamate, sulfur-containing amino acids (such as cysteine), 2-methylfuran-3-thiol, 2-methyldihydrofuran-3-thiol, 2,5-dimethylfuran-3-thiol, tetramethylpyrazine, propyl allyl disulfide, propyl allyl trisulfide, diallyl disulfide, diallyl trisulfide, di-propenyl disulfide, di-propenyl trisulfide, 4-methyl-2-[(methylthio)-ethyl]-1,3-dithiolane, 4,5-dimethyl-2-(methylthiomethyl)-1,3-dithiolane and 4-methyl-2-(methylthiomethyl)-1,3-dithiolane, hop oil and citrus oils (such as lemon oil, orange oil, lime oil and grapefruit oil). Detailed Description of the Invention

[0056] The inventors of the present invention have discovered a method for preparing a protein colloidal dispersion with a high solid content containing active ingredients, the dispersion having a controlled particle size and low viscosity, the method allowing the formation of stable microcapsules after drying. The resulting microcapsules are stable in liquid product formulations and also stable under the conditions required for preparing such liquid product formulations (e.g., pasteurization conditions in the case of beverage formulations).

[0057] The present invention relates to a method for preparing biodegradable microcapsules, the method comprising:

[0058] (a) A mixture comprising one or more plant-based proteins is formed in a solvent system, where the solvent system comprises miscible co-solvents; where a first co-solvent increases the solubility of the plant-based proteins, and a second co-solvent decreases the solubility of the plant-based proteins; where the co-solvents are added to the mixture in a concentrated or diluted form; and where the pH of the plant-based protein mixture is at least 0.5 pH units lower than the isoelectric point of the plant-based proteins;

[0059] (b) The plant-based protein mixture is subjected to shear treatment to form a plant-based protein hydrogel slurry;

[0060] (c) An active ingredient is dispersed in the plant-based hydrogel slurry to form a composition; and

[0061] (d) The composition is dried to form microcapsules.

[0062] Any suitable plant-based protein can be used in the present invention. In a preferred method of the present invention, the plant-based protein is obtained from broad beans, mung beans, peas, rice, potatoes, rapeseed, lentils, chickpeas, sunflower seeds, pumpkin seeds, flax, chia, canola, lupins, alfalfa, moringa, wheat, zein or sorghum; preferably, the plant protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, broad bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein. More preferably, the plant-based protein is pea protein and / or potato protein. Such proteins are considered to be low-allergenic proteins.

[0063] Suitable plant-based proteins further include:

[0064] - Brassica: including Brassica balearica: Mallorca cabbage, Brassica carinata: Abyssinian mustard or Abyssinian cabbage, Brassica elongata: elongated mustard, Brassica fruticulosa: Mediterranean cabbage, Brassica hilarionis: St Hilarion cabbage, Brassica juncea: Indian mustard, brown mustard and leaf mustard, Sarepta mustard, Brassica napus: rapeseed, canola, rutabaga, Brassica narinosa: broadbeaked mustard, Brassica nigra: black mustard, Brassica oleracea: kale, cabbage, collard greens, broccoli, cauliflower, Chinese kale, Brussels sprouts, kohlrabi, Brassica perviridis: tender green, mustard spinach, Brassica rapa (synonym: B. campestris): Chinese cabbage, turnip, rapini, komatsuna, Brassica rupestris: brown mustard, Brassica tournefortii: Asian mustard

[0065] - Solanaceae: including tomatoes, potatoes, eggplants, sweet peppers and chili peppers;

[0066] - Cereals: including corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, fonio;

[0067] - Pseudo-cereals: including amaranth (love-lies-bleeding, red amaranth, prince-of-Wales-feather), breadnut, buckwheat, chia, cockscomb (also known as quail grass or soko), pitseed Goosefoot, painted leaf goosefoot quinoa and wattleseed (also known as acacia seed);

[0068] - Leguminous plants: including Acacia alata (Winged Wattle), Acacia decipiens, Acacia saligna (which has various common names, including coojong, golden wreath wattle, orange wattle, blue-leaved wattle), Arachis hypogaea (peanut), Astragalus galegiformis, Cytisus laburnum (common names include laburnum, goldenchain or golden rain), Cytisus supinus, Dolichios lablab (common names include hyacinth bean, lablab-bean / bonavist bean / pea, dolichos bean, seim bean, lablab bean, Egyptian kidney bean, Indian bean, bataw and Australian pea), Ervum lens (Lentil), Genista tinctorial (common names include dyer's whin, waxen woad and waxen wood), Glycine max (Soybean), Lathyrus clymenum (peavine or vetchling), Lathyrus odoratus (peavine or vetchling), Lathyrus staivus (peavine or vetchling), Lathyrus Silvetris (peavine or vetchling), Lotus tetragonolobus (winged pea), Lupinus albus (Lupin), Lupinus angustifolius (Lupin), Lupinus luteus (Lupin), Lupinus polyphyllus (Lupin), Medicagosativa)(Alfalfa), Phaseolus aureus (Mung bean), Phaseolus coccineus (Runner bean), Phaseolus nanus (Greenbean / French bean), Phaseolus vulgaris (Greenbean / French bean), Pisum sativum (pea), Trifolium hybridum (Clover), Trifolium pretense (Red clover), Vicia faba (Broad bean), Vicia sativa (Vetch), Vigna unguiculate (cowpea);

[0069] - Non-leguminous plants: including: Acanshosicyos horrida, Aesculus hippocastanum (Conker tree / Horsechestnut), Anacardium occidentale (Cashew tree), Balanites aegyptica, Bertholletia excels (Brazil nut), Beta vulgaris (Sugar beet), Brassica napus (Rapeseed), Brassica juncea (Brown mustard), Brassica nigra (Black mustard), Brassica hirta (Eurasian mustard), Citrullus vulgaris (watermelon species), Citrus aurantiaca (Citrus), Cucurbita maxima (squash), Fagopyrum esculentum (knotweed), Gossypium barbadense (Extra-long staple cotton), Heianthus annuus (sunflower), Nicotiana sp. (tobacco plant), Prunus avium (cherry), Prunus cerasus (sour cherry), Prunus domestica (plum), Prunus amygdalus (almond), Rricinus communis (Caster bean / Castor oil plant), Sasamum indicum (Sesame), Sinapis alba (White mustard), Terlfalrea pedata (Oyster nut).

[0070] To avoid ambiguity, the plant-based microcapsules of the present invention do not contain plants in their natural state, such as naturally formed plant cells, organelles or vesicles are not the plant-based microcapsules of the present invention.

[0071] In step (a), the first co-solvent increases the solubility of the plant-based protein. The first co-solvent can be regarded as a solubility co-solvent. One or more solubility co-solvents can be present, and the solubility co-solvent can completely or partially dissolve the plant-based protein. The co-solvent can be added to step (a) in a highly concentrated form or a diluted form.

[0072] Examples of the solubility co-solvent are organic acids. Organic acids are organic compounds with acidity. Preferably, the organic acids are derived from natural plant-based or bio-based raw materials.

[0073] 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 can include β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-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.

[0074] Using organic acids can dissolve plant proteins and also allow mild hydrolysis of the proteins. For example, without wishing to be bound by theory, the dissolution of plant-based proteins in organic acids is possible because: i) protonation of the proteins and ii) the presence of an anionic solvation layer, which helps to reduce hydrophobic interactions. After preliminary dissolution in the organic acid, the protonation of the plant-based proteins can contribute to their stabilization in their non-solvent (such as water).

[0075] In step (a), compared with the first co-solvent, the second co-solvent reduces the solubility of the plant-based protein. The second co-solvent can be regarded as a desolvation co-solvent. One or more desolvation co-solvents can be present.

[0076] 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.

[0077] In a preferred method of the present invention, the solvent system contains a co-solvent ratio of the first co-solvent to the second co-solvent of about 10-90% v / v, preferably about 20-90% v / v, preferably about 20-80% v / v, preferably about 20-60% v / v, about 25-55% v / v, about 30-50% v / v, about 20%, about 30%, about 40%, about 50% or about 60% v / v, most preferably about 30-50% v / v.

[0078] According to the present invention, it is preferred that the first co-solvent is present in the mixture of step (a) at a concentration equal to or greater than the protein concentration based on weight %.

[0079] In a preferred method of the present invention, the pH of the plant-based protein mixture in step (a) is at least 0.5 pH units lower, more preferably at least 1.0 pH unit lower, than the isoelectric point of the plant-based protein.

[0080] In a preferred method of the present invention, the concentration of the plant-based protein in the solvent system is 25 - 200 mg / ml, more preferably 50 - 150 mg / ml.

[0081] The proportion of the organic acid can vary according to the protein concentration. For example, as the protein concentration increases, a higher proportion of the organic acid is used.

[0082] In a preferred method of the present invention, the degree of proteolysis (i.e., the percentage of peptide bonds cleaved in the protein hydrolysate) is controlled to alter the properties of the resulting hydrogel. For example, increasing the acid concentration present during formation will increase the degree of proteolysis. A higher degree of proteolysis results in the formation of a hydrogel with lower rigidity.

[0083] In a preferred method of the present invention, the degree of proteolysis is 0.1 to 10%, preferably 0.1 to 5%, even more preferably 0.1 to 2.5%.

[0084] To form a mixture comprising one or more plant-based proteins, it may be necessary to apply a physical stimulus to the protein / solvent system mixture to enable interaction between the solvent and the protein. Suitable physical stimuli include heating, sonication, stirring, high-shear mixing, high-shear homogenization, or other physical techniques. The preferred technique is heating, optionally followed by sonication.

[0085] Preferably, a physical stimulus of heating is applied to the protein / solvent system mixture, wherein the mixture is heated to about 70 °C or higher than 70 °C. More preferably, the protein / solvent system mixture is heated to about 75 °C or higher than 75 °C, about 80 °C or higher than 80 °C, about 85 °C or higher than 85 °C, or about 90 °C. More preferably, the protein / solvent system mixture is heated to 85 °C.

[0086] Preferably, a physical stimulus of heating the protein / solvent system mixture for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or greater than 30 minutes is applied. Optionally, subsequent sonication is performed on the heated protein / solvent system mixture.

[0087] In a preferred method of the present invention, the protein mixture is heated to a first temperature above the sol-gel transition temperature of one or more plant-based protein mixtures and then lowered to a second temperature below the sol-gel transition temperature of one or more plant-based protein mixtures to form a hydrogel, preferably between step (a) and step (b) or during step (b).

[0088] Heat the protein mixture such that the liquid mixture remains above the sol-gel transition of the protein. By changing the solvent system (e.g., by choosing an organic acid, the ratio of an organic acid to other solvents, or by other means), the sol-gel transition temperature of the protein can be altered. By appropriately selecting conditions, the sol-gel transition of the protein can be carefully controlled, thereby controlling the formation of the hydrogel.

[0089] Preferably, the protein mixture is heated to about 70 °C or higher. More preferably, the protein is heated to about 75 °C or higher, about 80 °C or higher, about 85 °C or higher, or about 90 °C. More preferably, the protein is heated to 85 °C.

[0090] The protein mixture can be held at the elevated temperature for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or 1 hour. The preferred period is at least 30 minutes to allow the protein to completely dissolve. It is possible to hold the protein mixture at the elevated temperature for a longer period.

[0091] After heating the protein mixture above the sol-gel transition temperature, the temperature of the protein mixture can be lowered to a second temperature below the sol-gel transition temperature to promote the formation of the hydrogel. The second temperature can be room temperature. The second temperature can be in the range of 5 to 25 °C, preferably in the range of 10 to 20 °C. The protein mixture can be held at the lowered temperature for a long period, such as several days, several weeks, and then subjected to the shearing treatment in step (b). The protein mixture can be held at the lowered temperature for a period of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or about 30 minutes. A specific lowered period is about 5 minutes.

[0092] The specific temperature will depend on the nature of the protein source, the solvent conditions used, and thus the sol-gel transition temperature. Optionally, the elevated and lowered temperatures can be relatively fixed (e.g., about 85 °C, then about room temperature), and the co-solvent mixing conditions can be adjusted to ensure that the selected plant-based protein has a suitable sol-gel transition temperature.

[0093] Without wishing to be bound by theory, it is believed that when plant proteins are added to a solvent system, the plant proteins form a dispersion of insoluble colloidal protein aggregates. The aggregate size can be measured by dynamic light scattering (DLS). A suitable instrument for measuring the aggregate size is a Zetasizer Nano S (Malvern).

[0094] It is believed that after heating the protein mixture to above the sol-gel transition temperature in the presence of a co-solvent system, the plant proteins partially unfold, resulting in the exposure of hydrophobic amino acids that were initially buried within the native protein structure. Once partially unfolded, the co-solvent is able to interact with the unfolded protein molecules. For example, organic acids are more likely to protonate amino acid residues and can form stable hydrophobic interactions with anionic salt bridges. In addition, when heated at elevated temperatures, protein-protein non-covalent intermolecular contacts are disrupted.

[0095] In addition, it is believed that applying mechanical agitation (such as sonication) disrupts large colloidal protein aggregates into smaller aggregates and disrupts the interactions between protein molecules.

[0096] In addition, it is believed that when the protein mixture is cooled to below the sol-gel transition temperature, protein-protein non-covalent intermolecular contacts can be achieved, thereby promoting the self-assembly of plant protein molecules into a hydrogel of interconnected protein aggregates.

[0097] It is believed that the method of the present invention allows plant proteins to aggregate into a supramolecular structure held together by intermolecular hydrogen bond interactions (and particularly between β-strands).

[0098] The method of the present invention is capable of forming a material in which there are high levels of intermolecular β-sheet interactions. Thus, in the method of the present invention, the plant-based protein has a protein secondary structure having at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% intermolecular β-sheet, where the percentage content of intermolecular β-sheet is measured by FTIR (Fourier transform infrared spectroscopy).

[0099] In a preferred method of the present invention, a solvent reduction step is carried out on the plant-based protein hydrogel, preferably a soluble solvent reduction step, preferably carried out between step (a) and step (b) or during step (b).

[0100] A solubility solvent means a solvent or solvent mixture in which the plant-based protein hydrogel dissolves. Examples include organic acids: such as acetic acid, lactic acid, formic acid, propionic acid, α-hydroxy acids, and / or β-hydroxy acids. The α-hydroxy acids can 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 can preferably be selected from β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-methylbutyric acid, 2-hydroxybenzoic acid, and carnitine.

[0101] In a preferred method of the present invention, the solvent reduction step involves:

[0102] (i) contacting the plant-based protein hydrogel with a non-solubility solvent;

[0103] (ii) separating the plant-based hydrogel from the non-solubility solvent to obtain a washed plant-based protein hydrogel; and

[0104] (iii) optionally repeating steps (i) and (ii).

[0105] Step (i) involves contacting the plant-based protein hydrogel with a non-solubility solvent. A non-solubility solvent means a solvent or solvent mixture in which the plant-based protein hydrogel is insoluble. Examples include water or a mixture of water and ethanol.

[0106] In a preferred method of the present invention, the shearing treatment includes one step (i.e., a single shearing step). The single shearing step can be a high-shearing step. Preferably, the single shearing step involves breaking the plant-based protein hydrogel into fragments.

[0107] In a preferred method of the present invention, the fragments produced in the single shearing step have a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns 50 , as determined by laser diffraction.

[0108] In a preferred method of the present invention, the single shearing step involves ultrasonic treatment (e.g., using equipment such as Bandelin HD4200 or Hielscher UIP1000hdT), high-shear mechanical stirring (e.g., using equipment such as a Silverson rotor-stator high-shear mixer), high-pressure homogenization, or cavitation, preferably ultrasonic treatment.

[0109] In a preferred method of the present invention, the single shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-based protein mixture. In a preferred method of the present invention, the first shearing step is carried out for a duration of at least 5 minutes, more preferably at least 1 minute.

[0110] 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 lower shearing step and the second shearing step may be a higher shearing step.

[0111] In a preferred method of the present invention, the first shearing step involves breaking the plant-based protein hydrogel into pieces. Preferably, at least 50 wt% of the pieces 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 wt% of the pieces 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 measured by optical microscopy or visual measurement.

[0112] 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-based protein mixture.

[0113] In a preferred method of the present invention, the first shearing step involves mechanical cutting. Mechanical cutting means cutting using a cutting edge (such as a knife, an extruder blade, etc.).

[0114] In an alternative preferred method of the present invention, the first shearing step involves extrusion. For example, the plant-based protein mixture formed in step (a) can be extruded into a non-solvent (such as water) to form a plant-based protein hydrogel in the form of large discrete pieces. For example, the large discrete pieces can be in the form of an extrudate having a thread or rope form. In this way, the pieces can directly undergo a solvent reduction step, as described in more detail below. The 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 (such as the diameter of the extrudate) of the large pieces to 1 mm to 100 mm. Preferably, at least 50 wt% of the pieces 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. More preferably, at least 80 wt% of the pieces 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. This can be measured by optical microscopy or visual measurement.

[0115] In a preferred method of the present invention, the second shearing step involves further fragmenting the plant-based protein hydrogel. Preferably, the fragments produced in the second shearing step have a d of 0.2 to 50 micrometers, preferably 1 to 40 micrometers, preferably 2 to 30 micrometers 50 , as determined by laser diffraction.

[0116] In a preferred method of the present invention, the particle size distribution of the hydrogel fragments in the plant-based protein hydrogel slurry can be adjusted by changing the nature and intensity of the second shearing step. In another preferred method, the particle size distribution of the hydrogel fragments in the plant-based protein hydrogel slurry can be adjusted by blending or combining two or more hydrogel slurries that have undergone different second shearing steps and have different particle size distributions.

[0117] In a preferred method of the present invention, the second shearing step is carried out at a temperature below the sol-gel transition temperature of the plant-based protein.

[0118] In a preferred method of the present invention, the second shearing step is carried out at a temperature below the protein denaturation temperature of the plant-based protein.

[0119] In a preferred method of the present invention, the second shearing step is carried out for a duration of at least 5 minutes, more preferably at least 1 minute.

[0120] In a preferred method of the present invention, the second shearing step involves sonication (e.g., using a device such as Bandelin HD4200 or Hielscher UIP1000hdT), high-shear mechanical stirring (e.g., using a device such as a Silverson rotor-stator high-shear mixer), high-pressure homogenization, or cavitation, preferably sonication.

[0121] In a preferred method of the present invention, step (b) further includes, between the first shearing step and the second shearing step, a solvent reduction step on the plant-based protein hydrogel slurry, preferably a solubility solvent reduction step.

[0122] In a preferred method of the present invention, the solvent reduction step includes the following steps:

[0123] (i) contacting the fragments of the plant-based hydrogel slurry with a non-solubility solvent;

[0124] (ii) separating the fragments of the plant-based hydrogel slurry from the non-solubility solvent to obtain a washed plant-based protein hydrogel; and

[0125] (iii) optionally repeating steps (i) and (ii).

[0126] The solubility solvent is as defined above.

[0127] Step (i) involves contacting the fragments of the plant-based protein hydrogel slurry with a non-solvent. The non-solvent is as defined above.

[0128] In a preferred method of the present invention, step (ii) involves sieving or centrifugation. More preferably, step (ii) involves sieving using a plurality of sieves with decreasing sizes.

[0129] Those skilled in the art will understand that if the fragments generated in the first shearing step are too small, then the solvent reduction step may be difficult because the fragments may eventually clog the sieve or the yield of collection is very low. However, if the fragments generated in the first shearing step are too large, then the solvent reduction step may take too much time due to the slow mass transfer of the solvent from the core of the fragments.

[0130] Without wishing to be bound by theory, it is believed that due to the porous nature of the hydrogel, the solvent reduction step can remove some or all of the solvent (such as organic acid) from the hydrogel through solvent exchange.

[0131] The strength of the protein hydrogel can be changed by varying the concentrations of the protein and the organic acid and other variables.

[0132] The strength of the hydrogel used to form the hydrogel slurry is useful within a certain range. This can be measured by oscillatory rheology. A suitable measure of the hydrogel strength is the storage modulus G' of the hydrogel. Suitable test conditions are at 20 °C with an oscillation frequency of 1 Hz and a strain of 1%. A suitable device is an Anton Paar MCR 92 rheometer with a 50 mm diameter, 1-degree angular cone and plate measurement geometry.

[0133] Thus, in a preferred method of the present invention, before washing, the plant-based protein hydrogel has a storage modulus (G') 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 at 10 rad / s. Those skilled in the art will understand that 2π rad / s is equivalent to 1 Hz.

[0134] In a preferred method of the present invention, before washing, the plant-based protein hydrogel has a storage modulus (G') less than 20,000 Pa, preferably less than 15,000 Pa, and more preferably less than 10,000 Pa at 10 rad / s.

[0135] In a preferred method of the present invention, before washing, the plant-based protein hydrogel has a storage modulus (G') of about 1000 to 20,000 Pa, preferably about 2000 to 15,000 Pa, and more preferably about 2000 to 10,000 Pa at 10 rad / s.

[0136] Further, in the preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') 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 at 10 rad / s.

[0137] In the preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') less than 5000 Pa, preferably less than 2500 Pa, preferably less than 1000 Pa, preferably less than 950 Pa, more preferably less than 900 Pa, more preferably less than 850 Pa, more preferably less than 800 Pa, more preferably less than 750 Pa at 10 rad / s.

