EP2217088A1

EP2217088A1 - Method for making protein-based encapsulates

Info

Publication number: EP2217088A1
Application number: EP08853315A
Authority: EP
Inventors: Aart Cornelis Alting; Theodorus Arnoldus Gerardus Floris; Fanny Chantal Jacqueline Weinbreck; Jeroen Grandia
Original assignee: NIZO Food Research BV; Nizo Food Res BV
Current assignee: NIZO Food Research BV; Nizo Food Res BV
Priority date: 2007-11-29
Filing date: 2008-12-01
Publication date: 2010-08-18
Also published as: WO2009070011A8; WO2009070011A1

Abstract

Microcapsules that contain a hydrophobic core and a protein-based shell are provided. Also a method for preparing protein-based encapsulates involving the steps of allowing activated protein aggregates in an aqueous solution containing a hydrophobic material to form a layer at the interface of the dispersed phase, and cross- linking of the activated protein aggregates. The protein encapsulated particles are particularly suited for food, feed, cosmetic and pharmaceutical applications.

Description

Method for making protein-based encapsulates

FIELD OF THE ESfVENTION

The present invention concerns a method for encapsulating a hydrophobic core using a protein-based encapsulation matrix. Encapsulated particles thus provided are suitable ingredients for various products, in particular food products.

BACKGROUND OF THE INVENTION

Encapsulation techniques have been developed to protect components against the detrimental effects of e.g. oxygen, moisture, heat, light or chemical reactions and further to control the release of encapsulated components under conditions of use. Acceptable encapsulating agents must be safe and non-hazardous to the consumer's health. For food products it should have a bland or no flavor. Suitable encapsulation agents for food applications include natural gums, carbohydrates, fats and waxes and some proteins. Whereas gum Arabic is one of the most widely used encapsulation agent in food applications the use of proteins is limited. The main protein that has been evaluated for encapsulation is gelatin. Gelatin has been successfully applied as encapsulation agent in the pharmaceutical industry. However, due to the high viscosity of aqueous gelatin solutions, gelatin has limited use in spray-drying processes. US 5,601,760 discloses a method for microencapsulation of a volatile or a nonvolatile core material in an encapsulation agent consisting essentially of a whey protein. It is described that whey protein isolate and whey protein concentrate, optionally in combination with milk-derived or non-milk derived carbohydrates, and also β- lactoglobulin and mixtures of β-lactoglobulin and α-lactalbumin were used in a spray- drying encapsulation process. One example describes encapsulation of anhydrous milk fat in whey protein isolate that has been heated at 80⁰C for 30 minutes. This treatment results in denaturation of whey proteins.

EP-A 1042960 describes a cappuccino creamer with advantageous foaming properties. The creamer is prepared by spray-drying a slurry that includes as essential constituents protein, lipid and carrier. The protein is partly denatured whey protein (concentrate or isolate). The product is said to contain buoyant, hydrated, insoluble, non-colloidal, irregularly shaped whey protein particles of approximately 10-200 microns in size, with an average particle size of about 60 microns.

US 6,841,181 B2 describes the encapsulation of active food components using spray-drying technology. The process consists of mixing active ingredients with non- activated proteins and polysaccharides which are spray-dried to form a capsule. The capsules are 1 - 200 μm and up to 90% core material.

However, methods for making encapsulated food-grade particles having predefined properties are still needed, as the prior art methods cannot fully control the particle size, particle content, particle uniformity, water solubility / insolubility, etc. It is an object of the invention to provide a method for producing encapsulated particles, whereby the production method allows for controlling the properties of the resulting particles.

SUMMARY OF THE INVENTION The inventors have discovered that the aforementioned objective can be realized by using a method for encapsulating particles containing hydrophobic material, involving the use of activated protein aggregates, allowing these aggregates to form layers around the hydrophobic material while being dispersed in aqueous phase, and cross-linking the activated protein aggregates by means of disulfide cross- links afterwards. The inventors have discovered that in a suspension comprising a dispersed hydrophobic phase and activated protein aggregates, the protein aggregates will spontaneously form a layer around the hydrophobic droplets and/or particles. Conveniently, encapsulation is initiated already at an early stage, whilst the components stay in aqueous environment. Thus, it is possible to encapsulate the hydrophobic droplets and/or particles in situ by cross-linking the protein aggregates in the interfacial layer.

Cross-linkages enhance the ability of the protein-based matrix to protect the encapsulated material against atmospheric oxygen. The ability improves if a protein is utilized that is capable of forming a plurality of disulfide cross-links per molecule. The cross-linking can be achieved by subjecting the suspension to a heat treatment or ultra high pressure. Alternatively, cross-linking may be brought about by contacting the activated aggregates in the interfacial layer with an oxidizing agent. Activation of protein particles is a special form of protein denaturation and is crucial for the formation of disulphide cross- links during subsequent encapsulation steps. In the present method the activated protein aggregates are formed by irreversible denaturation of dissolved protein molecules, resulting in exposure of thiol groups that have the ability and accessibility to form disulfide bridges. In the course of the activation process, the reactive thiol groups of denatured protein molecules react together to form disulfide bridges. Thus, aggregates comprising a multitude of cross- linked protein molecules are formed. In the present method it is crucial that these aggregates retain reactive thiol groups as these reactive thiol groups are required for the cross-linking of the activated aggregates.