[0138] In the preferred method of the present invention, the washed plant-based protein hydrogel has a storage modulus (G') 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 at 10 rad / s.

[0139] The preferred method of the present invention further comprises adding additional ingredients to the plant-based protein hydrogel slurry in step (b) or between steps (b) and (c), or to the composition of step (c). Preferably, the additional ingredients are selected from plasticizers, surfactants, rheological modifiers, light blockers, preservatives, pigments, carbohydrates, gums, polymers, and nanoparticles, or mixtures thereof.

[0140] In the preferred method of the present invention, the additional ingredient is a plasticizer. Preferably, the plasticizer is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oils, lecithin, waxes, and amino acids.

[0141] The preferred method of the present invention further comprises the step of changing the pH of the plant-based protein hydrogel slurry such that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

[0142] Preferably, the step of changing the pH of the plant-based protein hydrogel slurry is carried out after step (b). Optionally, the step of changing the pH of the plant-based protein hydrogel slurry is carried out in sequence with step (b).

[0143] During the adjustment of the pH of the plant-based protein hydrogel slurry, it is possible for the slurry to pass through the isoelectric point of the protein. Due to the lack of charge repulsion at the isoelectric point, the protein fragments dispersed in the plant-based protein hydrogel slurry can coagulate rapidly. To avoid this, a pH-adjusting substance can be used to quickly change the pH and thereby minimize the time the slurry spends at the isoelectric point. The isoelectric point of a specific plant-based protein can be determined using the method described in Helmick et al., Food Biophysics (2021) 16:474-483, or can be obtained in the literature such as Guldekin et al., Food Hydrocolloids (2023), 145:109029.

[0144] Thus, in a preferred method of the present invention, the step of changing the pH of the plant-based protein hydrogel slurry comprises adding a pH-adjusting substance to the plant-based protein hydrogel slurry. Preferably, the pH-adjusting substance is a solution containing monovalent metal ions, divalent metal ions or ammonium ions, preferably an alkaline aqueous solution containing monovalent metal ions, divalent metal ions or ammonium ions. More preferably, the pH-adjusting substance is an aqueous hydroxide solution, preferably sodium hydroxide, potassium hydroxide or ammonium hydroxide.

[0145] In a preferred method of the present invention, after the step of changing the pH of the plant-based protein hydrogel slurry, the pH of the plant-based protein hydrogel slurry is at least 1 pH unit lower than the isoelectric point of the plant-based protein.

[0146] In a preferred method of the present invention, after the step of changing the pH of the plant-based protein hydrogel slurry, the pH of the plant-based protein hydrogel slurry is at least 1 pH unit higher than the isoelectric point of the plant-based protein.

[0147] Those skilled in the art will understand that adding additional components to the plant-based protein hydrogel slurry in step (b) or between steps (b) and (c) or to the composition of step (c) may affect the pH of the slurry. Therefore, the step of changing the pH of the plant-based protein hydrogel slurry is preferably carried out after adding any additional components.

[0148] In a preferred method of the present invention, the composition formed in step (c) is a shear-thinning composition.

[0149] In a preferred method of the present invention, the composition formed in step (c) has a viscosity in the range of 1 to 10000 cP at 20 °C and 50 s -1 preferably in the range of 10 to 7500 cP at 20 °C and 50 s -1 preferably in the range of 15 to 5000 cP at 20 °C and 50 s -1 of the viscosity.

[0150] In a preferred method of the present invention, the composition formed in step (c) has a protein solids content in the range of 1 wt% to 25 wt%, preferably 2 wt% to 20 wt%, more preferably 4 wt% to 15 wt%, and even more preferably 5 wt% to 12 wt%, based on the total weight of the composition. The term "protein solids" refers to the dry dispersed 30 hydrogel solids derived from the plant protein added in step (a). Plant protein is typically a plant protein isolate, which mainly contains substances that can be chemically identified as proteins, but also contains low levels of other insoluble plant materials, such as fibers. The percentage of protein solids is measured according to the method described herein.

[0151] In a preferred method of the present invention, the active ingredient is selected from vitamins, minerals, flavors, fragrances, pro-flavours, pro-fragrances, flavor enhancers, malodour counteractants, nutraceuticals, live organisms (e.g., probiotics), pharmaceuticals, antimicrobials, antivirals, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments and adhesives, or combinations thereof.

[0152] In a particularly preferred method of the present invention, the active ingredient is at least one aroma substance or flavor substance. Preferably, at least one aroma substance or flavor substance is selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds and Schiff bases.

[0153] The aroma substances and flavor substances used in the present invention can be of natural origin (i.e., they are extracted from natural sources and are not synthetically modified in any way). Preferred aroma substances or flavor substances of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, citrus oil, orange flower extract, cedarwood, vetiver, lavender, ylang-ylang extract, tuberose extract, sandalwood oil, bergamot oil, rosemary oil, spearmint oil, peppermint oil, lemon oil, lavender oil, citronella oil, chamomile oil, clove oil, sage oil, neroli oil, labdanum oil, eucalyptus oil, verbena oil, mimosa extract, narcissus extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil, or a combination thereof. Preferably, the aroma substances or flavor substances of natural origin are of plant origin. Aroma substances or flavor substances of natural origin may be used alone or in combination, or in combination with synthetic aroma substances.

[0154] In a preferred method of the present invention, at least one fragrance or flavoring substance has a vapor pressure of greater than or equal to 0.00001 Torr at 25 °C.

[0155] In a preferred method of the present invention, at least one fragrance or flavoring substance has a logP of greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0.

[0156] In a preferred method of the present invention, at least one fragrance or flavoring substance has at least two Hansen solubility parameters selected from the following: an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bond (δH) of 2.5 to 11.

[0157] In a preferred method of the present invention, at least one fragrance or flavoring substance is part of a fragrance or flavoring agent.

[0158] Preferably, the fragrance or flavoring agent contains at least 20 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0.

[0159] Preferably, the fragrance or flavoring agent contains at least 40 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0160] Preferably, the fragrance or flavoring agent contains at least 50 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0161] Preferably, the fragrance or flavoring agent contains at least 60 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, more preferably greater than 4.0.

[0162] Preferably, the fragrance or flavoring agent contains at least 10 wt%, preferably at least 30 wt%, more preferably at least 50 wt%, more preferably at least 70 wt% of a fragrance or flavoring substance of natural origin.

[0163] Preferably, the fragrance or flavorant contains at least 10 wt% of a fragrance or flavor substance which, as measured according to ISO-14851 Edition 2019, has the following biodegradation percentages based on O2 consumption after 28 days: 60 to 100%, more preferably 65%, even more preferably 70 to 100%, even more preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100%, based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand. Due to its low water solubility, the fragrance or flavorant can be added to the biodegradation test carried out on an inert support, according to the method in ISO 10634 2018 Edition: Water quality - Preparation and handling of organic compounds with low solubility in water, for subsequent evaluation of their biodegradability in an aqueous medium. In addition, any inhibitory effect of the fragrance or flavorant on microorganisms can be checked as specified in this method.

[0164] Preferably, the fragrance or flavorant contains at least 80 wt%, preferably at least 90 wt%, more preferably at least 95 wt% of a fragrance or flavor substance, based on the total weight of the fragrance or flavorant, which has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) less than 8, and hydrogen bond (δH) of 2.5 to 11.

[0165] Preferably, the fragrance or flavorant contains only low levels of substances having an alcohol functional group (e.g., a primary alcohol functional group). In a preferred method of the present invention, the fragrance or flavorant contains less than 40% wt, more preferably less than 20% wt of alcohol-containing substances, based on the total weight of the fragrance or flavorant. In a particularly preferred method of the present invention, the fragrance or flavorant does not contain alcohol-containing substances. Without wishing to be bound by theory, it is believed that alcohols, especially primary alcohols having a straight-chain alkyl group, can easily diffuse through the shell of the microcapsule due to their structure, which means they may be difficult substances to encapsulate.

[0166] Preferably, the fragrance or flavor substance has a high odor impact. This is advantageous because it ensures that even low levels of the fragrance can be detected when released from the microcapsule.

[0167] In an alternative particularly preferred method of the present invention, the active ingredient is a vitamin or a mineral. Preferably, the active ingredient is a vitamin or a 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, linseed oil, ω3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus, or a mixture thereof. More preferably, the active ingredient is vitamin D.

[0168] In an alternative particularly preferred method of the present invention, additives may be included in the microcapsule core to act as active protectants, thereby reducing any potential degradation of the active ingredient during processing and storage. Preferably, the core additive is an antioxidant or a 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 having a mixture of antioxidants (such as polyphenols) and other compounds (such as olive oil and turmeric).

[0169] The vitamins and minerals employed in the present invention may be derived from vitamin- and / or mineral-containing materials. Examples of such materials are seaweeds.

[0170] In an alternative preferred method of the present invention, the active ingredient is an agrochemical, such as a pesticide, a herbicide, a fertilizer, a fungicide, an insecticide, an animal repellent or a combination thereof.

[0171] Preferably, the agrochemical is a natural alternative to synthetic materials and is based on plant extracts and / or plant essential oils (EO) or essential oil components, such as thymol. Preferably, the agrochemical is a biopesticide. Preferably, the agrochemical is suitable for use in formulations for plant care and production, which may be certified organic by organizations such as the USDA (United States Department of Agriculture) or Ecocert in Europe.

[0172] In a preferred method of the present invention, the active ingredient is part of a composition comprising an active ingredient and an active carrier phase. Preferably, the active carrier phase is a solvent, a fat or a wax.

[0173] In a preferred method of the present invention, the active carrier phase is a solvent. Preferably, the solvent is a solvent having low volatility (e.g., having a vapor pressure of less than 0.1 Torr at 25 °C, preferably having a vapor pressure of less than 0.01 Torr at 25 °C, preferably having a vapor pressure of less than 0.001 Torr at 25 °C).

[0174] Preferably, the solvent has a low odor or no odor.

[0175] Preferably, the solvent has at least two Hansen solubility parameters selected from the following: an atomic dispersion force (δD) of less than 20, a dipole moment (δP) of less than 8, and a hydrogen bond (δH) of less than 11. More preferably, the solvent has at least two Hansen solubility parameters selected from the following: an atomic dispersion force (δD) of less than 20, a dipole moment (δP) of less than 4, and a hydrogen bond (δH) of less than 5.

[0176] Preferably, the solvent has a density greater than 1.07 g / cm 3 . Solvents with this property can advantageously prevent the emulsion of the encapsulant from separating (e.g., in the final product formulation).

[0177] Preferably, the solvent contains only low levels of substances having an alcohol functional group (e.g., a primary alcohol functional group). In a preferred method of the present invention, the solvent contains less than 40% wt, more preferably less than 20% wt, of alcohol-containing substances based on the total weight of the solvent. In a particularly preferred method of the present invention, the solvent does not contain alcohol-containing substances.

[0178] In a preferred method of the present invention, the active carrier phase is a solvent selected from the following: carboxylic acid esters, fatty acid esters, phthalates, triols, diols, rosin resins, isoparaffins, terpenes, and vegetable oils, or combinations thereof.

[0179] Preferably, the solvent is selected from 840, 812N, 829, 829ECO, Coco 810, 810N, 128, 808, T-C7, 8810, PPG 810, OE, DO and 818, limonene, benzyl benzoate, diethyl phthalate, isopropyl myristate, triethyl citrate, dipropylene glycol and propylene glycol, glyceryl triacetate, glycerol, 1,3-propanediol, or combinations thereof, preferably 812N.

[0180] 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 given in the CTFACosmetic Ingredient Handbook, J.M. Nikitakis (ed.), 1st ed., The Cosmetic, Toiletryand Fragrance Association, Inc., Washington, 1988. Vegetable oils are oils from plant sources. Optionally, the solvent is derived from vegetable oils.

[0181] In a preferred method of the present invention, the active carrier phase is a fat or wax having a melting point below 60 °C, preferably below 45 °C, more preferably below 25 °C.

[0182] Preferably, the wax is selected from 100, 142 and 154 or a combination thereof, preferably 100.

[0183] Preferably, the vegetable oil, fat or wax is present in a weight ratio of plant protein to active carrier phase greater than 1:1, more preferably greater than 1.5:1, even more preferably greater than or equal to 2:1.

[0184] In the method of the present invention, the method for forming the composition in step (c) is not particularly limited. For example, step (c) may involve membrane emulsification, mechanical stirring (such as low or high shear mechanical stirring), sonication, high shear mechanical stirring, high pressure homogenization, and / or cavitation.

[0185] 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.

[0186] In a preferred method of the present invention, the composition formed in step (c) contains droplets of the active ingredient or droplets of the composition containing the active ingredient and the active carrier phase, the droplets having a d of 0.5 to 50 microns, preferably 1 to 40 microns, more preferably 2 to 30 microns 50 , as determined by laser diffraction.

[0187] In a preferred method of the present invention, additives, preferably water-soluble additives, are added to the composition in step (b) or between steps (b) and (c) or in step (c).

[0188] Preferably, the additive is selected from plasticizers, surfactants, rheological modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers, and nanoparticles, or mixtures thereof.

[0189] In a preferred method of the present invention, the additive is a plasticizer. Preferably, the additive is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oils, lecithin, waxes, and amino acids.

[0190] In a preferred method of the present invention, the additive is a polysaccharide, preferably maltodextrin, starch, or modified starch, such as starch or octenyl succinic anhydride (OSA) - modified starch. The use of the polysaccharide additive allows control of the matrix composition of the resulting microcapsules. For example, the use of the polysaccharide means that the amount of plant - based protein can be controlled.

[0191] 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 finally dried microcapsules are capable of delivering the additive in addition to the encapsulated active ingredient during the final use, for example, when the plant - based protein hydrogel matrix is enzymatically degraded in the oral cavity or digestive tract of a human or animal.

[0192] In a preferred method of the present invention, the weight ratio of the plant - based protein to the composition comprising the active ingredient and the active carrier phase in the composition formed in step (c) is in the range of 20:1 to 1:20.

[0193] In a preferred method of the present invention, the composition formed in step (c) is a homogeneous composition.

[0194] In a preferred method of the present invention, the composition formed in step (c) is a two - phase system (e.g., an aqueous two - phase system).

[0195] In the method of the present invention, step (d) involves drying the composition formed in step (c) to form microcapsules or microcapsule aggregates or larger objects. Those skilled in the art will be familiar with drying techniques and know how to select the necessary conditions to obtain dried microcapsules or larger objects. Suitable drying techniques include spray drying, electrostatic spray drying, spray freeze drying, and spray vacuum drying as used herein, wherein the slurry is sprayed into a chamber under reduced pressure so that the solvent can be removed at a lower temperature, thereby avoiding problems of thermal degradation. Advantageously, in the method of the present invention, step (d) can be carried out at a relatively low temperature (e.g., at a temperature below 100 °C). This helps to form microcapsules of small size and also prevents the loss or degradation of any volatile or heat-sensitive substances encapsulated. Advantageously, in the method of the present invention, step (d) can be carried out relatively quickly (e.g., with a residence time of a few seconds). This reduces the loss or degradation of any volatile or heat-sensitive substances encapsulated.

[0196] 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, thin 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 (e.g., fluidized bed drying). The typically extended drying time of fluidized bed drying generally allows the use of a lower drying temperature. Some designs of spray dryers incorporate this two-stage drying process as part of their design. When larger encapsulates are desired, some fluidized bed drying processes are preferred, which generally involve spraying or dispersing the composition of step (c) onto or into a fluidized bed of particles (e.g., encapsulates of the already dried composition (c)). The composition can be sprayed onto the fluidized bed or can be sprayed into the fluidized bed. Depending on the process conditions, the composition spray will agglomerate and / or coat (and thus accumulate) as it passes through the 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 readily define the conditions for controlling the size of the encapsulates in the fluidized bed drying process.

[0197] Other drying processes can be selected depending on the desired form of the dried composition in step (d). Thin film drying (wherein the composition is cast as a film onto a heated belt or drum) can be used to form an aesthetically pleasing shape containing the dried composition prepared in step (c). Belt drying (which can use conduction heating or infrared heating) can be used in combination with other drying techniques to complete the drying under controlled conditions.

[0198] During the drying process in step (d), the plant-based protein hydrogel slurry forms a plant-based protein carrier that encapsulates the active ingredient in the form of microcapsules. During the drying process in step (d), at least a portion of the first and second co-solvents is evaporated to enable the formation of microcapsules. For the two co-solvents, this evaporation does not occur at the same rate, such that during the process, the slurry generally becomes more concentrated in the least volatile co-solvent. The microcapsules formed during the drying process may still contain the first and second co-solvents trapped within the dried microcapsules, and their ratio may be different from their starting ratio in the mixture of step (a).

[0199] In a preferred method of the present invention, as measured according to ISO-14851 Edition 2019, the percentage of biodegradation of the plant-based protein carrier based on O2 consumption after 28 days is 60 to 100%, more preferably 65 to 90%, even more preferably 70 to 100%, even more preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand. ISO-14851 Edition 2019 describes a method that uses biological oxygen demand in a closed respirometer to determine the degree of biodegradation of a substance in a natural aqueous environment. This is achieved by exposing the substance in an aqueous standard test medium to an inoculum from non-previously exposed and unadapted activated sludge under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and if the percentage of its biodegradation at the end of the test is greater than 60%, the test is valid.

[0200] In a preferred method of the present invention, as measured according to ISO-14851 Edition 2019, the percentage of biodegradation of the microcapsules based on O2 consumption after 28 days is 60 to 100%, more preferably 65 to 90%, even more preferably 70 to 100%, even more preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand. ISO-14851 Edition 2019 describes a method that uses biological oxygen demand in a closed respirometer to determine the degree of biodegradation of a substance in a natural aqueous environment. This is achieved by exposing the substance in an aqueous standard test medium to an inoculum from non-previously exposed and unadapted activated sludge under laboratory conditions 15. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and if the percentage of its biodegradation at the end of the test is greater than 60%, the test is valid.

[0201] In a preferred method of the present invention, step (d) involves electrostatic spray drying.

[0202] In a preferred method of the present invention, the plant-based protein residues formed but not incorporated into the microcapsules in step (d) are recycled, preferably added back to step (a).

[0203] In a preferred method of the present invention, the microcapsules have a d of less than or equal to 250 μm, less than or equal to 200 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm 50 , as determined by laser diffraction.

[0204] In a preferred method of the present invention, the microcapsules have a diameter of less than or equal to 250 μm, less than or equal to 200 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm, as determined by optical microscopy.

[0205] The preferred method of the present invention further includes a post-treatment step for the microcapsules. Preferably, the post-treatment step includes a non-covalent crosslinking step, a covalent crosslinking step, or a coating formation step.

[0206] In a preferred method of the present invention, the post-treatment step includes a non-covalent crosslinking step. Preferably, the non-covalent crosslinking step includes treating the microcapsules with a non-covalent crosslinking agent selected from sodium tripolyphosphate (NaTPP), sodium hexametaphosphate, and phenolic compounds (such as tannic acid, caffeic acid, etc.).

[0207] In a preferred method of the present invention, the post-treatment step includes a covalent crosslinking step. Preferably, the covalent crosslinking step includes treating the microcapsules with a covalent crosslinking agent selected from genipin, epoxides, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and crosslinking resins, or combinations thereof.

[0208] In a preferred method of the present invention, the post-treatment step includes a coating formation step.

[0209] In a preferred method of the present invention, the coating formation step includes treating the microcapsules with a metal compound. Preferably, the metal compound is selected from silver compounds or gold compounds, preferably silver compounds.

[0210] In a preferred method of the present invention, the coating formation step includes a complex coacervation step for the microcapsules using a polysaccharide. Preferably, the polysaccharide is selected from xanthan gum, gellan gum, and chitosan, or combinations thereof.

[0211] In a preferred method of the present invention, the coating formation step includes treating the microcapsules with an aqueous solution of minerals to form a mineral coating. Preferably, the aqueous solution of minerals contains iron salts, calcium salts, phosphates, carbonates, titanium salts, or zinc salts, or a combination thereof.

[0212] In a preferred method of the present invention, the coating formation step includes 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 the most preferred. Those skilled in the art will understand that depending on which phase the silicon compound is present in, the silicon-based coating can be formed on the inner surface of the plant-based protein hydrogel (i.e., at the oil droplet interface) or on the outer surface of the plant-based protein hydrogel (i.e., forming a shell around the microcapsules). When coating the outer surface of the plant-based protein hydrogel, water-soluble silicate is most preferred. When formed on the inner surface of the plant-based protein hydrogel, organic silicate esters are most preferred.

[0213] Those skilled in the art will understand that multiple post-treatment steps can be carried out. For example, in a preferred method of the present invention, a non-covalent cross-linking step (e.g., using NaTPP) is performed on the microcapsules, 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, an electrostatic / non-covalent cross-linking step is performed on the microcapsules using sodium tripolyphosphate (NaTPP), followed by silica coating using poly-L-lysine as a deposition aid. In a preferred method of the present invention, a non-covalent cross-linking step is performed on the microcapsules using tannic acid, followed by a coacervate coating formation step using xanthan gum.

[0214] A preferred method of the present invention further includes adding a flowability aid to the microcapsules after step (d). Preferably, the flowability aid is fumed silica. Adding the flowability aid can prevent the newly formed microcapsules from caking.

[0215] A preferred method of the present invention includes further drying the microcapsules after step (d). Preferably, the further drying is selected from fluidized bed drying and / or tray drying.

[0216] A preferred method of the present invention further includes resuspending the microcapsules in an external phase, which is preferably an external aqueous phase, more preferably hard water or an acidic buffer solution.

[0217] A preferred method of the present invention further includes screening the microcapsules, for example, for size classification.

[0218] The present invention also provides biodegradable microcapsules obtained or obtainable by the above method.

[0219] The present invention also provides a method for preparing a biodegradable microcapsule composition, the method comprising:

[0220] (a) preparing biodegradable microcapsules according to the method described above; and

[0221] (b) suspending the biodegradable microcapsules in an external phase.

[0222] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0223] The preferred method of the present invention further comprises adding a suspending agent to the external phase.

[0224] Preferably, the suspending agent is selected from gum arabic, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil type 1, magnesium aluminum silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, 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 polyacrylate and cross-linked polyvinylpyrrolidone), and swelling clays (such as bentonite and laponite).

[0225] The present invention also provides a biodegradable microcapsule composition obtainable or capable of being obtained by the above method.

[0226] The present invention also provides a dried biodegradable microcapsule comprising an active ingredient and a plant-based protein carrier, the plant-based protein carrier comprising a plant-based protein, wherein the plant-based protein carrier encapsulates the active ingredient, and wherein when measured at 25 °C in an aqueous solution at pH 7 at a protein concentration of 5% w / w, the plant-based protein carrier has a solubility of less than 50%.

[0227] Protein solubility is determined using the following protocol: A known amount of spray-dried microcapsules is added to an aqueous solution, and then centrifuged to separate the soluble and insoluble fractions. After centrifugation, the liquid supernatant (the soluble fraction) is removed without obtaining any solid precipitating at the bottom (the insoluble fraction). The nitrogen content of the resulting supernatant is analyzed. Then, based on the nitrogen content, the protein content in the supernatant is calculated using a factor of 6.25. Protein solubility is defined as the amount of protein in the supernatant divided by the amount of protein in the entire aqueous solution.

[0228] In the preferred biodegradable microcapsules of the present invention, when measured at 25 °C in an aqueous solution at pH 7 at a protein concentration of 5% w / w, the plant-based protein carrier has a solubility of less than 30%, more preferably less than 10%.

[0229] In the preferred biodegradable microcapsules of the present invention, after incubation in water at 20 °C for 10 days, at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the initially encapsulated active ingredient remains within the microcapsules, as determined by high performance liquid chromatography (HPLC). In the alternative preferred spray-dried biodegradable microcapsules of the present invention, after incubation in water at 20 °C for 10 days, at least 25%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60% of the initially encapsulated active ingredient remains within the microcapsules, as determined by gas chromatography (GC).

[0230] More preferably, the water has been acidified such that it has a pH of less than 4.0, more preferably less than 3.0, more preferably less than 2.0, more preferably less than 1.0. Accordingly, the biodegradable microcapsules of the present invention are insoluble in water and are 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 prevail. For example, the biodegradable microcapsules can be added to beverage formulations that are typically acidic to provide the consumer with an active ingredient (such as a vitamin or mineral). The acid stability of the biodegradable microcapsules of the present invention also means that once ingested, the microcapsules can withstand the harsh acidic conditions of the stomach such that the active ingredient is released only when it reaches the small intestine and can be better absorbed. Accordingly, the biodegradable microcapsules of the present invention can provide controlled release of the encapsulated active ingredient. This is because the plant-based protein hydrogel matrix acts as an enteric coating.

[0231] In the preferred biodegradable microcapsules of the present invention, the plant-based protein is obtained from fava bean, mung bean, pea, rice, potato, rapeseed, lentil, chickpea, sunflower seed, pumpkin seed, flax, chia, canola, lupin, alfalfa, moringa, wheat, zein or sorghum; preferably, the plant protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein. More preferably, the plant-based protein is pea protein and / or potato protein. Such proteins are considered to be low allergenic proteins.

[0232] In the preferred biodegradable microcapsules of the present invention, the plant-based protein has been pretreated with an organic acid. Preferably, the organic acid is acetic acid, formic acid, propionic acid, α-hydroxy acid and / or β-hydroxy acid. Particularly preferably, the organic acid is acetic acid and / or lactic acid.

[0233] Preferred alpha-hydroxy acids include glycolic acid, lactic acid, acetic acid, malic acid, citric acid and / or tartaric acid, more preferably lactic acid or acetic acid. Preferred beta-hydroxy acids include beta-hydroxypropionic acid, beta-hydroxybutyric acid, beta-hydroxy beta-methylbutyric acid, 2-hydroxybenzoic acid and carnitine.

[0234] In the preferred biodegradable microcapsules of the present invention, the active ingredients are selected from vitamins, minerals, flavors, fragrances, flavor precursors, fragrance precursors, flavor enhancers, malodor counteractants, nutraceuticals, live organisms (e.g., probiotics), drugs, antimicrobial agents, antiviral agents, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin lighteners, emollients, skin moisturizers, wrinkle control agents, fabric softener actives, surface cleaning actives, skin conditioners, hair conditioners, dyes, pigments and binders, or combinations thereof.

[0235] In the specific biodegradable microcapsules of the present invention, the active ingredient is at least one aroma substance or flavor substance. Preferably, at least one aroma substance or flavor substance is selected from alcohols, aldehydes, ketones, esters, ethers, acetates, olefins, nitriles, nitrogen-containing heterocyclic compounds, sulfur-containing heterocyclic compounds and Schiff bases.

[0236] The aroma substances and flavor substances adopted by the present invention can be of natural origin (i.e. they are extracted from natural sources and are not synthetically modified in any way). Preferred aroma substances or flavor substances of natural origin include nutmeg extract, cardamom extract, ginger extract, cinnamon extract, patchouli oil, geranium oil, orange oil, citrus oil, orange flower extract, cedar, vetiver, eye-catching lavender, ylang-ylang extract, tuberose extract, sandalwood oil, bergamot oil, rosemary oil, spearmint oil, peppermint oil, lemon oil, lavender oil, citronella oil, chamomile oil, clove oil, sage oil, neroli oil, labdanum oil, eucalyptus oil, verbena oil, mimosa extract, narcissus extract, jasmine extract, frankincense extract, rose extract, vanillin, coffee extract, hop oil or its combination. Preferably, the aroma substances or flavor substances of natural origin are of plant origin. The aroma substances or flavor substances of natural origin can be used alone or in combination, or in combination with synthetic aroma substances.

[0237] In the preferred biodegradable microcapsules of the present invention, at least one aroma or flavor substance has a vapor pressure greater than or equal to 0.00001 Torr at 25°C.

[0238] In the preferred biodegradable microcapsules of the present invention, at least one aroma or flavor substance has a logP greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0.

[0239] In a preferred biodegradable microcapsule of the present invention, at least one fragrance or flavoring substance has at least two Hansen solubility parameters selected from the following: an atomic dispersion force (δD) of 14 to 20, a dipole moment (δP) of less than 8, and a hydrogen bond (δH) of 2.5 to 11.

[0240] In a preferred biodegradable microcapsule of the present invention, at least one fragrance or flavoring substance is part of a fragrance or flavoring agent.

[0241] Preferably, the fragrance or flavoring agent contains at least 20 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than or equal to 3.0, more preferably greater than or equal to 3.5, and even more preferably greater than or equal to 4.0.

[0242] Preferably, the fragrance or flavoring agent contains at least 40 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, and even more preferably greater than 4.0.

[0243] Preferably, the fragrance or flavoring agent contains at least 50 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, and even more preferably greater than 4.0.

[0244] Preferably, the fragrance or flavoring agent contains at least 60 wt% of a fragrance or flavoring substance based on the total weight of the fragrance or flavoring agent, and the fragrance or flavoring substance has a logP of greater than 3.0, more preferably greater than 3.5, and even more preferably greater than 4.0.

[0245] Preferably, the fragrance or flavoring agent contains at least 10 wt%, preferably at least 30 wt%, more preferably at least 50 wt%, and even more preferably at least 70 wt% of a fragrance or flavoring substance of natural origin.

[0246] Preferably, the fragrance or flavorant contains at least 10 wt% of a fragrance or flavor substance which, as measured according to ISO - 14851 Edition 2019, has the following biodegradation percentages based on O2 consumption after 28 days: 60 to 100%, more preferably 65%, even more preferably 70 to 100%, even more preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand. Due to its low water solubility, the fragrance or flavorant can be added to the biodegradation test carried out on an inert support according to the method in ISO 10634 2018 Edition: Water quality - Preparation and handling of organic compounds with poor water solubility to subsequently evaluate their biodegradability in an aqueous medium. Additionally, any inhibitory effect of the fragrance or flavorant on microorganisms can be checked as specified in this method.

[0247] Preferably, the fragrance or flavorant contains at least 80 wt%, preferably at least 90 wt%, more preferably at least 95 wt% of a fragrance or flavor substance based on the total weight of the fragrance or flavorant, and the fragrance or flavor substance has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) of 14 to 20, dipole moment (δP) less than 8, and hydrogen bond (δH) of 2.5 to 11.

[0248] Preferably, the fragrance or flavorant contains only low levels of substances having an alcohol functional group (e.g., a primary alcohol functional group). In the preferred biodegradable microcapsules of the present invention, the fragrance or flavorant contains less than 40 wt%, more preferably less than 20 wt% of alcohol-containing substances based on the total weight of the fragrance or flavorant. In the particularly preferred biodegradable microcapsules of the present invention, the fragrance or flavorant does not contain alcohol-containing substances.

[0249] Preferably, the fragrance or flavor substance has a high odor impact. This is advantageous as it ensures that even low levels of the fragrance can be detected when released from the microcapsules.

[0250] In an alternative particularly preferred biodegradable microcapsule of the present invention, the active ingredient is a vitamin or a mineral. Preferably, the active ingredient is a vitamin or a mineral selected from the following: 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, ω3 fatty acids, folic acid, thiamine, riboflavin, niacin, and phosphorus, or a mixture thereof. More preferably, the active ingredient is vitamin D.

[0251] In the optional particularly preferred biodegradable microcapsules of the present invention, additives can be included in the microcapsule core to act as active protectants, thereby reducing any potential degradation of the active ingredient during processing and storage. Preferably, the core additive is an antioxidant or a 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 having a mixture of antioxidants (such as polyphenols) and other compounds (such as olive oil and turmeric).

[0252] The vitamins and minerals employed in the present invention can be derived from vitamin- and / or mineral-containing materials. Examples of such materials are seaweeds.

[0253] In the preferred biodegradable microcapsules of the present invention, the active ingredient is part of a composition comprising the active ingredient and an active carrier phase. Preferably, the active carrier phase is a solvent, a fat, or a wax.

[0254] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent. Preferably, the solvent is a solvent having low volatility (e.g., having a vapor pressure of less than 0.1 Torr at 25 °C, preferably having a vapor pressure of less than 0.01 Torr at 25 °C, preferably having a vapor pressure of less than 0.001 Torr at 25 °C).

[0255] Preferably, the solvent has a low odor or no odor.

[0256] Preferably, the solvent has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) less than 20, dipole moment (δP) less than 8, and hydrogen bonding (δH) less than 11. More preferably, the solvent has at least two Hansen solubility parameters selected from the following: atomic dispersion force (δD) less than 20, dipole moment (δP) less than 4, and hydrogen bonding (δH) less than 5.

[0257] Preferably, the solvent has a density greater than 1.07 g / cm 3 . Solvents with this property can advantageously prevent the emulsion of the encapsulate from separating (e.g., in the final product formulation).

[0258] Preferably, the solvent contains only low levels of substances having an alcohol functional group (such as a primary alcohol functional group). In the preferred spray-dried biodegradable microcapsules of the present invention, the solvent contains less than 40% wt, more preferably less than 20% wt of alcohol-containing substances based on the total weight of the solvent. In the particularly preferred spray-dried biodegradable microcapsules of the present invention, the solvent does not contain alcohol-containing substances.

[0259] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a solvent selected from the group consisting of carboxylic acid esters, fatty acid esters, phthalic acid esters, triols, diols, rosin resins, isoparaffins, terpenes, and vegetable oils, or combinations thereof.

[0260] Preferably, the solvent is selected from 840, 812N, 829, 829ECO, Coco 810, 810N, 128, 808, T-C7, 8810, PPG 810, OE, DO and 818, limonene, benzyl benzoate, diethyl phthalate, isopropyl myristate, triethyl citrate, dipropylene glycol and propylene glycol, glyceryl triacetate, glycerol, 1,3-propanediol, or combinations thereof, preferably 812N.

[0261] 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 given in CTFA Cosmetic Ingredient Handbook, J.M. Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988. Optionally, the solvent is derived from a vegetable oil.

[0262] In the preferred biodegradable microcapsules of the present invention, the active carrier phase is a fat or wax having a melting point of less than 60°C, preferably less than 45°C, preferably less than 25°C.

[0263] Preferably, the wax is selected from 100, 142, 154, or combinations thereof, preferably 100.

[0264] In the preferred biodegradable microcapsules of the present invention, the plant-based protein carrier further comprises an additive, preferably a water-soluble additive.

[0265] Preferably, the additive is selected from plasticizers, surfactants, rheological modifiers, opacifiers, preservatives, pigments, carbohydrates, gums, polymers, and nanoparticles, or mixtures thereof.

[0266] In the preferred biodegradable microcapsules of the present invention, the additive is a plasticizer. Preferably, the additive is selected from glycerol, ethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, fatty acids, glucose, mannose, fructose, sucrose, lactic acid, citric acid, glycolic acid, malic acid, gluconic acid, tartaric acid, ethanolamine, urea, triethanolamine, vegetable oils, lecithin, waxes, and amino acids.

[0267] In the preferred biodegradable microcapsules of the present invention, the additive is a polysaccharide, preferably maltodextrin or OSA starch. The use of the polysaccharide additive allows control of the matrix composition of the resulting microcapsules. For example, the use of the polysaccharide means that the amount of plant-based protein can be controlled.

[0268] In the preferred biodegradable microcapsules of the present invention, the water-soluble additive is a water-soluble vitamin, preferably vitamin C. The presence of the 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 the final use process, for example, when the plant-based protein hydrogel matrix is enzymatically degraded in the oral cavity or digestive tract of a human or animal.

[0269] In the preferred biodegradable microcapsules of the present invention, the microcapsules have a d of less than or equal to 250 μm, less than or equal to 200 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm 50 , as determined by laser diffraction.

[0270] In the preferred biodegradable microcapsules of the present invention, the microcapsules have a diameter of less than or equal to 250 μm, less than or equal to 200 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm, as determined by optical microscopy.

[0271] In the preferred biodegradable microcapsules of the present invention, the plant-based protein has a protein secondary structure having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% intermolecular β-sheets, wherein the percentage content of intermolecular β-sheets is measured by FTIR.

[0272] The preferred biodegradable microcapsules of the present invention do not contain or substantially do not contain cross-linking agents.

[0273] In alternative preferred biodegradable microcapsules of the present invention, the plant-based protein carrier has been non-covalently modified by a non-covalent cross-linking agent. Preferably, the non-covalent cross-linking agent is selected from sodium tripolyphosphate, sodium hexametaphosphate, and phenolic compounds (such as tannic acid, caffeic acid, etc.).

[0274] In alternative preferred biodegradable microcapsules of the present invention, the plant-based protein carrier has been covalently modified by a covalent cross-linking agent. Preferably, the non-covalent cross-linking agent is selected from genipin, epoxides, glyceraldehyde, glutaraldehyde, formaldehyde, glyoxal, dialdehyde starch, microbial transglutaminase, and cross-linking resins, or combinations thereof.

[0275] In the preferred biodegradable microcapsules of the present invention, the plant-based protein carrier has a coating deposited thereon. Preferably, the coating is a metal coating, a coacervate coating, or a mineral coating.

[0276] In the preferred biodegradable microcapsules of the present invention, the metal coating is a silver coating or a gold coating, preferably a silver coating.

[0277] In the preferred biodegradable microcapsules of the present invention, the coating is a silicon-based coating formed from a silicon-containing compound. Preferably, the silicon-containing compound is selected from sodium metasilicate, tetraethyl orthosilicate, tetrapropyl orthosilicate, dimethyldiethoxysilane, and tetramethyl orthosilicate, or combinations thereof, preferably sodium metasilicate.

[0278] In the preferred biodegradable microcapsules of the present invention, the coacervate coating is formed from a polysaccharide. Preferably, the polysaccharide is selected from xanthan gum, gellan gum, and chitosan, or combinations thereof.

[0279] In the preferred biodegradable microcapsules of the present invention, the mineral coating is formed from an aqueous mineral solution. Preferably, the aqueous mineral solution contains an iron salt, a calcium salt, a phosphate, a carbonate, a titanium salt, or a zinc salt, or combinations thereof.

[0280] In the preferred biodegradable microcapsules of the present invention, the coating is a polymer coating, preferably formed from shellac.

[0281] The preferred biodegradable microcapsules of the present invention have a coating deposited on a covalently or non-covalently modified plant-based protein matrix. The preferred characteristics of the coating are as described above.

[0282] In particularly preferred biodegradable microcapsules of the present invention, the plant-based protein is pea protein and the active ingredient is vitamin D.

[0283] In the preferred biodegradable microcapsules of the present invention, as measured according to ISO-14851 Edition 2019, the biodegradation percentage of the plant-based protein carrier based on O2 consumption after 28 days is 60 to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, more preferably 65 to 100%, even more preferably 70 to 100%, even more preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100%. ISO-14851 Edition 2019 describes a method that uses biological oxygen demand in a closed respirometer to determine the degree of biodegradation of a substance in a natural aqueous environment. This is achieved by exposing the substance in an aqueous standard test medium to an inoculum from non-previously exposed and unadapted activated sludge under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and if its biodegradation percentage at the end of the test is greater than 60%, the test is valid.

[0284] In the preferred biodegradable microcapsules of the present invention, as measured according to ISO-14851 Edition 2019, the biodegradation percentage of the microcapsules based on O2 consumption after 28 days is 60 to 100% based on the ratio of biological oxygen demand (BOD) to theoretical oxygen demand, more preferably 65 to 100%, even more preferably 70 to 100%, even preferably 75 to 100%, even more preferably 80 to 100%, even more preferably 85 to 100%, and most preferably 90 to 100%. ISO-14851 Edition 2019 describes a method that uses biological oxygen demand in a closed respirometer to determine the degree of biodegradation of a substance in a natural aqueous environment. This is achieved by exposing the substance in an aqueous standard test medium to an inoculum from non-previously exposed and unadapted activated sludge under laboratory conditions. The measured value is calculated as a percentage of the theoretical oxygen demand calculated according to the molecular formula. An internal reference of microcrystalline cellulose is also tested, and if its biodegradation percentage at the end of the test is greater than 60%, the test is valid.

[0285] The present invention also provides a composition comprising the biodegradable microcapsules as described above and an external phase.

[0286] Preferably, the external phase is an external aqueous phase, preferably hard water or an acidic buffer solution.

[0287] The preferred composition of the present invention further comprises a suspending agent in the external phase.

[0288] Preferably, the suspending agent is selected from gum arabic, alginic acid, pectin, xanthan gum, gellan gum, carbomer, dextrin, gelatin, guar gum, hydrogenated vegetable oil type 1, magnesium aluminum silicate, maltodextrin, carboxymethyl cellulose, polymethacrylate, 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 polyacrylate, and cross-linked polyvinylpyrrolidone), and swelling clays (such as bentonite and laponite).

[0289] The present invention also provides a formulated product comprising the biodegradable microcapsules as described above.

[0290] The present invention also provides a method for preparing a formulated product, which includes:

[0291] (a) preparing the biodegradable microcapsules according to the method as described above; and

[0292] (b) mixing the biodegradable microcapsules with a product formulation.

[0293] Preferably, the formulated product is a food, beverage, cosmetic, household care product, personal care product, drug, industrial product (such as coatings, adhesives, sandpapers, tapes, etc.), medical device, biomaterial, or agrochemical.

[0294] The present invention also provides the use of the biodegradable microcapsules as described above in a formulated product.

[0295] The formulated products described in various aspects of the present invention can be in any suitable form for use. Preferably, the formulated product is in the form of a vapor spray, aerosol, emulsion, lotion, liquid, cream, gel, stick, ointment, paste, mousse, powder, granular product, matrix, or semi-solid. More preferably, the formulated product is a liquid, and even more preferably an aqueous liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0296] Figure 1a and 1b are 10X optical micrographs of the spray-dried microcapsules of Example 2B and 2E, respectively.

[0297] Figure 2a and 2b are 10X optical micrographs of the spray-dried microcapsules of Example 4A and 4B, respectively.

[0298] Figure 3 is a 10X optical micrograph of the control of Example 5 in water.

[0299] Figure 4a and 4bThey are SEM images of the microcapsules of Example 6A at magnifications of ×80 and ×300 respectively.

[0300] Figure 5a 、 5b and 5c are SEM images of the spray-dried microcapsules of Example 6B at magnifications of ×60, ×500 and ×1250 respectively.