Activated protein aggregates can be prepared by various methods, such as heating, high pressure treatment etc.. The resulting protein reactivity is determined by the overall treatment conditions (shear, protein concentration, type of protein, protein composition, type and concentration of salts, pH, other ingredients such as sugars and polysaccharides, fats). Typically, the activated aggregates used in the method of the invention exhibit a reactivity of at least 5.0 μmol thiol groups per gram of activated protein aggregates, as determined in the Ellman's assay (Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70-77). Methods for achieving activation of the proteins will be detailed below. Proteins that can suitably be cross-linked by disulfide links include proteins that contain amino acid residues with a thiol-group, notably cystein. Provided these cystein groups have been 'activated' in a suitable manner, cystein groups in different protein molecules can react with each other, thereby cross-linking these protein molecules by a disulfide cross-link. Not only cystein residues that have free thiol groups can participate in these cross-linking reactions, but also cystein residues that together form a disulfide bridge can react with a thiol group under the formation of a new disulfide bridge and the release of another free thiol group. This is why β-lactoglobulin can suitably be used as a cross-linkable protein even though this protein normally contains two pairs of cystein residues that form disulfide bridges and only one cystein residue that contains a free thiol group. Examples of proteins that are capable of forming disulfide cross- links include whey proteins, egg proteins, soy proteins, lupine proteins, rice proteins, pea proteins and wheat proteins.

Summarizing, the protein-based encapsulates of the present invention are produced by cross-linking activated protein aggregates in a "wet procedure" comprising the steps of:

• allowing the activated protein aggregates to form a layer at the interface of the dispersed phase of material to be encapsulated and the aqueous phase containing the activated protein aggregates; and • producing microcapsules by forming disulfide cross- links between the activated protein aggregates.

In this wet procedure, microcapsules are formed in situ in the dispersion.

Subsequent cross-linking makes the hydrophobic material become trapped within the cross-linked protein-based matrix. The resulting dispersion of microcapsules can be employed as such in the preparation of e.g. a foodstuff, a beverage or a nutritional supplement or animal feed. Alternatively, the dispersion may be dried to yield a dry encapsulate. However, the above makes it clear that particles are already formed before any drying step is performed. The flexibility and control of the "wet procedure" allows different types of encapsulated hydrophobic droplets and/or particles to be made. The particle size and properties can be controlled easily.

Also, the method of the present invention provides means for controlling the water-solubility of the particles to any extent desired, thus making it possible to distinguish from untreated reference material exhibiting 100 % water-solubility. For many applications, however, it is preferred that the particles are water-insoluble.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus pertains to a method of manufacturing microcapsules containing a hydrophobic core and a protein-based shell, said method comprising the steps of: a) preparing a dispersion of hydrophobic material in a continuous aqueous phase containing activated protein aggregates, said preparation comprising: al) providing an aqueous solution or suspension of a protein that is capable of forming disulfide cross-links; a2) submitting said aqueous solution or suspension to a protein activation treatment to produce an aqueous suspension of activated protein aggregates, said suspension having a reactivity of at least 5.0 μmol thiol groups per gram of protein as determined in the Ellman's assay; wherein the aqueous solution or suspension provided in step al) contains a dispersed phase of hydrophobic material and/or wherein hydrophobic material is dispersed throughout the suspension of activated protein aggregates in or after step a2), in any event prior to step b); b) allowing the activated protein aggregates to form a layer at the interface of the dispersed phase of hydrophobic material and the aqueous phase; and c) producing the microcapsules by forming disulfide cross-links between the activated protein aggregates contained in the interface layer by: cl) heating to a temperature in excess of 40⁰C; c2) pressurization to a pressure in excess of 50 MPa; and/or c3) contacting the activated protein aggregates with an oxidizing agent.

Preferably, the present method consists of the subsequent steps a) - c).

The terms "encapsulate" and "microcapsule" as used herein are interchangeable, and refer to the particulate or droplets of hydrophobic material obtained by the method of the present invention. The individual particles within the encapsulate can consist of clearly identifiable discrete particles, but they can also consists, for instance, of a cluster of (micro-)particles, e.g. as a result of agglomeration.

The term "comprising" is to be interpreted as specifying the presence of the stated parts, steps or components, but does not exclude the presence of one or more additional parts, steps or components. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The method will now be described in terms of its individual steps. al) Aqueous solution/suspension of a protein capable of forming disulfide cross- links In step al) of the method a protein is added to an aqueous solution, such as for example water. It should be understood that the aqueous solution of protein that is capable of forming disulfide cross-links can also contain non-dissolved protein and other non- dissolved components. Therefore, step al) results in a solution or suspension containing proteins which may be activated in subsequent steps. Where the text refers to "the aqueous solution containing a protein capable of forming disulfide cross- links", the reader is reminded that this includes suspensions containing proteins which are not readily dissolvable in water, such as many plant-derived proteins. The term "protein" as used herein refers to a polymer made of amino acids arranged in a chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Typically, the protein contains at least 10 amino acid residues. The protein employed in accordance with the present invention can be, for instance, an intact naturally occuring protein, a protein hydrolysate or a synthesised protein. The protein is preferably food-grade.

"Protein hydro lysates" refers to a mixture of proteins and/or peptides obtained by (partial) breakage of peptide bonds, e.g. through enzymatic hydrolysis or other treatments.

Suitable isolated proteins may be obtained from various sources. They may be extracted or purified from natural sources, such as plants, animal milk, animal tissue, microorganism, etc. using known methods or they may be obtained commercially. Suitable proteins or protein compositions (i.e. mixtures of different types of proteins and/or proteins from different sources) include for example total milk proteins, individual milk proteins, such as one or more whey proteins, e.g. β-lactoglobulin, α- lactalbumin, bovine serum albumin, etc., and/or one or more caseins such as α-caseins, β-caseins, κ-caseins and γ-caseins or total caseins or total whey proteins. Total whey proteins can for example be obtained from Davisco Foods, USA (e.g. BiPRO™).