[0301] Figure 6 is the SEM image of the spray-dried microcapsules of Example 7 at a magnification of ×1200.

[0302] Figure 7a and 7b are 10X optical microscope images of the microcapsules of Example 7 before ( Figure 7a ) and after ( Figure 7b ) extrusion to release the oil respectively.

[0303] Figure 8a 、 8b and 8c are 10X microscope images of the concentrated slurries of Example 15A, Example 15B and Comparative Example 15C.

[0304] Figure 9 is the 10X optical microscope image of the microcapsules of Example 20 in single-strength citrate buffer.

[0305] Figure 10a and 10b are photos of the inverted tubes of Example 21. Example

[0306] Material

[0307] Pea protein isolate (PPI) (80% protein, 4 wt% carbohydrate - ProEarth P16109) was purchased from Cambridge Commodities Ltd.

[0308] Lactic acid (85%, food grade) was purchased from Fisher Scientific.

[0309] Acetic acid (glacial acetic acid 99%) was purchased from Fisher Scientific.

[0310] Sodium benzoate was purchased from Fisher Scientific.

[0311] Vitamin D2 oil (1 MIU / g) was purchased from Prinova Europe, UK.

[0312] 812N was purchased from IOIOleochemical.

[0313] 100 was purchased from IOI Oleochemicals.

[0314] Vitamin D2 powder, Vitamin D2 100 CWS (100,000 IU / g), supplied by Prinova Europe, UK.

[0315] Prinova GA (gum arabic) Vitamin D2 capsule samples were supplied by Prinova Europe, UK.

[0316] Robinsons Double Strength No Added Sugar Squash Apple & Blackcurrant (produced by Britvic Plc and available commercially from supermarkets in the UK).

[0317] MiWadi single strength blackcurrant squash was produced by Britvic Plc and available commercially from supermarkets in the Republic of Ireland.

[0318] Thymol was purchased from Fisher Scientific, UK.

[0319] Calcium sulfate dihydrate (CaSO4·2H2O) was purchased from Fisher Scientific.

[0320] Dried magnesium sulfate (MgSO4) was purchased from Fisher Scientific.

[0321] Potassium chloride (extra pure, SLR, Eur.Ph., (KCl)) was purchased from Fisher Scientific.

[0322] Sodium bicarbonate, AR for analytical use, certified (NaHCO3) was purchased from Fisher Scientific.

[0323] Potassium hydroxide (KOH) (>85%) was purchased from Sigma Aldrich.

[0324] Absolute ethanol 99.8+%, (EtOH), AR for analytical use, certified, was purchased from Fisher Scientific.

[0325] Maltodextrin was purchased from Sigma - Aldrich Gillingham, UK.

[0326] Starch was supplied by Ingredion, Manchester, UK.

[0327] Polysorbate 80 was purchased from Sigma-Aldrich, Gillingham, UK.

[0328] All components of phosphate buffered saline (PBS) (pH 7.4) were purchased from Fisher Scientific, UK and prepared in the following ratio: 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4.

[0329] Sodium acetate trihydrate was purchased from Alfar Aesar, UK.

[0330] Calcium chloride dihydrate was purchased from Fisher Scientific, UK.

[0331] Methyl paraben was purchased from Alfar Aesar, UK.

[0332] Gellan gum (high acyl) was purchased from Special Ingredients Ltd, UK.

[0333] Flaxseed oil was purchased from Naissance, UK.

[0334] Shellac was purchased from A.F. Suter & Company Ltd, UK.

[0335] Tannic acid was purchased from Fisher Scientific, UK.

[0336] Xanthan gum was purchased from CP Kelco, UK.

[0337] Tetraethyl orthosilicate (TEOS) was purchased from Acros Organics, UK.

[0338] Olive oil was purchased from a supermarket in the UK.

[0339] Vitamin E in the form of DLα-tocopherol 97+% was purchased from Fischer Scientific, UK.

[0340] Curcumin in the form of 95% curcumin was purchased from Fisher Scientific.

[0341] β-carotene in the form of synthetic β-carotene ≥93% (UV) was purchased from Sigma Aldrich, USA.

[0342] Flavoring substances:

[0343]

[0344]

[0345] Composition of standard hard water and buffer solution:

[0346] 1) Hard water

[0347] This was prepared in deionized (DI) water with the following concentrations of salts: 192 mg / l of NaHCO3, 120 mg / l of CaSO4·2H2O, 120 mg / l of MgSO4, 8 mg / l of KCl. The pH was adjusted to 3 with 1M HCl.

[0348] 2) Single-strength buffer solution

[0349] 100 ml of single-strength buffer solution was prepared as follows:

[0350] i. Weigh 1.904 g of citric acid monohydrate and 0.276 g of trisodium citrate dihydrate;

[0351] ii. Make up the mixture to 80 g with DI water;

[0352] iii. Adjust the pH of the solution to 3 with 1M HCl and 1M NaOH solutions, and make up the volume of the solution to 100 g with DI water;

[0353] iv. Add 0.38 g of calcium chloride dihydrate; v. Add 0.2 g of sodium benzoate;

[0354] v. Place the solution in an ultrasonic bath at 80 °C for 5 - 10 min (i.e., until complete dissolution is observed);

[0355] vi. Cool the solution to 20 °C.

[0356] 3) Double-strength buffer solution

[0357] 100 ml of double-strength buffer solution was prepared in the same way as the single-strength buffer solution, but the levels of all substances were twice that, and the mixture was still made up to 80 g.

[0358] Measurement method

[0359] Analysis of vitamin D2 in dry powder samples of microcapsules: Version A

[0360] Vitamin D2 was extracted from the sample into dimethyl sulfoxide and then partitioned into hexane. Quantitative analysis of vitamin D2 was performed by normal-phase HPLC with UV detection. This method is applicable to the determination of high levels (usually 35 iu / g to 500,000 iu / g) of vitamin D2.

[0361] Analysis of vitamin D2 in dry powder samples of microcapsules: Version B

[0362] Suspend the powder sample (ca. 0.1 g) in 4 g of 50% w / v aqueous potassium hydroxide and 8 g of absolute ethanol. Add 0.1 g of sodium ascorbate. Place the sample in an ultrasonic bath for 1 to 1.5 h (without heating), cool on ice, then centrifuge at 14,500 rpm for 10 min. Subsequently, perform HPLC analysis on a reversed-phase column using 100% methanol as the eluent and conduct UV detection at 265 nm.

[0363] Analysis of vitamin D2 in diluted liquid systems containing microcapsules: Version A

[0364] Saponify the sample cold overnight with alcoholic potassium hydroxide solution (containing pyrogallol). Separate the unsaponified fraction by extraction into hexane. Then, concentrate the extracted sample and inject it onto a semi-preparative HPLC column. Collect the eluate fraction containing vitamin D2, evaporate to dryness, dissolve in methanol, and then separate vitamin D2 using reversed-phase HPLC with UV detection. This method is applicable for the determination of vitamin D2 at lower levels (usually 0.5 iu / g to 100 iu / g). Higher levels can be quantified by further dilution.

[0365] Analysis of vitamin D2 in diluted liquid systems containing microcapsules: Version B

[0366] Centrifuge the liquid sample (50 g) at 4500 rpm for 15 min to produce a precipitate. To measure the concentration of vitamin D2 in the supernatant, take approximately 5 g of the supernatant and add 2 g of 50% w / v aqueous potassium hydroxide, 3 g of absolute ethanol, and 0.05 g of sodium ascorbate, and mix the sample well. Place the sample in an ultrasonic bath for 1 to 1.5 h (without heating), but the final temperature may have risen to 50 - 60 °C. Examine the microcapsules using a microscope to check if they are completely ruptured. If not, subject the mixture to additional sonication. Then, cool the sample to room temperature using ice, and then take approximately 1 ml and centrifuge at 14,500 rpm for 15 min. Transfer the supernatant to an amber vial for HPLC analysis on a reversed-phase column using methanol / water (95:5) as the eluent and conduct UV detection at 265 nm. Report the vitamin D2 content per 100 g of the liquid.

[0367] To measure the concentration of vitamin D2 in the precipitate, carefully remove the supernatant using a pipette, leaving a small amount of supernatant to avoid losing any precipitate. This yields 1 to 2 g of wet precipitate, and follow the same procedure as described herein for 5 g of supernatant. This method is applicable for the determination of encapsulated vitamin D2 at lower levels (usually 0.5 iu / g to 100 iu / g). For the shelf-life study of encapsulated vitamin D2, only the precipitate values were reported as all vitamin D2 was found to be retained within the microcapsules. All vitamin D2 leaked from the microcapsules would be degraded.

[0368] Hydrogel slurry particle size

[0369] The particle size of the hydrogel was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. The measurement was carried out by diluting the hydrogel in an aqueous solution adjusted to the same pH with acetic acid or lactic acid within 1 h after completion of manufacturing. The test material was diluted to the required concentration to ensure the absence of aggregates and to have the optical density required for measurement (usually 5 - 15% obscuration). The cited d 50 refers to the volume distribution, as calculated by general analysis using Mie theory.

[0370] Droplet size of the composition for spray drying

[0371] The droplet size of the oil or wax containing the active ingredient in the composition for spray drying was measured using an Anton Paar laser diffraction particle size analyzer PSA 1190. The measurement was carried out by diluting the composition in an aqueous solution adjusted to the same pH with acetic acid or lactic acid within 1 h after completion of manufacturing. The test material was diluted to the required concentration to have the optical density required for measurement (usually 5 - 15% obscuration). The cited d 50 refers to the volume distribution, as calculated by general analysis using Mie theory.

[0372] Protein solid content

[0373] The solid content of the hydrogel slurry was measured by the mass remaining after 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 an oven at 40 °C and dried overnight. The dry mass was measured immediately after removing the dish from the oven, and the solid content of the protein hydrogel slurry was calculated as a percentage of the initial wet mass.

[0374] Viscosity of the composition for spray drying

[0375] Within 1 h after completion of the composition manufacturing, the viscosity of the composition for spray drying was measured using an Anton Paar MCR 92 rheometer. The rheometer was set 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.

[0376] Particle size of spray-dried microcapsules

[0377] 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 and diluted to the desired concentration to have the optical density required for measurement (usually 5 - 15% obscuration). The dispersion should be examined by optical microscopy to determine if there are any large microcapsule aggregates. If these microcapsule aggregates are visible, 0.5 wt% acetic acid can be added to ensure adequate dispersion of the primary particles. The cited d 50 refers to the volume distribution, as calculated by general analysis using Mie theory.

[0378] Optionally, the particle size can be measured using optical microscopy (e.g., using an open-frame microscope equipped with a CellCam200CR camera, an AuraPro phase-contrast illuminator, and 4×, 10×, and 20× universal plan fluorite objectives). The microcapsule powder was added to reverse osmosis water or single-strength buffer as needed. In this method, the particle size was measured based on the average size of 50 microcapsules for each corresponding sample. Then, the optical microscope was calibrated using the grid of a Hirschmann counting chamber (Fuchs Rosenthal). Using the line tool in ImageJ 1.53, the particle diameter was measured from two central edges, and the overlay text function was enabled to avoid double-counting capsules.

[0379] Scanning electron microscopy (SEM)

[0380] SEM images were obtained using a Hitachi TM3030.

[0381] Optical microscopy

[0382] Optical microscopy images were obtained using an open-frame microscope equipped with a CellCam 200CR camera, an Aura Pro phase-contrast illuminator, and 4×, 10×, and 20× universal plan fluorite objectives. First, the dry microcapsules were rehydrated in hard water with the following composition to prepare samples for optical microscopy:

[0383] <![CDATA[NaHCO3]]> <![CDATA[CaSO4.2H2O]]> <![CDATA[MgSO4]]> KCl mg / l 192 120 120 8

[0384] The microcapsules were left in the hard water or single-strength buffer for 5 minutes and then imaged microscopically. Then, a coverslip was placed on top of the rehydrated sample and an image was taken. Then, the slide was removed from the microscope stage and a strong manual pressure was applied to the coverslip, and then the sample was placed back on the stage and an image of the ruptured capsules was taken.

[0385] Total flavoring agent and flavor substance loading

[0386] The flavorants and flavor levels in the microcapsules can be determined by extracting the flavorants and injecting them into a GC column. Quantification is achieved by using a calibration curve of a given flavorant diluted in ethanol.

[0387] To measure the total level of flavorants in the encapsulate sample, it is first necessary to break the capsules by sonication. For example, 10 - 50 mg of dry encapsulate or 0.3 - 0.5 g of wet encapsulate is collected on a 40 μm cell filter in 3.0 g of deionized (DI) water and 10% KOH and vortexed until well mixed. Then, the solution is sonicated for 1 - 2 minutes with a Bandelin sonicator, small probe TS104, at an amplitude of 30% (total of approximately 1.5 - 3.0 kJ). During sonication, ice is used to keep the temperature below 20 °C to avoid any loss of flavorants by evaporation. The capsules are visually inspected using an optical microscope as detailed above to check if they are completely broken. If not, the sonication step is repeated. Ethanol is added to the mixture, which is then mixed and centrifuged. The supernatant is collected and retained as the first extract. The residue is mixed with ethanol and centrifuged. The supernatant is collected and added to the first extract. Then, the mixture is diluted as appropriate and injected into the GC for analysis.

[0388] For samples containing benzyl salicylate, it is quantified by measuring the benzyl alcohol formed by its hydrolysis.

[0389] Total thymol loading

[0390] Following the total flavorant loading method herein, the thymol level in the dry microcapsules is determined by extracting thymol and injecting it into a GC column.

[0391] Olfactory evaluation of microcapsule slurry

[0392] For any given encapsulate, the total flavorant level is quantified by GC as described herein. Based on the encapsulate loading, the material is diluted to deliver an appropriate level of flavorant for olfactory evaluation. Optionally, preservatives and suspending agents are added to the diluted encapsulate sample.

[0393] Using a low - shear micropipette tip, 50 μl of the diluted encapsulate is pipetted onto a standard microscope glass slide such that most of the surface area is covered and the edges are clear for easy handling of the slide. This operation is repeated at least four times.

[0394] Also, 50 μl of the diluted encapsulate slurry is pipetted onto a rectangular flavor evaluation card blotter (approx. 9 cm × 5 cm). This operation is repeated at least twice.

[0395] Trained evaluators evaluated the slides and blotting papers at different time points:

[0396] 1) Immediately after application;

[0397] 2) After being left overnight at 20 - 25 °C to dry;

[0398] 3) After being left overnight at 20 - 25 °C to dry and then rubbed 10 times (in either direction) on the glass slide with a gloved index finger;

[0399] 4) After being left overnight at 20 - 25 °C to dry and then pressed on the glass slide with another glass slide for 10 seconds and removed.

[0400] 5) After being left overnight at 20 - 25 °C to dry and then rubbed 10 times (in either direction) on the blotting paper with another clean blotting paper.

[0401] Evaluators graded the intensity according to the following scale and provided comments on the flavorant characteristics:

[0402] Odor intensity Odor intensity 5 Extremely strong 4 Strong 3 Medium 2 Weak 1 Extremely weak 0 Odorless

[0403] Moisture

[0404] The moisture content of the microcapsules was measured by weighing an appropriate mass of the dried microcapsules into an Ohaus MB23 moisture analyzer (Ohaus Europe GmbH, Switzerland) and evenly dispersing the sample throughout the pan. The sample was heated to 120 °C, and the moisture content was reported as weight %.

[0405] Microcapsule integrity

[0406] The physical integrity of the microcapsules was evaluated according to the following method. A microcapsule sample (0.1 g) was weighed into a 15 ml falcon tube. Hot deionized water (>70 °C) was added to the capsules to a total of 10 g, and manually shaken to redisperse the powder or agglomerates. Then, the sample was vortexed at 2000 rpm for 90 seconds to completely resuspend the microcapsules. The sample was examined by optical microscopy to check for uniform distribution, but if necessary, it could be vortexed for another 30 seconds. Then, the supernatant and precipitate were analyzed according to the vitamin D2 analysis method for the diluted liquid system containing microcapsules given herein: Version B. The integrity of the microcapsules was reported as the percentage of vitamin D2 leaked into the supernatant. For complete microcapsule integrity, this value was zero.

[0407] Example 1 - Preparation of protein hydrogel slurry with acetic acid

[0408] Preparation of protein hydrogel

[0409] Prepare 800 ml of a mixture consisting of 12.5% (w / v) pea protein isolate (PPI) in 30% (v / v) acetic acid solution.

[0410] Then, heat the mixture in an 85 °C water bath, followed by a sonication step to disrupt large colloidal aggregates (Hielscher UIP), after which a transparent solution is obtained. The energy applied is 200 kJ.

[0411] Then, pour the solution into a container to a depth of 1 cm and cool at 5 °C for 28 hours to obtain a self-standing protein hydrogel.

[0412] Applying shear to the protein hydrogel

[0413] Then, shear the hydrogel as follows. Cut the protein hydrogel into ~1 cm squares by a low shear cutting step. Place the squares into a 75 micron filter bag, then immerse it into a bucket containing 10 L of reverse osmosis water. This forms a crude protein hydrogel slurry inside the filter bag. Soak the hydrogel squares, stirring gently occasionally. Perform this step to reduce the acetic acid concentration in the hydrogel by diffusion into the continuous aqueous phase.

[0414] Transfer the filtered gel squares to a bottle with 4 wt% sodium benzoate added and expose to high shear, followed by exposure to ultra-high shear on ice (probe sonication using a Hielscher sonicator to 200 kJ) to form a homogeneous low-viscosity protein hydrogel slurry.

[0415] Example 2 - Preparation of spray-dried microcapsules containing vitamin D2

[0416] Preparation of wax / vitamin D2 mixture

[0417] Make a mixture of vitamin D2 oil (1 MIU / g) in 100 wax at 22 wt% (220,000 IU / g) and 11 wt% (110,000 IU / g). Melt the wax above 40 °C, add the vitamin oil, and manually agitate to make the mixture homogeneous. Keep the mixture above 40 °C until needed.

[0418] Example 2A: The ratio of acid-treated hydrogel slurry solids to wax is 5:1, adding vitamin D2 by sonication

[0419] Prepare 1500 g of protein hydrogel slurry according to Example 1, where the PPI and acetic acid mixture is heated for 20 minutes, and the gel squares are soaked for 2 hours, then sheared at 15,000 rpm high shear for 15 minutes, and then at 0.25 kJ / ml ultra-high shear to form the hydrogel slurry.

[0420] Preparation of the composition

[0421] Heat the protein hydrogel slurry to 35 °C and add 22 wt% wax / vitamin D2 molten mixture so that the weight ratio of the protein hydrogel slurry to wax / vitamin D2 reaches 98.3:1.7. Use an Ultra-Turrax disperser to perform mechanical high-shear on the resulting mixture at 15,000 rpm for 10 minutes, and then use a Hielscher sonicator to perform ultra-high shear on ice with an applied energy of 0.1 kJ / ml. This results in a composition having a viscosity of 27.3 cP and an average droplet size d 50 of the composition.

[0422] Spray drying of the composition

[0423] Spray-dry the composition according to the process settings in Table 1 to obtain microcapsules having an average particle size d 50 of 23.8 microns. Measure the vitamin D2 concentration in the microcapsule powder using Method Version A described herein and find it to be 100,250 μg / 100 g.

[0424] Example 2B: The ratio of acid-treated hydrogel slurry solids to wax is 2:1, and vitamin D2 is added by mechanical mixing

[0425] Prepare 1500 g of protein hydrogel according to Example 1, where the PPI and acetic acid mixture is heated for 20 minutes and the gel cubes are soaked for 2 hours, then high-sheared at 15,000 rpm for 15 minutes, and then ultra-high sheared at 0.25 kJ / ml to form a hydrogel slurry.

[0426] Preparation of the composition

[0427] Heat the protein hydrogel to 35 °C and add 11 wt% wax / vitamin D2 molten mixture so that the weight ratio of the protein hydrogel slurry to wax / vitamin D2 reaches 96:4. Use an Ultra-Turrax disperser to perform mechanical high-shear on the resulting mixture at 15,000 rpm for 10 minutes. This results in a composition having a viscosity of 31.4 cP and an average droplet size d 50 of 4.6 microns.

[0428] Spray drying of the composition

[0429] Spray-dry the slurry according to the process settings in Table 1 to obtain microcapsules having an average particle size d 50 of 35.9 microns. Measure the vitamin D2 concentration in the microcapsule powder using Method Version A described herein and find it to be 94,750 μg / 100 g.

[0430] Example 2C: The ratio of acid-treated hydrogel slurry solids to wax is 5:1, and vitamin D2 is added by mechanical mixing

[0431] Prepare 1500 g of protein hydrogel slurry according to Example 1, where the PPI and acetic acid mixture is heated for 20 minutes, and the gel cubes are soaked for 2 hours, then high-sheared at 15,000 rpm for 15 minutes, and then ultra-high-sheared at 0.25 kJ / ml to form a hydrogel slurry.

[0432] Preparation of the composition

[0433] Heat the protein hydrogel slurry to 35 °C and add a 22 wt% wax / vitamin D2 molten mixture such that the weight ratio of the protein hydrogel slurry to wax / vitamin D2 is 96:4. Mechanically high-shear the mixture at 15,000 rpm for 10 min using an Ultra-Turrax disperser. This results in a composition with a viscosity of 35.8 cP and an average droplet size d 50 of 6.2 microns.

[0434] Spray drying of the composition

[0435] Spray-dry the composition according to the process settings in Table 1. Using two different settings, samples Ci and Cii are obtained. The average particle size d of each of the Ci and Cii microcapsules is measured to be 31.3 microns 50 . Measure the vitamin D2 concentration in the Cii microcapsule powder using Method Version A described herein and find it to be 95,500 μg / 100 g.

[0436] Example 2D: The ratio of protein solids to wax in sonication-treated PPI (in 5 wt% acetic acid) is 5:1, and vitamin D2 is added by mechanical mixing

[0437] Preparation of PPI mixture

[0438] Prepare a mixture of 8 wt% pea protein isolate in a 5 wt% (v / v) acetic acid solution such that the solid concentration is equivalent to that of the protein hydrogel slurry prepared in Example 1. Ultra-high-shear the mixture using a Hielscher probe sonicator to an energy input of 1 kJ / ml. This results in a protein mixture with an average particle size d 50 of 2.9 microns. The pH of the protein mixture is measured to be 3.4.

[0439] Preparation of the composition

[0440] Heat the protein mixture to 35 °C and add a 22 wt% wax / vitamin D2 molten mixture such that the weight ratio of the protein mixture to wax / vitamin D2 is 98.3:1.7. Mechanically shear the mixture at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This results in a composition having a viscosity of 5.0 cP and an average droplet size d 50 of the composition.

[0441] Spray drying of the composition

[0442] Spray dry the composition according to the process settings in Table 1 to obtain microcapsules with an average particle size d 50 of 20.2 microns. Measure the vitamin D2 concentration in the microcapsule powder using Method Version A described herein and find it to be 86,500 μg / 100 g.

[0443] Comparative Example 2E: The ratio of protein solids to wax in sonicated PPI is 5:1, and vitamin D is added by mechanical mixing

[0444] Preparation of PPI mixture

[0445] Prepare a slurry of 8 wt% pea protein isolate in reverse osmosis water such that the solid concentration is comparable to that of the protein hydrogel slurry prepared in Example 1. Ultra-shear the mixture using a Hielscher probe sonicator to an energy input of 1 kJ / ml. This results in a protein mixture with an average particle size d 50 of 1.4 microns. The pH of the protein mixture is measured to be 6.85.

[0446] Preparation of the composition

[0447] Heat the protein mixture to 35 °C and add a 22 wt% wax / vitamin D2 molten mixture such that the weight ratio of the protein mixture to wax / vitamin D2 is 98.3:1.7. Mechanically shear the mixture at 15,000 rpm for 10 minutes using an Ultra-Turrax disperser. This results in a composition with a viscosity of 4.9 cP and an average droplet size d 50 of 4.6 microns.

[0448] Spray drying of the composition

[0449] Spray dry the composition according to the process settings in Table 1 to obtain microcapsules with an average particle size d 50 of 40.0 microns. Measure the vitamin D2 concentration in the microcapsule powder using Method Version A described herein and find it to be 93,000 μg / 100 g.

[0450] Spray drying conditions for Examples 2A to 2E

[0451] All the compositions prepared in Examples 2A to 2E were spray dried in a co-current spray dryer having a chamber with a 1 m diameter and a spray system with a FloMax nozzle. The inlet air flow rate 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.

[0452]

[0453] Table 1

[0454] The conditions in Table 1 show that, although the feed viscosities of Examples 2A, 2B, and 2C were higher compared to Examples 2D and 2E, the compositions were successfully spray dried using standard equipment and process conditions. This is considered to be due to the shear-thinning properties of the compositions of Examples 2A, 2B, and 2C.

[0455] All the compositions were successfully spray dried to produce free-flowing powders with an average particle size d 50 of 20 to 40 microns. Figure 1a and 1b are 10X optical micrographs of the spray-dried microcapsules of Examples 2B and 2E, respectively, showing the visual similarity of the resulting microcapsule powders.

[0456] Example 3 - Preparation of a protein hydrogel slurry with lactic acid

[0457] Preparation of protein hydrogel

[0458] 50.0 g of pea protein isolate was added to 212.5 g of reverse osmosis water in a bottle. The bottle was shaken to disperse the protein, and 187.5 ml of 85% (v / v) lactic acid was mixed in. The mixture was placed in an 85 °C water bath for 30 minutes and then sonicated with a Bandelin sonicator until 100 kJ was delivered. The hot protein solution was poured into a Petri dish and allowed to cool overnight at 5 °C to obtain a self-standing protein hydrogel.

[0459] Applying shear to the protein hydrogel

[0460] The hydrogel was cut into 1 cm squares and placed in a 75 micron nylon mesh bag in a bucket. 5 L of reverse osmosis water was added, and the squares were soaked for about 1.5 hours with occasional gentle stirring. Then, the water was drained from the bag and the same soaking procedure was repeated three more times until the pH was above 3.0.

[0461] Then, the hydrogel was drained and added to a 5 wt% sodium benzoate solution at 20 ml per kg of gel (pH was adjusted to 4 - 5 using lactic acid). The mixture was sonicated using a Bandelin sonicator until 0.1 kJ / ml was applied. The solid content of the hydrogel slurry was measured to be 5.1 wt% by the method described herein.

[0462] Example 4 - Preparation of Spray-Dried Microcapsules Containing Vitamin D2

[0463] Preparation of the composition

[0464] Vitamin D2 oil (1 MIU / g) was diluted in 812N or 100 wax to a concentration of 6000 μg / g. This concentration was chosen to produce spray-dried particles containing 1,000 μg / g of vitamin D2. In the case of 100 wax, the wax was melted above 40 °C, the vitamin oil was added, and the mixture was manually shaken to homogeneity. The mixture was kept above 40 °C until needed.

[0465] Then, the required mass of the oil or wax and vitamin D2 mixture was added to the protein hydrogel slurry prepared in Example 3 to achieve a mass ratio of hydrogel slurry solids to vitamin D2:wax / oil mixture of 5:1. The composition was formed by mixing at 5000 rpm for 15 minutes using an IKA Ultra-Turrax, followed by probe sonication with a Bandelin sonicator to 0.1 kJ / ml.

[0466] Spray drying conditions

[0467] Spray drying was carried out using a ProCept spray dryer in its three-column setup. This provided a column height of 1.8 m and a column diameter of 0.15 m. A two-fluid nozzle with a tip size of 0.8 mm was used. The atomizing air flow was 10 l / min. The fluid was pumped into the spray dryer at 6 ml / min using an injection pump. Table 2 below gives the other settings. An additional air flow of 100 l / min was set for the cyclone separator.

[0468]

[0469] Table 2

[0470] The results showed that protein hydrogel slurries formed using lactic acid could also be spray-dried using standard equipment and under standard conditions. The results also showed that a range of different carrier materials (e.g., oils and waxes) for the active ingredient (here vitamin D2) could be used.

[0471] The spray drying experiments produced two free-flowing powders. By laser diffraction measurement, the average particle size d of Example 4A 50 was 15.8 microns and the average particle size d of Example 4B 50 was 32.5 microns. The vitamin D2 concentration in the microcapsule powder was measured using Method Version A described herein, and the vitamin D2 concentrations in samples 4A and 4B were found to be 89,000 μg / 100 g and 86,500 μg / 100 g, respectively. Figure 2a and 2b are 10X optical microscope photographs of the spray-dried microcapsules of Example 4A and 4B, respectively, showing the visual similarity of the powders produced, in which the difference in particle size can be observed.

[0472] Example 5 - Accelerated Stability Testing of Vitamin D2 Microcapsules in Beverage Formulations

[0473] Preparation of the sample

[0474] The microcapsules of Example 4A and 4B were separately added to Robinsons Double Concentrate No Added Sugar Apple and Blackcurrant Juice Drinks. More specifically, 45 mg of microcapsules were added to 1 liter of juice drink to provide a vitamin D concentration in the product of 4.5 μg / 100 ml (assuming a vitamin D concentration in the microcapsules of 100,000 μg / 100 g). Comparative examples were also prepared by adding 9 mg of Prinova vitamin D2 powder to the same juice drink samples. Prinova vitamin D2 powder is a commercially available form of vitamin D2 powder. 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 simulate the temperature profile during flash pasteurization. The mixtures were then cooled to 20 °C.

[0475] Accelerated stability test

[0476] Two-hundred milliliter clear plastic PET bottles were filled with each of the three juice drink samples and placed in a Binder KBF P240-230V chamber that was set up for accelerated ambient light stability studies and whose standard configuration was with visible light and UV-A tubes. The nominal UV-A output was 1.1 W / m 2 . The temperature was set to 20 °C and laboratory humidity (usually 50% relative humidity (RH)) was used. The initial vitamin D2 load in the juice drinks was measured using Method Version A described herein and then measured again in the Binder chamber after 4 weeks. The percent reduction in vitamin D2 content over 4 weeks was calculated and recorded in Table 3. A significant degradation of vitamin D2 was expected upon exposure to UV within this time frame.

[0477]

[0478] Table 3

[0479] As can be seen from Table 3, after 4 weeks in fruit juice drinks, commercially available powdered vitamin D2 is almost completely degraded, with 95% of the initial vitamin D2 degraded.

[0480] Figure 3 Is a 10X microscopic image after adding Prinova vitamin D2 powder to cold tap water, showing that the material is finely dispersed and there are no microcapsules. Without wishing to be bound by theory, it is believed that the carrier material in this powder is highly soluble in fruit juice drinks, causing it to disintegrate in water and thus being unable to protect vitamin D2 in accelerated stability tests.

[0481] However, for Example 4A, after 4 weeks in fruit juice drinks, the vitamin D2 level is still approximately half of the original level, indicating that this microcapsule with a plant-based protein carrier and an oil-based active carrier phase protects vitamin D2 from exposure to UV, thereby reducing degradation. Compared with the commercially available materials tested in the comparative experiment, the presence of the insoluble plant-based protein carrier clearly contributes to protecting the encapsulated vitamin D2. In addition, for Example 4B, after 4 weeks in fruit juice drinks, the vitamin D2 level shows essentially no change compared to the original level, indicating that this microcapsule with a plant-based protein carrier and a wax-based active carrier phase can completely protect vitamin D2 from degradation. Without wishing to be bound by theory, it is believed that using a solid wax carrier for vitamin D2 means that the material around the vitamin is more opaque and thus provides better UV protection for the vitamin.

[0482] Example 6 - Preparation of Spray-Dried Flavor Microcapsules

[0483] Preparation of flavoring agent / solvent mixture

[0484] Prepare a mixture of δ-ionone and 812 in a weight ratio of 80:20 by gentle mixing.

[0485] Preparation of spray-dried microcapsules

[0486] The protein hydrogel slurry was prepared according to Example 1, where the mixture of PPI and acetic acid was heated for 30 minutes, the gel cubes were soaked for 1 hour, then high-sheared at 25,000 rpm for 15 min using an IKA Ultra-Turrax, and then ultra-high-sheared to 1.0 kJ / ml using sonication (Hielscher sonicator) to form the hydrogel slurry.

[0487] Measure the average particle size d of the hydrogel slurry according to the method described herein 50, and it was found to be 2.5 microns. The average solid content of the hydrogel slurry was measured according to the method described herein and found to be 8.6 wt%.

[0488] The desired 5 μm dispersed droplet size was achieved by pouring the flavorant / solvent mixture into the hydrogel slurry and mixing with an Ultra-Turrax at 15,000 rpm for 5 - 15 seconds, thereby preparing two samples having the

[0489] composition shown in Table 4.

[0490]

[0491] Table 4

[0492] Example 6A was prone to phase separation, resulting in a transparent liquid top layer and a hydrogel fragment bottom layer, but it could be made uniform again by vigorous mixing before spray drying. The well-mixed slurry had a viscosity of 150 cP at 50 / s.

[0493] 1.5 mg of the droplets of Example 6A were suspended on a fine wire connected to a microbalance and placed in the middle of a pipe with an inner diameter of 2.5 cm. Air at a temperature of 97 °C and a velocity of 0.85 m / s was passed around the droplets, and the weight of the droplets was recorded over 11 minutes. At the end of the experiment, the dried droplets were carefully separated from the wire and placed on a HitachiTM 3030 SEM mounting pad for examination.

[0494] As Figure 4a and 4b the SEM images show, the resulting particles had an internal matrix structure and an external smooth epidermis.

[0495] Example 6B was homogeneous, viscous, and semi-solid, with a viscosity of 1000 cP at 50 / s.

[0496] 2.5 mg of the droplets of Example 6B were suspended on a fine wire connected to a microbalance and placed in the middle of a pipe with an inner diameter of 2.5 cm. Air at a temperature of 85 °C and a velocity of 0.85 m / s was passed around the droplets, and the weight of the droplets was recorded over 3.5 minutes. At the end of the experiment, the dried droplets were carefully separated from the wire and placed on a HitachiTM 3030 SEM mounting pad for examination.

[0497] As Figure 5a , 5b and 5c of the SEM images show, the resulting particles had a matrix structure and a porous surface.

[0498] Example 7 - Preparation of Spray-Dried Flavorant Microcapsules

[0499] Preparation of flavoring agent / solvent mixture

[0500] Prepare a mixture of δ-damascone and 812 with a volume ratio of 80:20 by gentle mixing.

[0501] Preparation of spray-dried microcapsules

[0502] Prepare the protein hydrogel slurry according to Example 1. Dilute the hydrogel slurry with acetic acid to an average protein solid content of 8.0 wt%. Stir 221 g of the batch for 2 minutes at 8000 rpm using an UltraTurrax laboratory mixer. Then, add 131 g of the flavorant:oil mixture, which contains 80% δ-damascone and 20% 812 oil. Table 5 below gives the composition.

[0503]

[0504]

[0505] Table 5

[0506] Use an UltraTurrax laboratory mixer set at 8000 rpm to perform high-shear on the batch for 5 minutes to prepare a fine emulsion. The expected droplet size is about 5 microns.

[0507] Then, spray-dry the sample using a ProCepT R&D spray dryer in its three-column setup. This provides a column height of 1.8 m and a column diameter of 0.15 m. The air inlet temperature is 130 °C and the flow rate is 550 l / min. Use a two-fluid nozzle with a tip size of 1.0 mm. The atomizing air flow rate is 10 l / min. Use an injection pump to pump the fluid into the spray dryer at 6 ml / min. Introduce an additional air flow of 140 l / min below the drying chamber to transport the air to the cyclone separator.

[0508] Collect the dried microcapsule powder formed in Example 7 from the cyclone separator and also collect the dried microcapsule powder from the inner surface of the equipment to which it adheres (e.g., in the delivery pipe leading to the cyclone separator).

[0509] Examine the microcapsules of Example 7 under a Hitachi TM3030 SEM, and mainly observe spherical flavorant encapsulates with a diameter of about 30 μm or less ( Figure 6 ). The microcapsules show a pitted surface, indicating the presence of very little free flavorant and that the sample is reasonably dry.

[0510] Rehydrate the microcapsules of Example 7 in hard water and take optical micrographs ( Figure 7a)。 Then, squeeze the same capsule to rupture it to release the oil inside. Figure 7b )。 The oil droplets are clearly visible around the ruptured microcapsules, and there is a very distinct dark interface between the oil and water.

[0511] Analyze the microcapsules of Example 7 by the method described herein to determine its total flavorant loading. It was found to be 28.8%.

[0512] Example 8 - Preparation of Spray-Dried Flavorant Microcapsules

[0513] Preparation of flavoring agent / solvent mixture

[0514] Prepare a mixture of damascenone and 812 in a volume ratio of 80:20 by gentle mixing.

[0515] Preparation of spray-dried microcapsules

[0516] Prepare the protein hydrogel slurry according to Example 1. Dilute the hydrogel slurry with acetic acid to an average protein solids content of 8.0 wt%. Stir 221 g batches with an UltraTurrax laboratory mixer at 8000 rpm for 2 minutes. Then, add 33.1 g of the flavorant:oil mixture, the oil mixture containing 80% damascenone and 20% 812 oil. Table 6 below gives the composition.

[0517]

[0518] Table 6

[0519] Prepare the flavorant emulsion according to Table 6, using manual shaking instead of high-shear mixing to form the emulsion. The expected droplet size is about 20 microns.

[0520] Then, spray-dry the sample using a ProCepT R&D spray dryer in its three-column setup. This provides a column height of 1.8 m and a column diameter of 0.15 m. The air inlet temperature is 120 °C and the flow rate is 400 l / min. Use a two-fluid nozzle with a tip size of 1.0 mm. The atomizing air flow rate is 10 l / min. Use an injection pump to pump the fluid into the spray dryer in ml / min. Introduce an additional air flow of 1450 l / min below the drying chamber to convey the air to the cyclone separator.

[0521] Collect the formed dry microcapsule powder from the cyclone separator and also collect the dry microcapsule powder from its adhered inner surfaces of the equipment (e.g., in the delivery pipe leading to the cyclone separator).

[0522] The microcapsules of Example 8 were analyzed by the method described herein to determine their total flavorant loading. It was found to be 18.7%.

[0523] Example 9 - Preparation of Spray-Dried Flavorant Microcapsules

[0524] A flavorant composition as shown in Table 5 was prepared according to Example 7, but an emulsion was formed by manual shaking instead of high-shear mixing with an UltraTurrax. The expected droplet size was about 20 microns.

[0525] Then, the sample was spray-dried using a ProCepT R&D spray dryer in its three-column setup. This provided a column height of 1.8 m and a column diameter of 0.15 m. The air inlet temperature was 120 °C and the flow rate was 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. A syringe pump was used to pump the fluid into the spray dryer at 2 ml / min. An additional air flow of 150 l / min was introduced below the drying chamber to convey the air to the cyclone separator.

[0526] The formed dry microcapsule powder was collected from the cyclone separator and also from the inner surface of the equipment to which it adhered (e.g., in the transfer pipe leading to the cyclone separator).

[0527] The microcapsules of Example 9 were analyzed by the method described herein to determine their total flavorant loading. It was found to be 22.9%.

[0528] Example 10 - Coating Spray-Dried Flavorant Microcapsules with Shellac

[0529] In a 250 ml tall-form glass beaker, 30 g of a 25 wt% shellac solution was diluted in 120 ml of deionized water. Mechanical stirring was started at 350 rpm using an anchor stirrer at room temperature.

[0530] Additionally, hard water at pH 3 was added dropwise to 1.5 g of the dry powder from Example 8 in a 50 ml Falcon tube, with vortexing between drops to obtain a slurry paste. When the paste was homogeneous, more hard water at pH 3 was added, still using vortexing to ensure uniform dispersion of the particles. The microcapsule slurry was further adjusted to a 50% (v / v) slurry in hard water at pH 3.

[0531] The diluted microcapsules were added dropwise to the stirred shellac solution using a 3 ml pipette. The mixture was stirred for 30 minutes and then stored in the refrigerator for 3 days to allow the formation of the coating.

[0532] Then, the crosslinked microcapsules are subjected to a washing process, suspended in a separatory funnel, and sieved through a 250 μm sieve stacked with a 38 μm sieve to eliminate any aggregates that may have formed during the crosslinking step. The capsules collected on top of the 38 μm sieve are resuspended in hard water in a clean separatory funnel for an additional washing step. The decanted capsules are filtered through a 40 μm cell strainer for 5 minutes. Any excess moisture is blotted with a paper towel. The filtered capsules are diluted with single-strength buffer to prepare a 50 wt% slurry.

[0533] The coated microcapsules of Example 10 are analyzed by the method described herein to determine their total flavorant loading. It is found to be 0.8%.

[0534] Example 11 - Crosslinking Spray-Dried Flavorant Microcapsules with Tannic Acid and Complex Coacervation with Xanthan Gum

[0535] A 0.1 wt% xanthan gum solution is prepared in a water bath set at 60 °C with magnetic stirring until complete dissolution is observed. The solution is stored in the refrigerator overnight to allow the xanthan gum to hydrate fully. The next day, the xanthan gum solution is adjusted to pH 3 using 1 M HCl.

[0536] A 10 wt% tannic acid solution is prepared by manually shaking and vortexing for several seconds using a Fisherbrand TM ZX4 IR vortex mixer, and then immersing it in an ultrasonic bath set at 50 °C for 5 - 10 min until complete dissolution.

[0537] Additionally, hard water at pH 3 is added dropwise to 1.5 g of the dry powder from Example 8 in a 50 ml Falcon tube, vortexing between drops to obtain a slurry paste. When the paste is homogeneous, further hard water at pH 3 is added, still using vortexing to ensure uniform dispersion of the particles. The microcapsule slurry is further diluted in a total of 380 ml of hard water and adjusted to pH 3 using 1 M HCl.

[0538] While mechanically stirring at 170 rpm at 50 °C, 20.2 g of the tannic acid solution is added to the microcapsule suspension and stirred for 5.5 hours to allow crosslinking to occur. Thereafter, the suspension is stored in the refrigerator overnight. Then, the crosslinked microcapsules are subjected to the washing process as in Example 10, resulting in a brown encapsulation, indicating that crosslinking has occurred.