In order to prepare a protein-based encapsulation matrix that exhibits a high level of disulfide cross- linking it is advisable to employ a protein containing at least three cross-linkable groups. Accordingly, in a preferred embodiment a protein is employed that in its native form comprises at least three cystein residues per molecule, even more preferably at least 4, most preferably at least 5 cystein residues per molecule. The whey proteins β-lactoglobulin and α-lactoglobulin contain 5 and 8 cystein residues per molecule, respectively. Here the term "in native form" refers to the natural state of the protein in the material it is sourced from. The term "cystein residue" also encompasses cystein residues that are bound to other cystein residues by means of a disulfide bond. In another embodiment the protein preferably comprises at least about 1 or even

2 cystein residues per 500, especially per 400 amino acids, more preferably at least 1 or even 2 cystein residues per 300 or 200 amino acids, even more preferably per 100, 30 or 20 amino acids. The average molecular weight of the protein capable of forming disulfide cross-links is preferably at least 5, 10, 15, 20, 50, 100, 200, 250 or more kDa as determined by SDS-PAGE analysis.

In its native form, the protein employed in step al) advantageously contains at least 30 amino acid residues, more preferably at least 50 amino acid residues and most preferably at least 60 amino acid residues.

Examples of proteins that are capable of forming disulfide cross- links and that can suitably be employed in the method of the invention include whey proteins and plant proteins, such as wheat proteins, soybean proteins, pea proteins, lupine proteins, canola or oilseeds rape proteins, maize proteins, rice proteins, and many others. Similarly, animal proteins such as bovine serum albumin, one or more blood proteins and/or one or more egg proteins may be used. In addition, also certain microbial proteins such as one or more bacterial proteins and/or fungal proteins (including yeast proteins) can be used.

The aqueous phase provided in step al) preferably contains one or more proteins selected from one or more of the group consisting of whey proteins, egg proteins, soy proteins, lupine proteins, rice proteins, pea proteins, wheat proteins and combinations thereof. Even more preferably, said cross-linked protein is selected from the group consisting of whey proteins, soy proteins and combinations thereof. Most preferably, the protein is a whey protein or a combination of whey proteins. Most preferably, all proteins provided in step al) fall within the aforementioned ranges. The dispersion of hydrophobic material further containing activated protein aggregates submitted to step b) contains preferably 0.1 - 50 wt%, more preferably 0.2- 25 wt%, most preferably 0.5 - 15 wt% of protein(s) capable of forming disulfide cross- links, based on the weight of the continuous aqueous phase. The weight of any dispersed hydrophobic material is not incorporated in these numbers.

Step a2) activation treatment In the present method, the aqueous protein solution/suspension is submitted to a protein activation treatment. The nature of this treatment is not essential, provided that the protein becomes sufficiently activated for further use. Thus, although the activation treatment is preferably a heat treatment, other methods may also be suitable for achieving the same degree of protein activation, such as application of high pressure, shear forces, etc. Examples of suitable methods for achieving adequate protein reactivity are microwave treatment, high pressure, shear, unfolding with urea, and combinations thereof. The skilled person can easily determine whether the treatment results in sufficiently activated (reactive) protein aggregates.

When heat treatment is used to activate the proteins, the temperature and time required for obtaining the minimum reactivity depends on the types of protein used and other conditions, such as applied shear, pH of the solution, salts, etc. For example, heat treatment of a solution of 9%wt. whey proteins (BiPRO™; Davisco, USA) in demineralized water for 30 minutes holding time at 90⁰C in a water bath without stirring resulted in a reactivity of more than 15 μmol per gram of protein. Such a heat treatment preferably comprises heating the aqueous solution or suspension containing protein to a temperature of at least 60 ⁰C and less than 200 ⁰C, more preferably less than 160 ⁰C, for a period of time equal to t , which period of heating t is governed by the following formula: t = (500/(T-59)) - 4 in which t is the duration of heating (in seconds) and T is the heating temperature (in ⁰C). More preferably, the heating conditions complied are governed by the following formula: t = (90000/(T-59))-900, in which t and T have the aforesaid meaning. Heat treatment preferably involves a period of 0.1 sec to 24 hour. It is particularly preferred that the heating time ranges from 10 s - 1 hour, more preferably from at least 10 minutes. The preferred corresponding minimum and maximum temperatures may be calculated from the above formulae.

For any given type of protein and protein-comprising solution/suspension, a skilled person can easily determine conditions which are suitable for obtaining the minimum reactivity required by varying these conditions and measuring the reactivity of the protein aggregates obtained after treatment. Whatever treatment is used for activation, the treatment should be sufficient to result in protein aggregates having a reactivity of at least 5.0 μmol thiol groups per gram of protein. Preferably, the reactivity achieved is at least 10 μmol/g, more preferably at least 15 μmol/g, even more preferably at least 20 μmol/g and most preferably at least 25 μmol/g.

Reactivity is required to enable covalent cross-linking of the protein aggregates. The reactivity is defined as the number of reactive thiol groups per gram of protein. Exposure of reactive thiol groups, which leads to their reactivity, can be achieved by e.g. heat-treatment. Preferably, the activated protein aggregates submitted to step b) have a volume- weighted average diameter in the range of 1-1000 nanometers, more preferably in the range of 2 - 250 nanometers, even more preferably in the range of 2 - 100 nanometers.