[0539] Then, the crosslinked microcapsules are filtered and suspended in 400 ml of hard water and adjusted to pH 3 using 1 M HCl. The suspension is mechanically stirred at 350 rpm at room temperature. While mixing, 545.7 g of the xanthan gum solution is slowly added and allowed to coacervate for 1.5 hours.

[0540] Then, the final microcapsules are subjected to the washing process outlined in Example 10.

[0541] The microcapsules of Example 11 were analyzed by the method described herein to determine their total fragrance load. It was found to be 1.5%.

[0542] Example 12 - Coating Spray-Dried Fragrance Microcapsules with Silica by Direct Immersion in TEOS

[0543] 300 mg of the dry powder from Example 9 was directly added to 2 g of TEOS and manually mixed to form a suspension. The microcapsule suspension was mixed at 200 rpm for 6 hours, ensuring that the capsules did not precipitate during the reaction. Then, the microcapsules were precipitated by centrifugation at 4500 rpm for 5 min and most of the supernatant was removed. The precipitated microcapsules were resuspended in the remaining TEOS and pipetted onto filter paper for air drying overnight to remove the excess TEOS.

[0544] The microcapsules of Example 12 were analyzed by the method described herein to confirm their total fragrance load. It was found to be 6.3%.

[0545] Example 13 - Preparation of a Protein Hydrogel Slurry with Acetic Acid

[0546] Preparation of protein hydrogel

[0547] 1120 g of reverse osmosis (RO) water was added to a 2-liter stainless-steel container and 216 g of pea protein isolate was added. The container was placed in a 92 °C water bath and mixed with a overhead stirrer at 1500 rpm. After stirring for 3 minutes, 480 g of glacial acetic acid was added. The mixture was stirred at 1500 rpm for 15 minutes and then at 1200 rpm for 30 minutes, ensuring that the temperature of the mixture exceeded 85 °C for at least 10 minutes. The mixture was poured into a tray to a depth of approximately 10 mm and left at room temperature overnight.

[0548] Applying shear to the protein hydrogel

[0549] Then, shear is applied to the hydrogel as follows. The protein hydrogel is cut into approximately 1 cm squares by a low-shear cutting step. The squares are divided into two 75-micron filter bags and then immersed separately into buckets containing 16 L of RO water. This forms a crude protein hydrogel slurry inside the filter bags. The hydrogel squares are soaked and stirred at 600 - 800 rpm for 90 - 150 min by a overhead stirrer. This step is carried out to reduce the acetic acid concentration in the hydrogel by diffusion into the continuous aqueous phase. Then, the pH of the wash water is measured and if it is higher than 3.2, soaking is continued for 30 minutes. If it is lower than 2.9, half of the water is drained and replaced with fresh RO water and then soaking is continued for 30 minutes. Then, the filter bags are hung above the buckets to drain for 5 minutes. The washed gels in the two filter bags are transferred to 5 L beakers and homogenized at 5000 rpm for 5 minutes, at 6000 rpm for 5 minutes, and at 7000 rpm for 5 minutes using a Silverson mixer. Then, the smooth slurry is transferred to 1 L Nalgene bottles (800 g each) and cooled on ice, exposed to high-shear ultrasonication (Hielscher UP500Hdt) until 250 kJ is applied, with oscillation every 75 kJ. Then, the hydrogel slurry is passed through a 200-micron sieve before use.

[0550] Comparative Example 14 - Preparation of protein slurry without using acid

[0551] 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 using an overhead stirrer at 1500 rpm. The mixture was stirred at 1500 rpm for 15 minutes and then at 1200 rpm for 30 minutes, ensuring that the temperature of the mixture exceeded 85 °C for at least 10 minutes. Contrary to the experiments using acetic acid, no gel was formed when the mixture was cooled to ambient temperature. Then, shear was applied at 8000 rpm for 10 minutes using a Silverson mixer and then shear was applied using an ultrasonic generator (Hielscher UP500Hdt) until 156 kJ was applied. For comparison, 0.3125 kJ / g was used for the protein slurry prepared with organic acid. Then, before use, the mixture was passed through a 200-micron sieve.

[0552] Example 15 - Preparation of spray-dried microcapsules containing vitamin D2

[0553] Preparation of the composition

[0554] Vitamin D2 oil (1 MIU / g) was in Dilute it to a concentration of 6000 μg / g in 100 wax. The purpose of selecting this concentration is to produce spray-dried particles containing vitamin D2 at a concentration of 1,000 μg / g. Melt the wax at a temperature above 40 °C, add the vitamin oil, and manually agitate to homogenize the mixture. Keep the mixture at a temperature above 40 °C until needed.

[0555] Then, add the required mass of the wax and vitamin D2 mixture to the protein hydrogel slurry of Example 13 and mix it with a Silverson L5M-A homogenizer at 7000 rpm for 10 minutes to form a composition having a total solids content (including vitamin D2 and wax) of 11.9 wt%. Then, further dilute this composition with an aqueous 3.0% acetic acid solution to produce two samples: Example 15A, which has a total solids content of vitamin D2 and wax of 10.0% (8.9 wt% protein solids); and Example 15B, which has a total solids content of vitamin D2 and wax of 5.0% (4.4 wt% protein solids).

[0556] Add the required mass of the wax and vitamin D2 mixture also to the protein slurry of Comparative Example 14 and mix it with a Silverson L5M-A homogenizer at 7000 rpm for 10 minutes to form a composition. Then, further dilute this composition with an aqueous 3.0% acetic acid solution to produce Comparative Example 15C, which has a total solids content of vitamin D2 and wax of 11.7 wt% (10 wt% protein solids).

[0557] Spray drying conditions

[0558] For Examples 15A and 15B, spray drying is carried out using a ProCept spray dryer in its three-column setting. This provides a column height of 1.8 m and a column diameter of 0.15 m.

[0559] For Example 15A, a two-fluid nozzle with a tip size of 1 mm is used. The atomizing air flow rate is 10 l / min. The fluid is pumped into the spray dryer at 6 ml / min using an injection pump. An additional air flow of 150 l / min is set for the cyclone separator.

[0560] For Example 15B, an ultrasonic nozzle is used. The atomizing air flow rate is 10 l / min. The fluid is pumped into the spray dryer at 6 ml / min using an injection pump. An additional air flow of 150 l / min is set for the cyclone separator.

[0561] For Comparative Example 15C, spray drying was carried out using a Buchi B290 spray dryer with a two-fluid nozzle having a tip size of 1.4 mm. The aspirator flow rate was set to 100%, and the Q-flow was set to 40. The fluid was pumped into the spray dryer using a peristaltic pump at a speed set to 35% (about 10 ml / min). The formed dry microcapsule powder was collected from the collection tank.

[0562] Other process settings are given in Table 7 below.

[0563]

[0564] Table 7

[0565] The spray drying experiments produced three free-flowing powders. For Examples 15A, 15B, and Comparative Example 15C, the average particle size d measured by laser diffraction according to the method therein 50 was 28.9 μm, 40.8 μm, and 9.1 μm, respectively. It should be noted that for the acid-treated plant protein encapsulates, the more dilute compositions dried with an ultrasonic nozzle produced larger microcapsules. For the non-acid-treated plant proteins with similar solid contents, the powders had much smaller average particle sizes.

[0566] The vitamin D2 concentration in the microcapsule powder was measured using Method Version B described herein, and the vitamin D2 concentrations of Examples 15A, 15B, and Comparative Example 15C were found to be 963 μg / g, 919 μg / g, and 1149 μg / g, respectively. This is close to the expected target of 1000 μg / g microcapsules, indicating minimal loss of vitamin D2 during the spray drying process.

[0567] Example 16 - Accelerated Stability Test of Vitamin D2 Microcapsules in Beverage Formulations

[0568] Preparation of beverage sample

[0569] The microcapsules of Examples 15A, 15B, and Comparative Example 15C were separately prepared into concentrated slurries so that they could subsequently be added to Miwadi blackcurrant single-strength fruit juice beverage.

[0570] First, 0.2 g of each microcapsule was suspended in 30 g of boiling water, shaken vigorously, and then vortexed at 2000 rpm for 1 minute to simulate the temperature profile during instant pasteurization. It should be noted that the concentrated slurry prepared with Comparative Example 15C was more turbid and appeared finer than those of Examples 15A and 15B. Figure 8a 、 8bAnd 8c are 10X microscope images of the concentrated slurries of Example 15A, 15B and Comparative Example 15C. Microcapsules of Example 15A and 15B can be seen and they clearly withstood the processing. For Comparative Example 15C, the image clearly lacks intact microcapsules, indicating that heating and shearing them resulted in rapid disintegration of the microcapsules. Instead, smaller protein aggregates and extremely small wax and vitamin D2 particles are seen.

[0571] Accelerated stability test

[0572] Each concentrated slurry of calculated mass was added to 2 liters of fruit juice beverage and mixed with a overhead mixer at 800 rpm for 5 minutes to ensure uniformity and then transferred to bottles.

[0573] 200 ml clear plastic PET bottles were filled with each of the three fruit juice beverage samples and the initial vitamin D2 load was measured using Method A described herein. Table 7 gives the initial beverage vitamin D load of Example 15A and 15B. However, in Comparative Example 15C, the vitamin D2 load of the initially measured beverage could not be detected. Without wishing to be bound by theory, it is believed that this is because in the concentrated slurry product, the microcapsules were suspended and heated and they were destroyed to release vitamin D2. The vitamin D2 oil floated to the top of the bottle and was not sampled.

[0574] Then, the filled PET bottles of Example 15A and 15B were placed in a Binder KBF P 240 - 230V chamber which was set up for accelerated ambient light stability studies and its standard configuration was with visible light and UV-A tubes. The nominal UV-A output was 1.1 W / m 2 ². The temperature was set at 20 °C and the laboratory humidity (usually 50% relative humidity (RH)) was used. After 4 weeks in the Binder chamber, the vitamin D load was measured again using Method A described herein. The % reduction in vitamin D2 content was calculated and recorded in Table 8.

[0575]

[0576] Table 8

[0577] After 4 weeks in a fruit juice drink, the vitamin D2 in the encapsulates of the present invention was protected from degradation. Thus, for Example 15A, only 22.5% of the initial vitamin D2 was lost, and for Example 15B, only 5.5% of the initial vitamin D2 was lost. Unencapsulated vitamin D2 was expected to degrade significantly when exposed to UV over this period, and this low level of loss demonstrates the protection provided by the microcapsules of the present invention. It can also be seen that the lowest losses were achieved with larger average encapsulate sizes. Without wishing to be bound by theory, it is believed that the larger the average particle size of the encapsulate, the smaller the surface area available per mass of vitamin D2 when exposed to UV, and thus the less degradation.

[0578] Example 17 - Encapsulation of Natural Agrochemical Active Substances

[0579] Example 17A: Thymol protein microcapsule preparation

[0580] Preparation of protein hydrogel and applying shear to it

[0581] Prepare and shear the protein hydrogel shell as described in Example 13.

[0582] The protein solids content was measured to be 9.3 wt.%. Dilute acetic acid (3 wt% in DI water) was added to reduce the protein solids content to 8.0%.

[0583] Preparation of spray-dried microcapsules

[0584] Homogenize the diluted dispersion with a Silverson L5M-A high-shear mixer (for 2 minutes at 8000 rpm). Add 16.81 g of thymol diluted to 40 wt% in Miglyol 812N to 103.25 g of the diluted dispersion. Further homogenize the mixture with the Silverson at 8000 RPM for 5 minutes. The expected droplet size was approximately 5 microns.

[0585] Then, spray-dry the sample using a Buchi B290 spray dryer. The air inlet temperature was 120 °C and the Q-flow was set to 40. A two-fluid nozzle with a tip size of 1.4 mm was used. The aspirator flow was set to 100%. A peristaltic pump was used to pump the fluid into the spray dryer at a speed setting of 13% (4 - 5 ml / min).

[0586] Collect the formed dry microcapsule powder from the collection tank. Analyze the microcapsules to determine their total thymol loading. The total thymol loading was found to be 4.9 wt%. The particle size was measured by laser diffraction and was found to have a d 50 .

[0587] Example 17B: Comparative maltodextrin thymol microcapsule preparation

[0588] Dissolve 45 g of maltodextrin in 105 g of deionized water and mix with a magnetic stir bar until a homogeneous slurry is formed. In addition to 0.14 g of polysorbate 80, 15.23 g of thymol (diluted at 40 wt% in Miglyol 812N) is added to the slurry. The mixture is homogenized for 5 minutes at 8000 rpm using a Silverson L5M-A high-shear mixer.

[0589] Then, the sample is spray-dried using a Buchi B290 spray dryer. The inlet air temperature is 130 °C and the Q-flow is set to 40. A two-fluid nozzle with a tip size of 1.4 mm is used. The aspirator flow is set to 100%. The fluid is pumped into the spray dryer using a peristaltic pump at a speed set to 13% (4 - 5 ml / min).

[0590] The formed dry microcapsule powder is collected from the collection tank. The microcapsules are analyzed to determine their total thymol loading. The total thymol loading is found to be 3.4 wt%. Since these microcapsules are too fragile, their particle size cannot be measured. When placed on a microscope slide with a coverslip, it can be observed that the weight of the coverslip is sufficient to break them.

[0591] Example 17C: Aqueous environment release test

[0592] According to Table 9, the thymol microcapsule powder of Example 12A is suspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben and in a solution of sodium acetate and calcium chloride such that the sample contains 4 mg of thymol. Duplicate samples are prepared so that the samples can be analyzed at incubation times t = 0, 24 hours, 72 hours, 1 week, and 2 weeks.

[0593]

[0594] Table 9 - Composition of the test samples

[0595] On day 0, the samples are mixed and placed in a 37 °C dark incubator, except for the t = 0 sample which is analyzed immediately. At each time point, the samples are centrifuged at 4900 RPM for 30 min and the supernatant is transferred to a 50 ml Falcon tube. Ethanol is added to achieve a ten-fold dilution of the supernatant sample. The concentration of thymol in the supernatant is measured by gas chromatography, which is used to calculate the amount of thymol released from the microcapsules.

[0596] Add 2.9 ml of DI water and 0.1 ml of 10% KOH solution to the pelletized material after centrifugation of each sample. Sonicate the mixture at 30% amplitude (Bandelin Sonopuls HD4200, with probe TS104) for 2 minutes, vortex, and centrifuge at 4900 rpm for 5 minutes. Dilute 100 μl of the supernatant of each sample tenfold with ethanol, and then measure the concentration of thymol by gas chromatography. This is used to calculate the amount of thymol remaining in the sample at the end of the experiment, which is regarded as the amount of thymol that was not initially released in the first supernatant. The results are shown in Table 10.

[0597]

[0598] Table 10

[0599] This release study shows that most of the active ingredient (more than 90%) remains encapsulated after 2 weeks. This slurry is suitable for storage before application to fields and crops.

[0600] Example 17D: Comparative maltodextrin - thymol release test

[0601] According to Table 11, suspend the thymol microcapsule powder of Comparative Example 17B in phosphate buffered saline (PBS) (pH 7.4) containing 0.2% w / v methylparaben such that the sample contains 4 mg of thymol, the same level as in Example 17A. In the absence of low levels of NaOAc and CaCl2, the suspension composition has only minor differences, but this is immaterial in this example.

[0602]

[0603] Table 11

[0604] Prepare the sample, mix, and incubate for one hour. Centrifuge the sample at 4900 RPM for 10 min, and transfer the supernatant to a 50 ml Falcon tube. Add ethanol to achieve a tenfold dilution of the supernatant sample. Measure the concentration of thymol in the supernatant by gas chromatography, which is used to calculate the amount of thymol released from the capsules in the buffer water.

[0605] Add 2.9 ml of deionized water and 0.1 ml of 10% KOH solution to the agglomerated material after centrifugation of the sample. Sonicate the mixture at 30% amplitude (Bandelin Sonopuls HD4200, with probe TS104) for 2 minutes, vortex and centrifuge at 4900 rpm for 5 minutes. Dilute 100 μl of the supernatant tenfold with ethanol, and then measure the concentration of thymol by gas chromatography. It is used to calculate the amount of thymol remaining in the sample at the end of the experiment, which is regarded as the amount of thymol that was not initially released in the first supernatant. The results are shown in Table 12.

[0606]

[0607] Table 12

[0608] This release study shows that for the maltodextrin microcapsules in Comparative Example 17B, a large amount of the active ingredient (more than 40%) is released in the aqueous buffer after 1 hour. This slurry is not suitable for storage before application to the fields and crops. Once prepared as an aqueous composition, or when water is present in the environment (such as rain), it will prematurely release the active ingredient, thus showing uncontrolled release characteristics.

[0609] Example 18 - Preparation of Spray-Dried Microcapsules Containing Flavoring Oils

[0610] Flavoring agent / solvent mixture

[0611] Prepare two different flavorings (18A and 18B) according to Table 13. Then, blend each flavoring with one of the two solvents at a flavoring:solvent weight ratio of 80:20: 812N or isopropyl myristate (IPM).

[0612] Flavoring 18A contains only medium-volatile flavor substances with similar volatilities, and the hydrophobicity of these substances ranges from very low to very high. Flavoring 18B contains substances with similar hydrophobicities, and these substances have a full range of volatilities, including low-volatile, medium-volatile, and high-volatile substances. Both flavorings have flavor substances with an HSP parameter range of δD from 15 to 20, δP from 1 to 7, and δH from 2.5 to 8.

[0613]

[0614] Table 13

[0615] Preparation of spray-dried microcapsules

[0616] Prepare the protein hydrogel slurry according to Example 13.

[0617] The first batch of flavorant compositions was prepared as follows. 100 to 120 g of a batch of the protein hydrogel slurry of Example 13 was diluted by 20% with 3% acetic acid solution to obtain a slurry with a protein solids content of 8.0 wt%. Then, in a 400 ml Nalgene beaker, the diluted slurry was stirred with a Silverson laboratory mixer at 8000 rpm for 2 minutes. Then, 31.5 wt% (based on the weight of the slurry) of flavorant 18B / 812N blend was added to the slurry, and further emulsified with a Silverson mixer at 8000 rpm for 5 min to form a fine emulsion. The fine emulsion was aged at room temperature for about 2 days and then spray-dried. This mixture was used for the second round of TTB.

[0618] The second batch of flavorant compositions was prepared as described above, except that the flavorant blend was emulsified by gentle manual shaking just prior to spray drying instead of using a Silverson mixer. This mixture was used for the third round of TTB.

[0619] The third batch of flavorant compositions was prepared according to the method of the first batch, except that isopropyl myristate (IPM) was used instead of 812N to form a flavorant:solvent blend. The flavorant and slurry mixture was further emulsified with a Silverson mixer at 8000 rpm for 5 min to form a fine emulsion. The fine emulsion was aged at room temperature for about 2 days and then spray-dried. This mixture was used for the sixth round of TTB.

[0620] The fourth batch of flavorant compositions was prepared using the same composition and in the same manner as the materials used in the sixth round of TTB, and was used for the seventh round of CPB and the eighth round of TTB.

[0621] The samples were spray-dried using a ProCepT R&D spray dryer in its three-column configuration. This provided a column height of 1.8 m and a column diameter of 0.15 m.

[0622] The inlet air temperature was varied to obtain the dried powder. It was kept as low as possible to reduce the loss of volatile flavor substances. The dry inlet air flow rate was kept constant at 0.4 m 3 / min. A two-fluid nozzle of the spray system with a tip size of 1.0 mm was used. The atomizing air flow rate varied between runs as shown in Table 14 below. For all runs, a syringe pump was used to pump the fluid into the spray dryer at 4 ml / min. An additional air flow of 150 l / min was introduced below the drying chamber to convey the air to the cyclone separator.

[0623] The formed dry microcapsule powder is collected in a collection tank and also from the inner surface of the equipment to which it adheres (e.g., in the delivery pipe leading to the cyclone separator). The particle size of the microcapsules is measured by laser diffraction.

[0624] As given in Table 14, flavorant 18B was successfully encapsulated.

[0625]

[0626]

[0627] Table 14

[0628] When drying at a lower temperature (inlet temperature about 69 °C), the maximum particle size was 30.1 microns, produced from a fresh coarse emulsion prepared with 812N as the solvent just prior to spray drying. Changing the flavorant solvent in the aged fine emulsion from 812N to isopropyl myristate had a minimal effect on the particle size.

[0629] Reducing the atomizing air flow produced larger particles (compare Run 8 TT with Run 7 CP).

[0630] The flavor composition of these microcapsules was analyzed using the flavorant loading quantification method by GC described herein. The results are given in Table 15.

[0631]

[0632] Table 15

[0633] Samples dried at a lower air inlet temperature generally had a higher total flavorant loading than those dried at a higher air inlet temperature.

[0634] When drying at a lower temperature (inlet temperature about 69 °C), for the fine emulsion, isopropyl myristate resulted in a higher total loading than 812N. Additionally, under these drying conditions, the coarse emulsion resulted in a higher loading.

[0635] When drying at a higher temperature (inlet at least 80 °C) and reducing the atomizing air flow, a higher total loading than with high air atomization was achieved.

[0636] Flavorant 18B shows that for flavor substances with similar hydrophobicity, substances with a range of volatilities can be encapsulated. However, as expected, the least volatile flavor substances are enriched in the encapsulate, where benzyl salicylate accounts for 48.4% to 56.3% of the flavorant, while it accounted for 20% of the flavorant before encapsulation. Drying at a lower temperature and using a coarse emulsion improves the encapsulation of more volatile flavor substances (such as limonene), from 0.6% of the flavorant to 1.5% of the flavorant.

[0637] As given in Table 16, flavorant composition 18A was also successfully encapsulated.

[0638] The first batch of flavorant composition was prepared as follows. 100 g to 120 g of a batch of the protein hydrogel slurry of Example 13 was diluted by 20 wt% with 3% acetic acid solution to obtain a slurry with a protein solid content of 8.0 wt%. Then, the diluted slurry was stirred in a 400 ml Nalgene beaker with a Silverson laboratory mixer at 8000 rpm for 2 minutes. Then, 28.3 wt% (based on the weight of the slurry) of the composition 18A / 812N blend was added to the slurry and further emulsified with a Silverson mixer at 8000 rpm for 5 min to form a fine emulsion. The fine emulsion was aged at room temperature for about 2 days and then spray-dried. This mixture was used for the 1st round of TTA.

[0639] The second batch of flavorant composition was prepared as described above. Immediately before use, 120 g of the emulsion was diluted with 60 g of 3% acetic acid solution and gently stirred. This mixture was used for the 9th round of TTA.

[0640] The third batch of flavorant composition was prepared in the same manner as the first batch above, except that the flavorant blend was sheared and emulsified immediately before spray-drying instead of aging it. This mixture was used for the 3rd round of TTA.

[0641] The fourth batch of flavorant composition was prepared as described above, except that the flavorant blend was emulsified by gently manually shaking immediately before spray-drying instead of using a Silverson mixer. This mixture was used for the 2nd round of TTA.

[0642] The fifth batch of flavorant composition was prepared in the same manner as the first batch, but now isopropyl myristate (IPM) was used instead of 812N. This mixture was used for the 4th round of TTA.

[0643] The sixth batch of flavorant composition was prepared in the same manner as the fifth batch, and this mixture was used for the 5th round of CPA.

[0644] Use the same equipment as in the previous run, where the dry air flow rate, emulsion flow rate, and transport air velocity are the same as those of flavor composition 18B. Again, collect the formed dry microcapsule powder in the collection tank and also collect the dry microcapsule powder from the inner surface of the equipment to which it adheres (e.g., in the delivery pipe leading to the cyclone separator). Measure the particle size of the microcapsules by laser diffraction.

[0645]

[0646] Table 16

[0647] Using Fresh crude emulsion prepared with 812N as the flavor solvent produced the largest particle size.

[0648] Using The aged fine emulsion using 812N as the flavor solvent produced the smallest microcapsule particle size. Dilution of the aged fine emulsion and operation at lower drying temperatures did not affect the particle size. Changing the flavor solvent in the aged fine emulsion from 812N to isopropyl myristate had a minimal effect on the particle size.

[0649] The aged fine emulsion spray-dried after aging had a smaller particle size than the same freshly prepared emulsion.

[0650] Analyze the flavor composition of these microcapsules using the flavor loading quantification method by GC described herein. Table 17 gives the results.

[0651]

[0652]

[0653] Table 17

[0654] All samples had similar total flavor loadings, but the aged fine emulsion sample with IPM sprayed at a lower air atomization rate had the highest loading, at 30%. This is significantly lower than flavor composition 18B, which had a maximum total flavor loading of 51%. Without wishing to be bound by theory, this may be due to the presence of flavor substances with very low logP in flavor 18A.

[0655] Flavor composition 18A contains 4 flavor substances with similar medium volatilities and different hydrophobicities and shows the ability to encapsulate substances with a range of hydrophobicities. For all examples, the composition of the encapsulated flavor is richer in the most hydrophobic flavor substance, dodecanenitrile. This is least evident in the crude emulsion samples.

[0656] Although δ-damascenone has a higher logP than geraniol and methyl-decenol, it is the least enriched fragrance substance. Both of these alcohols are well encapsulated, especially geraniol, indicating that it is still possible to encapsulate these relatively hydrophilic fragrance substances within the fragrance agent.

[0657] Olfactory evaluation of spray-dried microcapsules

[0658] Prepare a concentrated slurry of the microcapsules of the 3rd round of TTA, and then prepare a dilution preparation for olfactory evaluation. Dropwise add some single-strength buffer solution with preservatives to the dry microcapsules in a 50 ml Falcon tube, vortexing at 2000 rpm between drops to obtain a slurry paste. When the paste appears to be homogeneous, add more buffer solution for further dilution, still using vortexing as a means to ensure the uniform dispersion of the microcapsules. Let the suspension stand for 1 h to allow for possible swelling of the microcapsules, and then filter through a 40 μm cell strainer for 5 minutes. Absorb any excess moisture with a paper towel.

[0659] The total fragrance agent level by GC analysis according to the method herein is 12.6 wt%. This is lower than the total fragrance agent level of the spray-dried microcapsules. Without wishing to be bound by theory, it is believed that since the hydrogel shell has hydrated and swollen, this has led to an increase in the mass of the shell. In addition, a small amount of surface fragrance agent may have been lost to the aqueous phase.

[0660] Prepare a dilution slurry of the microcapsules containing gellan gum to contain 0.1 wt% total fragrance agent for olfactory evaluation. Gellan gum helps to keep the microcapsules well suspended. Prepare a gellan gum solution by mixing 9.5 mg of gellan gum with 15 g of deionized water in a 50 ml Falcon tube. Heat the mixture to above 90 °C for 20 minutes and stir at 500 rpm using an Eppendorf Thermomixer C set at 100 °C. After cooling to room temperature, add 15 g of double-strength buffer solution. Using a micropipette with a low-shear tip, add the concentrated encapsulation slurry to the gellan gum solution, close the sample tube and invert repeatedly to mix.

[0661] The pH of the dilution slurry is 3.05. Evaluate the odor of the dilution slurry according to the method described herein, and the results are shown in Table 18.

[0662]

[0663] Table 18

[0664] Upon initial application, there is a strong characteristic odor of the fragrance, indicating that some of the fragrance oil associates with the surface of the hydrogel shell. After drying for 24 hours, the odor is very faint, indicating that most of the surface fragrance has evaporated. When rubbed or pressed on glass, a strong characteristic odor is noted as the force ruptures the microcapsules and releases the fragrance. When rubbed on a card, the odor is of medium intensity and the fragrance characteristics are slightly different from those on glass, indicating only partial release of the encapsulated fragrance.

[0665] Example 19 - Crosslinking of Spray-Dried Fragrance Microcapsules

[0666] Prepare spray-dried microcapsules as shown in the 3rd round of TTA in Example 18.

[0667] Add hard water adjusted to pH 3 dropwise to 0.8 g of dried microcapsules in a 50 ml Falcon tube, vortexing at 2000 rpm between drops to obtain a slurry paste. When the paste appears to be homogeneous, add more hard water at pH 3 to further dilute, still using vortexing as a means to ensure uniform dispersion of the microcapsules. Dilute the suspended microcapsules further in a total of 390 ml of hard water and adjust the pH to 3.0 ± 0.5.

[0668] Prepare a 10 wt% tannic acid solution by manually shaking and vortexing for several seconds using a Fisherbrand TM ZX4 IR vortex mixer, then immersing it in an ultrasonic bath set at 50 °C for 5 - 10 min until completely dissolved.

[0669] With mechanical stirring at 170 rpm at 50 °C, add 10.6 g of tannic acid solution to the microcapsule suspension and stir for 5.5 hours. After that, store the suspension in the refrigerator for approximately 36 hours. Pour the crosslinked microcapsule suspension onto a 38 μm sieve, wash with hard water at pH 3, and resuspend in hard water at pH 3 in a clean separatory funnel. Collect the decanted capsules a second time on a 38 μm sieve, wash with a single-strength buffer solution with a preservative, and resuspend in this buffer solution in the separatory funnel. Collect the capsules as a concentrated slurry after decantation, and they appear as brown microcapsules, indicating that crosslinking has occurred.

[0670] Filter the crosslinked capsule slurry directly through a 40 μm cell filter for 5 minutes and absorb any excess moisture with a paper towel. Analyze the total fragrance level by gas chromatography by the method herein to be 4.1 wt%. This is lower than the total fragrance level of the microcapsules before crosslinking. Without wishing to be bound by theory, it is thought that this is due to the removal of surface and loosely associated fragrance during the crosslinking process through the washing procedure. Measure the particle size by laser diffraction to obtain a D of 47.9 μm 50This is larger than the particle size of the original spray-dried encapsulates, indicating that the hydrogel shell has hydrated and the encapsulates have swollen.

[0671] Olfactory evaluation of spray-dried and cross-linked microcapsules

[0672] Prepare a dilute slurry of microcapsules with gellan gum such that it contains 0.1 wt% total flavorant. Gellan gum helps keep the microcapsules well suspended. This is done according to the method of Example 18. The pH of the dilute slurry is 3.04. The odor of the dilute slurry is evaluated according to the method described herein, and the results are shown in Table 19.

[0673]

[0674] Table 19

[0675] At initial application, there is a moderate characteristic odor of the flavorant, indicating that a small amount of the flavorant is associated with the surface of the hydrogel shell. After 24 hours of drying, the odor is very faint, indicating that most of the surface flavorant has evaporated. When rubbed or pressed on glass, a strong characteristic odor is noted as the microcapsules rupture under the force, releasing the flavorant. When rubbed on a card, a moderate odor is noted and the flavor profile is similar to that on glass.

[0676] Example 20 - Preparation of Spray-Dried Microcapsules Containing Flaxseed Oil

[0677] Preparation of protein hydrogel and applying shear to it

[0678] Prepare a protein hydrogel slurry as described in Example 13. The protein solids content is measured to be 9.5 wt%.

[0679] Preparation of spray-dried microcapsules

[0680] Flaxseed oil is a dietary supplement used because of its high level of omega-3 fatty acids, which are also a vegetarian alternative to fish oil. The slurry is homogenized for 2 minutes at 8000 rpm using a Silverson L5M-A high shear mixer. Flaxseed oil is added to the slurry at an oil:protein solids weight ratio of 1:1, and the mixture is further homogenized for 5 minutes at 8000 RPM using a Silverson to produce a fine emulsion.

[0681] Using a two-fluid nozzle with a tip size of 1.4 mm, the slurry is spray-dried using a Buchi B290 spray dryer within a day of preparation. The aspirator flow is set to 100% and the Q-flow is set to 40. A peristaltic pump is used to pump the fluid into the spray dryer at a speed setting of 13% (4 - 5 ml / min). The air inlet temperature varies between 183 and 180 °C, and the air outlet temperature varies between 126 and 122 °C.

[0682] Dry microcapsule powder was formed, indicating that the oil was effectively encapsulated. The particle size was measured by laser diffraction according to the method of the present invention and found to have a d50 of 45.3 microns. The powder had no putrid odor, indicating that the temperature in the dryer did not degrade the flaxseed oil. It had a light nutty flavor and a very slight vinegar flavor, indicating that only low levels of surface oil were present.

[0683] Figure 9 A 10X optical microscope image of Example 20 suspended in single-strength citrate buffer is shown, where it can be seen that the encapsulation remains intact.

[0684] The microcapsule powder of Example 20 was also vortexed at 2000 rpm for 1 minute and suspended in Miwadi blackcurrant single-strength concentrate at a level of 12.5 g / L. The encapsulation did not impart any negative odor to the characteristic red fruit flavor of the original product, indicating that the encapsulation was insoluble and the flaxseed oil remained encapsulated, thus having a minimal impact on the product flavor. In contrast, the same level of flaxseed oil could not be incorporated into the beverage without an emulsifier and would impart a very distinct nutty odor and a characteristic effect on the beverage taste.

[0685] Example 21 - Demonstration of Protein Gel Formation

[0686] Reverse osmosis (RO) water and pea protein isolate were added to a 1 L stainless steel container and placed in a 92 °C water bath and mixed with an overhead stirrer at 1300 rpm. After stirring for 3 minutes, glacial acetic acid was added. The mixture was stirred at 1300 rpm for a total of 45 minutes, ensuring that the temperature remained above 80 °C. Then, the mixture was subjected to high shear for 10 minutes at 8000 rpm using a Silverson L5M-A homogenizer.

[0687] Twenty-milliliter aliquots of the hot liquid were pipetted into 50 ml Falcon tubes, sealed, and refrigerated overnight in the normal upright position. High levels of organic acids were tested, as given in Table 20a.

[0688] Five-gram aliquots of the hot liquid were weighed into 14 ml glass vials, sealed, and refrigerated overnight in the normal upright position. Lower levels of organic acids were tested, as given in Table 20b.

[0689]

[0690]

[0691] Table 20a

[0692] Example 21D 21E 21F 21G 21H Mass of glacial acetic acid (g) 0 5 10 15 25 Mass of pea protein isolate (g) 50 50 50 50 50 Mass of water (g) 450 445 440 435 425 wt% of acetic acid 0 1 2 3 5 Weight ratio of acetic acid to water 0 1:445 1:220 1:145 1:85 Weight ratio of acetic acid to protein solids 0 1:10 1:5 3:10 1:2 pH of the mixture after homogenization 6.21 4.16 3.87 3.66 3.50

[0693] Table 20b

[0694] After refrigeration for at least 12 hours, the formation of the gel was evaluated by inverting the vial.

[0695] As can be seen from Figure 10a it, the use of high levels of acid (30% and 10% glacial acetic acid) resulted in the formation of a solid gel that did not fall under gravity. No gel was formed without the use of acid and it fell into the lid of the inverted Falcon tube.

[0696] As can be seen from Figure 10b it, the use of 5%, 3% and 2% glacial acetic acid resulted in the formation of a solid gel that did not fall under gravity (within a 20-minute observation period). The use of 1% glacial acetic acid and no acid did not form a gel and it fell into the lid of the inverted vial.

[0697] The gel test results were correlated with the pH measurements of the hot slurry after homogenization. The mixtures that formed gels had a pH more than 0.5 units lower than the isoelectric point of pea protein isolate, which is 4.5 according to Guldekin et al., Food Hydrocolloids (2023), 145:109029.

[0698] Example 22 - Preparation of Protein Slurry

[0699] A: Preparation of protein slurry with acetic acid and sonication

[0700] 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 a overhead stirrer with a propeller impeller at 1700 rpm for about 1 to 2 minutes to ensure uniform wetting of the PPI powder. 269 g of an 80% (v / v) acetic acid solution was added to the stirred mixture, the lid was placed on the container and stirring was continued for an additional 45 minutes. The temperature of the mixture was checked. If the temperature was below 85 °C, stirring was continued until this temperature was reached.

[0701] Then, the hot mixture was transferred to a Silverson mixer and homogenized at 8000 rpm for 15 min. Then, the container was covered and cooled until the mixture temperature was below 40 °C, which may take several hours, usually overnight.

[0702] Once this temperature was reached, the mixture was gently stirred with an overhead mixer with an anchor impeller at 200 rpm for 15 min to make the mixture pourable and it was divided into smaller 1-litre batches in bottles.

[0703] Each bottle containing approximately 860 - 890 g of the mixture was sonicated using a Hielscher UIP500hdT in an ice bath to ensure that the mixture temperature remained below 40 °C. This operation was carried out at 100% amplitude over a period of 22 - 25 minutes until the total energy applied was 175 kJ. Sonication was stopped at the quarter and half points of the process and the bottles were manually shaken for one minute to ensure a homogeneous mixture. The mixture was then passed through a 200 - micron sieve to produce a uniform low - viscosity liquid. The viscosity was measured at 81.8 mPas at 50 s⁻¹, and the pH was 3.5.

[0704] B: Preparation of protein slurry with acetic acid and high-pressure homogenization

[0705] Follow the method of Example 22A until the mixture cools below 40 °C. Use a pressure - cell homogenizer SPCH - EP model FPG12805 (Homogenising System Ltd) equipped with a piston - gap valve (HPVS - 1) with a contact diameter of 1 mm to process the sample. The slurry was passed through twice at 100 MPa. Add 0.1 wt% methyl p - hydroxybenzoate preservative. At 50 s -1 the viscosity was measured at 390.8 mPas, and the pH was 3.6.

[0706] C: Comparative example: Preparation of protein slurry without using acid

[0707] Follow the method of Example 22B, with the variation that in the formulation, 2744.0 g of reverse - osmosis water was used and 256 g of reverse - osmosis water was added instead of acetic acid. At 50 s -1 the viscosity was measured at 188.3 mPas, and the pH was 6.5.

[0708] D: Comparative example: Preparation of protein slurry with HCl

[0709] Follow the method of Example 22B, with the variation that in the formulation, 2658.7 g of reverse - osmosis water was used and 341.3 g of 1M HCl was added instead of acetic acid. At 50 s -1 the viscosity was measured at 204.8 mPas, and the pH was 3.1.

[0710] Example 23 - Preparation of spray - dried microcapsules containing vitamin D2 with a modified slurry

[0711] Preparation of protein slurry and applying shear to it

[0712] Prepare the protein slurry as described in Example 22B and Comparative Examples 22C, 22D. The protein solid content was calculated to be 10.07% wt in all cases.

[0713] Preparation of spray-dried microcapsules

[0714] Dilute vitamin D2 oil (1 MIU / g) in the carrier to a concentration of 6000 μg / g in 100 wax. Melt the wax above 40 °C, add the vitamin D2 oil, and homogenize the mixture by manual shaking. Keep the mixture above 40 °C until needed.

[0715] Then, add the required mass of vitamin D2 and wax to each of the protein slurries 22B, C, and D at a protein:carrier solid weight ratio of 5:1. Then, mix it with a Silverson L5M-A homogenizer at 8000 rpm for 10 minutes to form emulsions of mixtures 23B, C, and D, which have a total solids content (including vitamin D2 and wax) of 11.5 wt%.

[0716] Use a Buchi B290 spray dryer to spray-dry each emulsion within one day of preparation using a two-fluid nozzle (with a tip size of 1.4 mm). The aspirator flow rate is set to 100%, and the Q-flow rate is set to 40. The air inlet temperature is maintained at 180 °C. Use a peristaltic pump to pump the fluid into the spray dryer at a speed set to 6.7 - 23 g / min to bring the air outlet temperature to 85 to 120 °C.

[0717] Table 21 gives the process conditions and results.

[0718]

[0719] Table 21

[0720] Dry microcapsule powders are formed, indicating that the wax and vitamin D are encapsulated. The moisture content, particle size determined by laser diffraction, and vitamin D2 content (method version B) are all measured using the methods described herein.

[0721] All three examples have very similar vitamin D2 contents. The large differences in the operating rate and outlet temperature between Examples 23B and 23C and 23D are due to differences in viscosity, as the emulsion samples without added organic acid have lower viscosities than those prepared using organic acids. This is driven by the fact that they are prepared from protein slurries with very different viscosities.

[0722] Example 24 - Preparation of spray-dried microcapsules containing vitamin D2 using different carriers Preparation of protein slurry And applying shear to it

[0723] Prepare a protein hydrogel slurry as described in Example 22A. The protein solid content is measured to be 11.6 wt%.

[0724] Preparation of spray-dried microcapsules

[0725] Dilute vitamin D2 oil (1 MIU / g) in a carrier 100 wax to a concentration of 6000 μg / g. Melt the wax above 40 °C, add the vitamin D2 oil, and homogenize the mixture by manual shaking. Keep the mixture above 40 °C until needed.

[0726] Additionally, dilute vitamin D2 oil (1 MIU / g) separately in the carrier olive oil to a concentration of 6000 μg / g, and homogenize the mixture by manual shaking. Keep the mixture at room temperature until needed.

[0727] Then, add the desired mass of vitamin D2 and the carrier to the protein hydrogel slurry of Example 22A at a protein:carrier solid weight ratio of 5:1 and 2:1. Then, mix it with a Silverson L5M-A homogenizer at 7000 rpm for 10 minutes to form an emulsion.

[0728] Using a rotary disk atomizer, spray-dry the slurry using a pilot-scale co-current spray dryer. The drying chamber has a diameter of approximately 1 m and a height of approximately 2.1 m. The nominal evaporation capacity is 3 kg H2O / hr at 150 °C, and the nominal gas flow rate is 125 m 3 / hr. Pump the fluid into the spray dryer using a peristaltic pump and adjust the flow rate to achieve the target outlet temperature. The air inlet temperature varies between 179 and 181 °C, and the air outlet temperature varies between 92 and 95 °C. The processing conditions and results are given in Table 22.

[0729]

[0730]

[0731] Table 22

[0732] Form dry microcapsule powder, indicating that vitamin D2 and the carrier are encapsulated. The moisture content, particle size determined by laser diffraction, and vitamin D2 content (method version B) are all measured using the methods described herein.

[0733] The vitamin D2 content of the microcapsule powder is higher than the target value of 1000 μg / g in all cases. In Examples 24B and C, the elevated levels may be due to uneven distribution of oil and protein between samples obtained from the collection container and smaller particles not recovered by the cyclone separator.

[0734] Example 25 - Preparation of Spray-Dried Microcapsules Containing Vitamin D2 and Core Additives Protein hydrogel Of preparation and applying shear to it

[0735] Prepare a protein hydrogel slurry as described in Example 22A. The protein solids content was measured to be 11.8 wt%.