For any given type of protein and protein-comprising solution/suspension, a skilled person can easily determine conditions which are suitable for obtaining the minimum reactivity required by varying these conditions and measuring the reactivity of the protein aggregates obtained after treatment. Whatever treatment is used for activation, the treatment should be sufficient to result in protein aggregates having a reactivity of at least 5 μmol thiol groups per gram of protein. Preferably, the reactivity achieved is at least 10 μmol/g, more preferably at least 15 μmol/g, even more preferably at least 20 μmol/g and most preferably at least 25 μmol/g.

Reactivity is required to enable covalent cross-linking of the protein aggregates. The reactivity is defined as the number of reactive thiol groups per gram of protein. Exposure of reactive thiol groups, which leads to their reactivity, can be achieved by e.g. heat-treatment. Preferably, the activated protein aggregates submitted to step b) have a volume- weighted average diameter in the range of 1-1000 nanometers, more preferably in the range of 2 - 250 nanometers, even more preferably in the range of 2 - 100 nanometers. Ellman's assay

The aforementioned reactivity can suitably be determined at pH 7 according to the Ellman's assay (Ellman,1959 vide supra). In this assay the number of thiol groups is determined using e(412 nm) = 13,600 M^"1 cm^"1 for 2-nitro-5-mercaptobenzoic acid (DTNB) and expressed as the amount of thiol groups (μmol) per gram of protein (aggregates). The absorbance is measured at 20-25 ⁰C. The value after 30 minutes of incubation with DTNB is taken to calculate the reactivity. The reactivity is measured by determining the concentration of thiol groups (in mM) in a 2 wt% protein solution. The numbers on the molar amount of thiol groups per gram protein as used throughout the text can easily be calculated there from.

A convenient way to perform the Ellman's assay is described in Alting et al. (Formation of disulphide bonds in acid- induced gel of preheated whey protein isolate. J. Agric. Food Chem. 48 (2000) 5001-5007). Typically, 0.25 ml of a 1 mg/ml DTNB solution in 50 mM imidazol-buffer pH 7 (pH adjusted with HCl), 0.2 mL protein solution (2 wt% protein solution) and 2.55 ml imidazol-buffer pH 7 are mixed. The assay is preferably performed in the absence of detergents such as urea or SDS.

It is preferred that the suspension of activated protein aggregates is subjected to cooling to preferably lower than 30 °C, more preferably ambient conditions prior to any subsequent steps.

Step a) Hydrophobic material

As mentioned before, before, during or after activation the aqueous solution/suspension is provided with one ore more hydrophobic materials. In other words, the hydrophobic materials may be provided to the aqueous solution/suspension before adding the cross- linkable proteins, after adding but before activating the cross-linkable proteins, or even after activating the cross-linkable proteins. Inherent to the process of the invention, the hydrophobic material should at least be dispersed throughout the aqueous solution/suspension prior to step b). However, in a preferred embodiment the hydrophobic material is added as a dispersed phase in the aqueous solution after the activation step a2), as a step a3). The term "hydrophobic material" stands for all components incorporated in the dispersed phase. It includes oils, waxes, oil-soluble vitamins, fatty acids (e.g. PUFAs), lipophilic drugs, active pharmaceutical ingredients (APIs), oil-soluble flavors, oil- soluble colorants, peptides, minerals, vitamins, bioactive components, hormones etc. However, this list is non-limiting, as any component, preferably food-grade, which benefits from protection against the environment, such as oxygen, moisture, acid conditions, interaction with food matrix, temperature etc. may be used as well as any other component that is to be separated from its environment simply to prevent the escape thereof, e.g. volatile components as well as gases, in particular air. In a preferred embodiment of the invention, a method as defined herein before is thus provided, wherein a component is encapsulated, said component being selected from the group consisting of vitamins, minerals, peptides, polyphenols, fatty acids, oils, drugs, bioactive components, flavors, colorants, fibres, gas and combinations thereof and combinations thereof. The hydrophobic material may be present in liquid or in solid form, or as a mixture thereof, thus forming an emulsion or suspension, respectively, containing hydrophobic material dispersed in water.

The aqueous phase provided in step a) may contain as much hydrophobic material as possible, provided that the aqueous phase thus formed remains the continuous phase, and the hydrophobic material stays dispersed therein. It is preferred that the aqueous phase, submitted to step b), contains 1-50 wt%, more preferably 5 - 45 wt%, most preferably 10 - 40 wt% of the dispersed hydrophobic material, based on the total weight of the dispersion submitted to step b).

The ratio (w/w) of hydrophobic material to protein in the aqueous dispersion submitted to step b) is preferably comprised between 1:8 to 200:1, preferably in a weight ratio of 1 :4 to 10:1.

Oils

In one embodiment, the hydrophobic material comprises one or more oils, preferably at least 60 wt%, more preferably at least 70 wt% of the hydrophobic material dispersed in the aqueous phase submitted to step b). It is preferred that less than 80 wt% of the hydrophobic phase is represented from oils. Here Although the term "oil" is often used in the art to characterise fats which are in liquid form at room temperature, in the context of the invention the terms "fat" and "oil" are considered interchangeable. Both fats and oils may be applied, thus resulting in oil droplets or fat particles, or combinations thereof.