[0736] Prepare a protein hydrogel slurry as described in Example 13. The protein solids content was measured to be 10.2 wt%.

[0737] Preparation of spray-dried microcapsules

[0738] Dilute vitamin D2 oil (1 MIU / g) in 100 wax to a concentration of 6000 μg / g. Melt the wax above 40 °C, add vitamin D2 oil, and homogenize the mixture by manual shaking. Add a solution of 10 mg / g curcumin in absolute ethanol to the mixture, where curcumin is 0.46% wt of the mass of vitamin D2 in the emulsion. Homogenize mixture 25A by manual shaking and keep it above 40 °C until needed.

[0739] Dilute vitamin D2 oil (1 MIU / g) in 100 wax to a concentration of 6000 μg / g. Melt the wax above 40 °C, add vitamin D2 oil, and homogenize the mixture by manual shaking. Add a solution of 10 mg / g curcumin in absolute ethanol to the mixture, where curcumin is 0.62% wt of the mass of vitamin D2 in the emulsion. Homogenize mixture 25B by manual shaking and keep it above 40 °C until needed.

[0740] Dilute vitamin D2 oil (1 MIU / g) in 100 wax to a concentration of 6000 μg / g. Melt the wax above 40 °C, add vitamin D2 oil, and then add vitamin E, where vitamin E is 10% wt of the mass of vitamin D2 in the emulsion. Homogenize this mixture 25C by manual shaking and keep it above 40 °C until needed.

[0741] Dilute vitamin D2 oil (1 MIU / g) in 100 wax to a concentration of 6000 μg / g. Melt the wax above 40 °C, add vitamin D2 oil, and homogenize the mixture by manual shaking. Add a solution of 100 mg / g β - carotene in absolute ethanol to the mixture, where β - carotene is 6.2% wt of the mass of vitamin D2 in the emulsion. Homogenize this mixture 25D by manual shaking and keep it above 40 °C until needed.

[0742] Then, mixtures 25A, C, and D of the desired quality were added separately to the protein hydrogel slurry of Example 22A, and mixture 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. Then, it was mixed with a Silverson L5M-A homogenizer at 7000 rpm for 10 minutes to form an emulsion, where the emulsion of mixtures 25A, C, and D had 13.8 wt% total solids (including vitamin D2, wax, and additives), and the emulsion of mixture 25B had 11.9 wt% total solids (including vitamin D2, wax, and additives).

[0743] Using a Buchi B290 spray dryer with a two-fluid nozzle (tip size 1.4 mm), the slurry was spray-dried within a day of preparation. First, the slurry was heated to 40 °C to 45 °C. The aspirator flow was set to 100%, and the Q-flow was set to 40. The fluid was pumped into the spray dryer using a peristaltic pump at a speed set to 35% (13 - 14 ml / min). The air inlet temperature varied between 181 and 179 °C, and the air outlet temperature varied between 127 and 111 °C. The processing conditions and results are given in Table 23.

[0744]

[0745] Table 23

[0746] Dry microcapsule powder was formed, indicating that vitamin D and the carrier were encapsulated. It should be noted that the additives slightly altered the overall buttery color of the powder. Examples 25A and B were light yellow, Example 25C was whiter, and Example 25D was light orange. The moisture content, particle size determined by laser diffraction, and vitamin D2 content (method version B) were all measured using the methods described herein.

[0747] Example 26 - Integrity of Spray-Dried Microcapsules Containing Vitamin D2

[0748] The integrity of the microcapsules was determined according to the methods described herein, and the results are given in Table 24. For each capsule, 1% of the dry powder was added to water to ensure that the vitamin D level was within the method detection limit.

[0749] Comparative tests were performed using commercially available polysaccharide vitamin D microcapsules Prinova GA. The vitamin D content in the powder was measured to be 2727.4 μg / g using Method B described herein, and the moisture content was measured to be 7.4 wt% using the methods described herein.

[0750]

[0751] Table 24

[0752] The Prinova GA microcapsules are highly soluble in water, so they do not form visible precipitates. Since they are highly soluble, they release most of the encapsulated vitamin D2 into the water. The leakage was measured to be 75%.

[0753] On the other hand, the inventive compositions of Examples 23B and 24C are highly insoluble in water and maintain their integrity. 0% of the encapsulated vitamin D2 leaks out.

[0754] Comparative Examples 23C and 23D have 15% and 8% leakage, indicating the importance of treating vegetable proteins with organic acids.

[0755]

[0756] Table 25

[0757] The inventive compositions of Examples 25A, B, C, and D maintain their integrity and have 0% leakage, indicating that the use of the active protectant has no adverse effect on the integrity of the microcapsules, as shown in Table 25.

[0758] Example 27 - Accelerated stability test of vitamin D2 microcapsules in beverage formulations

[0759] Preparation of beverage sample

[0760] The microcapsules of Examples 24A and B and Examples 25A, B, C, and D were separately prepared into concentrated slurries so that they could subsequently be added to MiWadi blackcurrant single strength fruit juice drink.

[0761] First, 0.2 g of each microcapsule was suspended in 30 g of boiling water, shaken vigorously, and then vortexed at 2000 rpm for 1 minute to simulate the temperature profile during flash pasteurization.

[0762] Accelerated stability test

[0763] The calculated mass of each concentrated slurry was added to 2 liters of fruit juice drink and mixed with an overhead mixer at 500 rpm for 5 minutes to ensure homogeneity, and then transferred to bottles.

[0764] Fill 200 ml clear plastic PET bottles with each of the three fruit juice beverage samples, and measure the initial vitamin D2 load using Method B in this article. Then, place the filled PET bottles in a Binder KBF LQC 720 chamber, which is set to operate using only visible light bulbs. The total light dose studied was determined by the expected visible light intensity in a typical supermarket (usually 600 lux), operating 24 hours a day for 6 months or 9 months. This corresponds to 2.6 MLUXh (representing 6 months on the shelf) or 3.9 MLUXh (representing 9 months on the shelf) at 35 °C and laboratory humidity (usually 50% relative humidity (RH)). Use the photometric function on the Binder KBF LQC 720 to achieve the required dose. After the equivalent of 6 months and 9 months on the shelf, measure the vitamin D load again using Method B described in this article. Calculate the percent reduction in vitamin D2 content and record it in Table 26 (for Examples 24A and B) and Table 27 (for Examples 25A, B, C, D).

[0765]

[0766] Table 26

[0767] The results in Table 26 show that after the equivalent of 9 months of shelf life in the beverage, the vitamin D2 in the encapsulates of the present invention was protected from degradation. Within this time frame, unencapsulated vitamin D2 was expected to degrade below the method detection limit when exposed to light. For Examples 24A and 24B with a protein to wax ratio greater than 1:1, the losses of the initial vitamin D2 were only 48% and 46% respectively. This low loss level demonstrates the protection provided by the microcapsules of the present invention during long-term storage.

[0768]

[0769] Table 27

[0770] The results in Table 27 show that after a shelf life equivalent to 9 months in the beverage, although the encapsulates of the present invention have a very small average particle size due to the spray dryer used, vitamin D2 in the encapsulates of the present invention is still protected from degradation. Within this time frame, unencapsulated vitamin D2 is expected to degrade below the method detection limit when exposed to light. Example 25B had the lowest vitamin D2 loss, at 41%, and without wishing to be bound by theory, this is thought to be due to the combination of the protective core additive of curcumin with a larger average particle size. Examples 25A, C, and D all had slightly higher losses than Example 25B, which is thought to be due to the very small average particle size. Nevertheless, the core additives provided additional protection, so their stability performance was consistent with that of Example 24A, which had a much larger average particle size. This low loss level demonstrates the protection provided by the microcapsules of the present invention during long-term storage. Without wishing to be bound by theory, it is thought that the larger the average particle size of the encapsulate, the lower the surface area available per mass of vitamin D2 when exposed to light, and thus the less degradation.

[0771] Example 28—Preparation of Spray-Dried Microcapsules Containing a Flavorant with a Shell Additive Flavoring agent / solvent Mixture

[0772] Flavorant 28A was prepared according to Table 28 and blended with 812N at a weight ratio of flavorant:solvent of 80:20. Flavorant 28A contains substances with similar hydrophobicity and a full range of volatilities, including low-volatility, medium-volatility, and high-volatility substances. The flavorant has flavor substances with HSP parameter ranges of δD from 15 to 20, δP from 1 to 7, and δH from 2.5 to 8.

[0773]

[0774] Table 28

[0775] Preparation of spray-dried microcapsules

[0776] The first batch of flavorant composition was prepared as follows. A batch of the protein hydrogel slurry of Example 13 was homogenized for 2 minutes at 8000 rpm using a Silverson laboratory mixer. Then, 5.1 wt% (based on the weight of the slurry) of the flavorant 28A / 812N blend was added to the slurry and emulsified for 2 minutes at 800 rpm using an overhead stirrer with a 4-blade impeller to form a coarse emulsion. This emulsion was used for Example 28B.

[0777] The second batch of the flavorant composition was prepared as follows. A batch of the protein hydrogel slurry of Example 13 was mixed with 2.0 wt% (based on the weight of the slurry) of glycerol. The mixture was homogenized for 2 minutes at 8000 rpm using a Silverson laboratory mixer. Then, 6.1 wt% (based on the weight of the slurry) of the flavorant 28A / 812N blend was added to the slurry and emulsified for 2 minutes at 800 rpm using an overhead stirrer with a 4 - blade impeller to form a coarse emulsion. This emulsion was used in Example 28C.

[0778] The third batch of the flavorant composition was prepared as follows. A batch of the protein hydrogel slurry of Example 13 was homogenized for 2 minutes at 8000 rpm using a Silverson laboratory mixer. Then, 4.9 wt% (based on the weight of the slurry) of the flavorant 28A / 812N blend was added to the slurry and further emulsified for 5 minutes at 8000 rpm using a Silverson mixer to form a fine emulsion. This mixture was used in Example 28D.

[0779] The fourth batch of the flavorant composition was prepared as follows. A batch of the protein hydrogel slurry of Example 13 was heated to 70 °C in a water bath and combined with 3.6 wt% (based on the weight of the slurry) of starch. The mixture was homogenized for 2 minutes at 8000 rpm using a Silverson laboratory mixer. The mixture was cooled to ambient temperature overnight. Then, 6.4 wt% (based on the weight of the slurry) of the flavorant 28A / 812N blend was added to the slurry and further emulsified for 5 minutes at 8000 rpm using a Silverson mixer to form a fine emulsion. This emulsion was used in Example 28E.

[0780] Using a pilot - scale co - current spray dryer, each emulsion was spray - dried using a rotary atomizer. The drying chamber had a diameter of approximately 1.2 m and a height of approximately 2.1 m. The nominal evaporation capacity was 3 kg H2O / hr at 150 °C, and the nominal air flow rate was 125 m 3 / hr. The fluid was pumped into the spray dryer using a peristaltic pump, and the flow rate was adjusted to achieve the target outlet temperature. The air inlet temperature varied between 129 and 135 °C, and the air outlet temperature varied between 69 and 70 °C.

[0781] In Examples 28B, C, D, and E, the flavorant 28A was successfully encapsulated, as shown in Table 29.

[0782]

[0783] Table 29

[0784] In all four examples, dry microcapsule powders were formed, indicating that the flavoring agent was encapsulated. The particle size measured by laser diffraction and the flavoring agent content were measured using the methods described herein. Adding glycerol or starch had little effect on the flavoring agent loading or the particle size. Using a fine emulsion resulted in a larger average encapsulate particle size.

[0785] The individual flavoring substances in the oil of Example 28A were measured using the method in this article, as shown in Table 30.

[0786]

[0787] Table 30

[0788] All flavoring agent components were encapsulated, but each component was not exactly 20 wt% the same, indicating a permitted degree of selectivity. The differences between the four encapsulates were minimal, indicating that the shell modification did not affect any flavoring agent selectivity during the encapsulation process.

[0789] δ-Damascenone and dipentene had the lowest encapsulation levels. δ-Damascenone is the flavoring substance with the lowest logP. Without wishing to be bound by theory, it is expected that substances with a higher logP will be more easily emulsified and thus preferentially encapsulated. Dipentene is the most volatile substance and thus is most likely to be lost by evaporation during the drying process. However, it is still present at approximately three-quarters of the expected level.

[0790] The olfactory performance of the encapsulates was also evaluated according to the olfactory evaluation method in this article. Example 28C performed as well as Example 28B, with an intensity rating of 3.5 when rubbed and pressed on glass and an intensity rating of 4.5 when rubbed on blotting paper. Example 28E performed as well as Example 28D, with an intensity rating of 3.5 when rubbed on glass and an intensity rating of 4.0 when pressed on glass and rubbed on blotting paper. This confirmed that adding glycerol or starch did not prevent the dry encapsulates from rupturing when rubbed or pressed, and thus releasing the flavoring agent so that it could be smelled.

Claims

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

2. The method according to claim 1, further comprising inducing at least a portion of the plant-based proteins in the mixture to form a plant-based protein hydrogel by one or more of cooling, adding salt, adding an anti-solvent, solvent reduction, adding a cross-linking agent, electrostatic cross-linking, preferably by cooling and / or solvent reduction, most preferably by cooling.

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

4. The method according to 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. The method according to claim 1, wherein the plant-based protein is selected from pea protein, potato protein, rapeseed protein, lentil protein, chickpea protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupin protein, alfalfa protein, moringa protein and / or rice protein, preferably pea protein and / or potato protein.

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

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

10. The method according to any one of claims 3 to 9, wherein the sol-gel transition is achieved by heating the protein mixture to a first temperature above the sol-gel transition temperature of the one or more plant-based protein mixtures and then lowering it to a second temperature below the sol-gel transition temperature of the one or more plant-based protein mixtures to form a hydrogel.

11. The method according to any one of claims 1 to 10, wherein the shearing treatment comprises a single shearing step involving breaking the plant-based protein hydrogel into fragments, preferably, wherein the fragments have a d of 0.2 to 50 microns, preferably 1 to 40 microns, preferably 2 to 30 microns as determined by laser diffraction 50 .

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. The method according to claim 12, wherein the first shearing step involves breaking the plant-based protein hydrogel into fragments, preferably, wherein at least 80 wt% of the fragments produced in the first shearing step have a particle size in the range of 1 mm to 100 mm, preferably 1 mm to 50 mm, preferably 1 mm to 30 mm, more preferably 10 mm to 30 mm, more preferably 15 mm to 30 mm, even more preferably 20 mm to 30 mm as determined by optical microscopy.

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

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

16. The method according to claim 15, wherein the solvent reduction step comprises the steps of: (i) contacting the fragments of the plant-based hydrogel slurry with a non-solubility solvent; (ii) separating the fragments of the plant-based hydrogel slurry from the non-solubility solvent to obtain a washed plant-based protein hydrogel slurry; and (iii) optionally repeating steps (i) and (ii).

17. The method according to any one of claims 1 to 16, further comprising a step of changing the pH of the plant-based protein hydrogel slurry such that it differs from the isoelectric point of the plant-based protein by more than 1 pH unit.

18. The method according to claim 17, wherein the step of changing the pH of the plant-based protein hydrogel slurry is carried out after step (b) or in sequence with step (b).

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

20. The method according to any one of claims 1 to 19, wherein the composition formed in step (c) has a viscosity in the range of 1 to 10,000 cP at 20 °C and 50 s -1 preferably in the range of 10 to 7,500 cP at 20 °C and 50 s -1 more preferably in the range of 15 to 5,000 cP at 20 °C and 50 s -1 viscosity.

21. The method according to any one of claims 1 to 19, wherein the composition formed in step (c) has a protein solids content in the range of 1 wt% to 25 wt%, preferably 2 wt% to 20 wt%, more preferably 4 wt% to 15 wt%, even more preferably 5 wt% to 12 wt% based on the total weight of the composition.

22. The method according to any one of claims 1 to 21, wherein the active ingredient is selected from vitamins, minerals, flavorings, fragrances, flavor precursor, fragrance precursor, flavor enhancers, malodor abatement agents, nutritional preparations, living organisms (such as probiotics), drugs, antimicrobial agents, antiviral agents, anti-inflammatory agents, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle control agents, fabric softening active substances, surface cleaning active substances, skin conditioners, hair conditioners, dyes, pigments and binders, or combinations thereof.

23. The method according to claim 22, wherein the active ingredient is at least one fragrance or flavoring, and wherein: The at least one fragrance or flavoring substance has a vapor pressure of greater than or equal to 0.00001 Torr at 25 °C; and / or The at least one fragrance or flavoring substance has a logP of greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.

0.

24. The method according to claim 22, wherein the active ingredient is at least one fragrance or flavoring substance, and the at least one fragrance or flavoring substance is part of a fragrance or flavoring agent. Preferably, the fragrance or flavoring agent contains at least 20 wt% of fragrance or flavoring substances having a logP of greater than or equal to 3.0, more preferably greater than or equal to 3.5, and more preferably greater than or equal to 4.0, based on the total weight of the fragrance or flavoring agent.

25. The method according to claim 22, wherein the active ingredient is a vitamin or a mineral, and the vitamin or mineral is 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, ω3, folic acid, thiamine, riboflavin, niacin, and phosphorus, or a mixture thereof, and more preferably is vitamin D.

26. The method according to any one of claims 1 to 25, wherein the active ingredient is part of a composition comprising the active ingredient and an active carrier phase.

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

28. The method according to any one of claims 1 to 27, wherein the composition formed in step (c) comprises droplets of the active ingredient or droplets of the composition comprising the active ingredient and the active carrier phase, and the droplets have a d of 0.2 to 50 µm, preferably 1 to 40 µm, preferably 2 to 30 µm, as determined by laser diffraction 50 .

29. The method according to any one of claims 1 to 28, wherein the plant-based 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 according to any one of claims 1 to 29, further comprising a post-treatment step for the microcapsules. Preferably, the post-treatment step comprises a non-covalent crosslinking step, a covalent crosslinking step, or a coating formation step, and the coating formation step comprises: (i) treating the microcapsules with a metal compound or a silicon-containing compound; and / or (ii) performing a complex coacervation step on the microcapsules using a polysaccharide; and / or (iii) treating the microcapsules with an aqueous solution of a mineral, preferably, the post-treatment step comprises a non-covalent crosslinking step using tannic acid, followed by a complex coacervation step using xanthan gum for (ii).

31. A biodegradable microcapsule obtained by or obtainable by the method according to any one of claims 1 to 30.

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

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

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

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

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

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

38. The biodegradable microcapsule according to any one of claims 34 to 37, wherein the active ingredient is selected from vitamins, minerals, flavorings, fragrances, flavoring precursors, fragrance precursors, flavor enhancers, malodor neutralizers, nutritional preparations, living organisms (such as probiotics), drugs, antimicrobials, antivirals, anti-inflammatories, pesticides, herbicides, fertilizers, fungicides, insecticides, animal repellents, anti-acne agents, skin brighteners, emollients, skin moisturizers, wrinkle controllers, fabric softening active substances, surface cleaning active substances, skin conditioners, hair conditioners, dyes, pigments, and adhesives, or combinations thereof.

39. The biodegradable microcapsule according to claim 38, wherein the active ingredient is at least one fragrance or flavoring, and wherein: the at least one fragrance or flavoring has a vapor pressure of greater than or equal to 0.00001 Torr at 25 °C; and / or the at least one fragrance or flavoring has a logP of greater than or equal to 3.0, preferably greater than or equal to 3.5, more preferably greater than or equal to 4.

0.

40. The biodegradable microcapsule according to claim 38, wherein the active ingredient is at least one fragrance or flavoring, and the at least one fragrance or flavoring is part of a flavoring or fragrance, preferably, wherein the flavoring or fragrance contains at least 20 wt% of a fragrance or flavoring having a logP of greater than or equal to 3.0, more preferably greater than or equal to 3.5, more preferably greater than or equal to 4.0 based on the total weight of the flavoring or fragrance.

41. The biodegradable microcapsule according to claim 38, wherein the active ingredient is a vitamin or a mineral, and the vitamin or mineral is 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, ω3 fatty acids, folic acid, thiamine, riboflavin, niacin and phosphorus, or a mixture thereof, more preferably vitamin D.

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

44. The biodegradable microcapsule according to any one of claims 34 to 43, wherein, as determined by optical microscopy, the microcapsule has a diameter of less than or equal to 250 μm, less than or equal to 200 μm, less than or equal to 150 μm, less than or equal to 100 μm, less than or equal to 50 μm, less than or equal to 30 μm, less than or equal to 10 μm.

45. The biodegradable microcapsule according to any one of claims 34 to 44, wherein the plant-based protein has a protein secondary structure having at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% intermolecular β-sheets, wherein the percentage content of intermolecular β-sheets is measured by FTIR.

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

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

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

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

50. A method for preparing a formulated product, comprising: (a) preparing biodegradable microcapsules according to the method of any one of claims 1 to 30; and (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 a formulated product.

52. The method according to claim 50 or the use according to claim 51, wherein the formulated product is a food, beverage, cosmetic, household care product, personal care product, drug, industrial product (such as paint, adhesive, sandpaper, tape, etc.), medical device, biomaterial or agrochemical.

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