The term "oil" as used herein encompasses any lipid substance that contains one or more fatty acid residues. Thus, the term oil encompasses, for instance, triglycerides, diglycerides, monoglycerides, free fatty acids and phospholipids. Not all need be present necessarily. The oil employed in accordance with the present invention can be a solid, a liquid or a mixture of both. The term "fatty acid" as used herein encompasses free fatty acids as well as fatty acid residues. Whenever reference is made herein to a weight percentage of fatty acids, this weight percentage includes free fatty acids as well as fatty acid residues (e.g. fatty acid residues contained in triglycerides). Preferably, the oil droplets/particles consist of an oil having a melting point of less than 40 ⁰C, more preferably of less than 30⁰C and most preferably of less than 15 ⁰C.

In one embodiment, the oil droplets/particles of the dispersed phase preferably contain at least 50 wt%, more preferably at least 70 wt% and most preferably at least 80 wt% of a lipid material selected from the group consisting of triglycerides, diglycerides and mixtures thereof.

In one embodiment, the oil comprises one or more flavor oils. Any fat or oil may be suitable, in particular food-grade fats or oils, such as plant derived oil (e.g. sunflower oil, canola oil, palm oil, soybean oil, flax oil, safflower oil, peanut oil, maize oil, olive oil, pumpkin oil, etc.). They may also be processed fats and oils or synthesized fats and oils which are subjected to hardening, fractionation, interesterifϊcation and the like.

In another embodiment, especially oils and fats rich in poly unsaturated fatty acids (PUFA) are used. The term "polyunsaturated fatty acid" (PUFA) as used herein encompasses any fatty acid containing 2 or more double bonds in the carbon chain. The advantageous properties of the present encapsulates are particularly pronounced in case the dispersed hydrophobic phase contains high levels of PUFAs. Accordingly, in a preferred embodiment, the dispersed hydrophobic phase contains at least 5 wt%, more preferably at least 10 wt%, most preferably at least 20 wt% of PUFAs.

Examples of PUFAs that can advantageously incorporated in the dispersed hydrophobic phase include linoleic acid, linolenic acid, conjugated linoleic acid, ω3- unsaturated fatty acids, ω6-unsaturated fatty acids and combinations thereof. According to a particularly preferred embodiment, the dispersed hydrophobic phase submitted to step b) contains at least 3 wt%, more preferably at least 5 wt% and most preferably at least 10 wt% of one or more PUFAs selected from the group consisting OfC₁₈-C₂₄ ω3- fatty acids, C₁₈-C₂₄ ω6-fatty acids and combinations thereof, based on the total weight of the hydrophobic phase. According to a particularly preferred embodiment, the latter PUFAs are selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid, gamma-linoleic acid, conjugated linoleic acid (CLA) and combinations thereof. Most preferably, the PUFAs are selected from the group consisting of DHA, EPA, CLA and combinations thereof. The PUFA in the dispersed hydrophobic phase may suitably be provided by a vegetable oil or a marine oil. Examples of such oils include fish oil, algae oil and linseed oil. Naturally, the present invention also encompasses encapsulates in which the oil droplets/particles comprise a blend of a high PUFA oil and one or more other lipid components.

Additives

In the present method one or more (food-grade) additives may be added to the aqueous solution, either before the activation treatment and/or after the activation treatment, but prior to the cross-linking treatment. Preferably the additives are not reactive towards the activated protein aggregates, i.e. non-cross-linkable by forming disulfide bonds, e.g. the additives do not react with free thiol groups as this would interfere with subsequent cross-linking of the protein. The exception to this concerns cross- linkers which will assist in cross-linking the activated protein aggregates, as addressed below.

Suitable additives are e.g. carbohydrates (e.g. fibers, inulin, maltodextrin, lactose, sucrose, glucose, galactose), hydrocolloids (e.g. gum Arabic, alginate, pectin, starch, xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, gellan gum), polyols (e.g. glycerol, xylitol), plasticizers (e.g. glyceryl triacetate and/or di-(2- ethylhexyl)adipate) and proteins that are not able to form disulfide cross-links (e.g. gelatin).

Step b) Layer formation in wet conditions

It is the inventors' findings that the activated protein aggregates form a layer at the interface of the dispersed phase of hydrophobic material and the aqueous phase. Although the layer forms spontaneously, the process may be promoted by mechanical agitation, e.g. stirring or homogenization.

The protein layer formed at the interface of the hydrophobic phase with the continuous aqueous phase preferably has a thickness in the range of 0.01 - 50 μm, more preferably 0.01 - 25 μm, even more preferably 0.01 - 5 μm.

The dispersed phase containing hydrophobic material thus obtained preferably has a mass-weighted average diameter of 0.1 - 5000 μm. If the dispersed phase involves hydrophobic droplets, it is preferred that the mass-weighted average diameter is preferably 1 - 500 μm, most preferably 1 - 100 μm.

Step c) Cross-linking

The microcapsules or encapsulates are ultimately formed by forming disulfide crosslinks between the activated protein aggregates contained in the interface layer by heating, pressurization and/or contacting the activated protein aggregates with an oxidizing agent.

If cross-linking is to be achieved by heating, it should involve heating to a temperature of a least 40 ⁰C for at least 5 milliseconds and/or by pressurizing the suspension droplets to a pressure of at least 50 MPa. More preferably said step comprises heating the suspension droplets to a temperature within the range of 50-150 ⁰C, most preferably within the range of 60-120 ⁰C, preferably for 1-86,000 seconds, more preferably for 20-86,000 seconds.

Alternatively or in addition, cross-linking may be established by pressurization to a pressure within the range of 50-1000 MPa, most preferably within the ranges of 100-600 MPa. Said pressures may typically be applied for at least 0.1 second, preferably for 1-7200 seconds.

Yet another method of cross-linking the activated protein aggregates at the interface of the dispersed droplets in the emulsion is by oxidizing, using an oxidizing agent. The oxidizing agent according to the invention has the ability to oxidize the free thiol groups in the protein aggregates to form disulfide cross-links. Any oxidizing agent having this ability may be used in accordance with the invention. Preferably, the oxidizing agent is selected from the group consisting of salts, oxides or ligands of transition metals, reactive oxygen compounds (e.g. hydrogen peroxide) and oxidizing enzymes (oxidoreductases) and combinations thereof.

Preferred examples of transition metals that can be used in the form of salts or ligands in the present method are selected from the group consisting of copper, iron, manganese, zinc, ruthenium, cobalt and combinations thereof. Most preferably, the transition metal is selected from the group consisting of copper (Cu(II)), iron (Fe(III)), manganese, nickel, zinc and combinations thereof. According to another preferred embodiment, the present method employs a salt of a transition metal, e.g. a transition metal oxide or a transition metal halide. The term "salt" as used herein also encompasses the use of dissociated salts.

Furthermore, it is preferred that the protein aggregates are contacted with the one or more transition metals in an aqueous medium containing at least 0.001 mM of the said transition metals, more preferably 0.001-500 mM, most preferably 0.01-100 mM. Preferably, said transition metals are contained in the aqueous medium in the form of cations having a valency of at least 2.

Oxidoreductases (i.e. enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the Interantional Union of Biochemistry and Molecular Biology (IUBMB)) are enzymes catalyzing redox reaction. Suitable examples include laccases or related enzymes which act on molecular oxygen and yield water; oxidases, which act on molecular oxygen and yield peroxide; and peroxidases which act on peroxide and yield water. Hence, in a preferred embodiment of the invention, a method is provided as defined herein before, wherein the oxidizing agent is an enzyme selected from the group consisting of oxidases, peroxidases, laccases and combinations thereof. More preferably the oxidizing enzyme is selected from the group consisting of glutathione peroxidase, horseradish peroxidase, microperoxidase, coprinus cinereus oxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase and combinations thereof. Most preferably, the oxidizing enzyme is selected from the group consisting of glutathione peroxidase, horseradish peroxidase, coprinus cinereus oxidase, manganese peroxidase and combinations.

It is clear from the above that the present invention also encompasses the formation of encapsulates in which protein in the protein-based matrix has not only been cross-linked by disulfide bonds, but in which additional cross-linking mechanisms have been used to cross- link the protein molecules. Examples of alternative cross- linking techniques include the aforementioned use of gluteraldehyde and/or transglutaminase as cross- linking agents. Preferably, however, the method of the only involves disulfide cross-linking. The aforementioned characteristics of the protein layer thickness and the volume- weighted average diameter of the dispersed phase formed in step b) are not expected to change significantly during cross-linking.

Dispersion of protein-based encapsulated particles/droplets Also provided are a dispersion of hydrophobic particles and/or droplets in a continuous aqueous phase, as obtainable by the method. The protein coating formed has unique properties, as do the particles and droplets themselves. The dispersed hydrophobic phase is enclosed by a protein-based layer containing at least 10 wt% of a protein that has been cross-linked by means disulfide cross-links. It is preferred that at least 50 wt% of this cross-linked protein dissolves when dithiothreitol (DTT) is incorporated in the aqueous phase in a concentration of 2% by weight of water.

The dispersion formed in step c) of the present method can be either an o/w- emulsion or an aqueous suspension, depending on the state of the dispersed material. Numbers on amounts of proteins and hydrophobic material, and the relative weight ratio of oil to protein as provided in steps al) and a) are also applicable here.

The size of the hydrophobic particles and droplets in the encapsulates formed in step c) can vary within a wide range. According to a large estimate, the encapsulates have a volume-weighted average size of 0.1 - 5000 microns. If the encapsulates contains hydrophobic droplets, these are preferably characterized by a volume- weighted averaged size within the range of 0.1 - 1000 μm, especially within the range of 1 - 500 μm, particularly ranging 1-100 μm. According to a particularly preferred embodiment, the averaged particle size is equal to or below 50μm in diameter, such as equal to or less than 25, 20, 15, 10 or 5 μm. Size and shape can be analyzed using microscopy (e.g. light microscopy or electron microscopy) or light scattering techniques. Preferably at least 80%, 85%, 90% or more of the particles have a diameter of 50 μm or less, such as 40, 30, 25, 15, 10 μm or less. In case of oil droplets, the volume- weighted diameter of the oil droplets is preferably within the range of 0.1 - 10 μm, most preferably 0.2 - 5 μm. In another embodiment, the numbers in the foregoing paragraph apply to the mass- weighted average size. The particle (droplet) size distribution of the encapsulate can suitably be determined in a manner well known to a skilled person using a set of sieves with different well-defined mesh sizes. In the present encapsulate the average number of hydrophobic droplets/particles per encapsulate can vary widely, e.g. from 1.0 tolO⁹. Preferably, the encapsulate contains 1.0 to 10⁶ of hydrophobic droplets/particles per encapsulate.

Advantageously, a substantial fraction of the cystein residues in the cross- linked protein is actually participating in disulphide cross-links, i.e. in the cystein-cystein cross-links. According to a preferred embodiment, the encapsulation matrix contains at least 20 wt%, most preferably at least 40 wt%, even more preferably at least 60 wt% of a protein that has been cross- linked by means of disulfide cross- links, based on the weight of total protein in protective protein-based matrix In another embodiment, the protein that has been cross-linked by disulfide cross-links exhibits a number weighted average degree of polymerisation of at least 500 more preferably of at least and most preferably of at least 1000. Here the degree of polymerisation equals the total number of protein molecules that are linked together in a single cross-linked macromolecule.

Dependent on the actual cross-linking step c), encapsulates may be prepared in which the proteins have not been cross-linked in any other way than by disulfide cross-links.

Further processing

After producing the dispersion of microcapsules in accordance with steps a) - c) as laid down above, water can be removed in an optional step d) to yield a free flowing powder. It is preferred that the dried encapsulates contain less than 20 wt%, preferably less than 15 wt% of water. Even more preferably the water content does not exceed 10 wt%.

Preferably, the coated particles/droplets obtained in step c) may be subjected to an additional step of spray-drying, resulting in a dried powder comprising the encapsulates. Spray-drying can be carried out as known in the art, for example as described in US 6,223,455 or the "Spray Drying Handbook", K. Masters, 5th ed.,

Longman Scientific & Technical Publishers, 1991, pp. 329-337 and 346-349.

Preferably, the encapsulates are sprayed and dried using e.g. fluidized bed or spouted bed equipment. Such equipment is available in the art, see e.g. Fluid bed coater GPCG 1.1 with Wurster insert (Glatt GmbH).

Naturally, it is also feasibly to produce a dispersion containing a high concentration of microcapsules by removing a fraction of the water contained in the dispersion, preferably after the cross-linking step c).

Also, the encapsulates, either in dispersion as obtained after step c), or concentrated or dried after an optional step d), may be subjected to additional coating steps. This way, multi-layered encapsulates are made. According to a particularly preferred embodiment the present encapsulates contain at least on coating layer that contains at least 60 wt% of one or more components selected from the group consisting of lipids (including waxes and low PUFA fats), polyols (such as: glycerol, xylitol), menthol, glyceryl triacetate, di-(2-ethylhexyl) adipate, plasticizers (such as glycerol, glyceryl triacetate and/or di-(2-ethylhexyl) adipate, or others), or mixtures of two or more plasticizers; sugars (such as for example: lactose, sucrose, glucose, galactose), hydrocolloids (such as for example: gum Arabic, alginate, pectin, starch, xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, gellan gum), salts (such as for example: sodium salts, calcium salts, potassium salts:, enzymes (such as for example: proteases, peptidases, oxidases, hydrolases, esterases, lyases), cross-linkers (such as for example: tannins, transglutaminase, formaldehyde, glutaraldehyde), antioxidants, and additional layers of activated protein aggregates.

These additional outer layers may be provided for using any suitable coating method known in the art. In case multiple layers are produced, the ranges given throughout the text apply to the core and the first protein-based shell layer in direct contact therewith only, obtainable by the method involving steps a) - c) as explained above, unless stated otherwise.

In a particularly preferred embodiment, the protein-based encapsulates are characterized in that they exhibit a low amount of hydrophobic material at the surface, since the method of the invention ensures that the hydrophobic phase is completely enveloped by the protein-based matrix. In accordance with this embodiment, the surface hydrophobic content of the encapsulate is less than 10%, more preferably less than 5% and most preferably less than 2%. This may be determined by weighing 10 gram powder and adding 10OmL tetrachloromethane, at room temperature. After 15 minutes of contact time, the mixture is filtered using 502 Schleicher & Schuell filter. 50 mL filtrate is weighed and subsequently the tetrachloromethane is evaporated by drying. After drying the dry amount is weighed again. Form these measurements the amount of unbound hydrophobic material, particularly oil, can be determined.

Water-solubility

As mentioned herein before, the protein-based hydrophobic encapsulates according to the present invention exhibit the special property that the protein contained in the protein-based matrix exhibits very poor water solubility as a result of the disulfide cross-linking. Without these cross-links the protein-based matrix normally has a much higher water solubility. This poor water solubility is also apparent within a broad pH range at near ambient conditions. Consequently, in a preferred embodiment, the protein-based matrix is characterized in that less than 70 wt.%, more preferably less than 40 wt.% and most preferably less than 25 wt.% of the protein contained in the protein-based matrix can be dissolved when 75 mg of the encapsulate is dispersed in 50 ml distilled water having a temperature of 25 ⁰C at any pH within the range of 3.0-7.0. Preferably, the weight percentage of the protein that can be dissolved is at least a factor 1.3 higher when in the aforementioned procedure the distilled water is replaced by an aqueous solution of 2 wt.% dithiothreitol (DTT). According to a particularly preferred embodiment, the protein-based matrix is characterised in that: i. less than 40 wt.%, more preferably less than 25 wt.% of the protein contained in the protein-based matrix can be dissolved when 75 mg of the encapsulate is dispersed in 50 ml distilled water having a temperature of 25 ⁰C at any pH within the range of 3.0-7.0; ii. the weight percentage of the protein that can be dissolved is at least a factor 2 higher when in the aforementioned procedure the distilled water is replaced by an aqueous solution of 2 wt.% DTT.

According to yet another preferred embodiment, the protein-based matrix is characterized in that less than 40 wt.%, more preferably less than 25 wt.% of the protein-based matrix can be dissolved when 75 mg of the encapsulate is dispersed in 50 ml distilled water having a temperature of 25 ⁰C at any pH within the range of 1.0-8.0. According to a particularly preferred embodiment, the protein-based matrix is characterised in that: i. less than 40 wt.%, more preferably less than 25 wt.% of the protein contained in the protein-based matrix can be dissolved when 75 mg of the encapsulate is dispersed in 50 ml distilled water having a temperature of 25 ⁰C at any pH within the range of 1.0-8.0; ii. the weight percentage of the protein that can be dissolved is at least a factor 2 higher when in the aforementioned procedure the distilled water is replaced by an aqueous solution of 2 wt.% DTT.

In the above mentioned solubility tests the pH of the distilled water or the DTT solution is adjusted with the help of HCl and solubility is measured 16 hours after the encapsulate was dispersed in the liquid. During this period the mixture is continuously gently stirred in order to prevent 'clumping' of the encapsulate particles. In both the solubility test i) and ii) pH is adjusted to achieve maximum protein solubility within the pH range of 3.0-7.0.

The invention also encompasses a further step of combining the microcapsules formed by the method of the present invention, either in dispersion as obtained after step c), or concentrated or dried after an optional step d), with one or more other edible ingredients, to produce a food, a beverage, a nutritional supplement or animal feed, that is subsequently packaged.

Uses

The protein-based encapsulates of the present invention can for instance advantageously be employed as a vehicle for delivering biologically active ingredients to an animal or a human. In particular protein-based encapsulates that are stable under gastric conditions may suitably be used to deliver biologically active ingredients that are not stable under gastric conditions. Thus, one aspect of the invention relates to the of use the present protein-based encapsulates in therapeutic or prophylactic treatment, said treatment comprising oral administration of the protein-based encapsulates. Typically, the protein encapsulated particles are orally administered in an amount of 0.1 to 40 g per administration event. In accordance with this aspect of the invention, the biologically active ingredient may be a pharmaceutically active ingredient or a nutrient (including micronutrients such as vitamins). Another aspect of the present invention relates to a foodstuff, a beverage, a nutritional supplement or animal feed containing from 0.3-80 wt.% of a dried or concentrated encapsulate as defined herein before (as obtained in step d) or from 0.5-98 wt.% of the dispersion as obtained in step c) of the method of the invention. The invention also pertains to a method of these applications, involving combining the dried microcapsules of step d) or the dispersion of step c) of the present method with conventional ingredients, to obtain the aforesaid numbers.

Food products comprising the particles include for example the following: cold or warm drinks, such as coffee, chocolate, tea, fruit or vegetable juices; soups; sauces; spreads, batters, ready-to-eat meals, dairy products (milk, milk-based drinks, yoghurt, cheese, butter, margarine, ice cream), pasta, fruit or vegetable products, meat or fish products, meat replacers, bread, pastries, deserts, sweets, candy-bars, confectionary, food- or drink- additives (such as coffee or tea creamers, sweeteners), powders such as instant coffee or tea, milk-powder, soup powder, ice-cream, etc. Feed products include any type of animal feed, such as feed for farm animals (cows, horses, pigs, chicken, etc.), pets (dogs, birds, fish, cats, rabbits, rodents, etc), wild animals, etc.

Also, the invention relates to the use of encapsulates obtainable by the method of the present invention in pharma applications and cosmetic products.

The invention is further illustrated by means of the following examples.

EXAMPLES

Example 1 : preparation of spray-dried orange oil capsules Protein solution was prepared by mixing 54 g of whey protein isolate (Bipro; Davisco,

USA) in 546 g of demineralized water at room temperature (stirred for 2 h).

Reactive protein aggregates were prepared by heating the whey protein isolate solution at 68.5⁰C during 2 hours in a water bath. The solution was further cooled down in ice and then brought to room temperature. The reactivity of the particles was determined using the DTNB-method as described before. The reactivity was about 17 μmol thiol groups per gram protein.

A pre-emulsion was prepared by mixing 128 g of demineralized water and 400 g of a solution of 9% of reactive protein aggregates and 72 g of orange oil (lot nr 04723BC-137 Sigma Aldrich) using an Ultra-turrax (2 min at power 9.5). The mixture was then homogenized using a two-stage high pressure homogenizer (Model NSlOOlL - PANDA, Niro Soavia S.P.A., Italy; flow 10 L/h) (400/40 bar). The suspension of orange oil capsules with activated protein aggregates was subsequently heated for at 90⁰C during 30 minutes hours in a water bath. The emulsion was further cooled down in ice and then brought to room temperature.

Prior to spray drying, 90 g of maltodextrin was added to 500 g of emulsion. The mixture was slowly stirred.

In the next step the emulsion was spray-dried using a Buchi lab-scale spray- dryer (inlet temperature 160⁰C, outlet temperature 90⁰C). By spray-drying a powder was obtained.

Example 2: Measurement of the payload of the spray-dried orange oil capsules: Spray-dried orange oil capsules were prepared as described in example 1. The payload of the oil contained in the powder after spray drying was measured using nucleic magnetic resonance (NMR, Brucker Minispec MQ20 NMR analyser). The maximum payload based on the solid content of the suspension before spray drying is 33 %wt. of the orange oil contained in the powder after spray drying of capsules.

The capsules prepared with reactive protein aggregates and subsequent cross- linking showed a payload of 25 %.

Example 3: preparation of an aqueous suspension of fish oil capsules with activated protein aggregates

Protein solution was prepared by mixing 54 g of whey protein isolate (Bipro; Davisco, USA) in 546 g of demineralized water at room temperature (stirred for 2 h).

Reactive protein aggregates were prepared by heating the whey protein isolate solution at 68.5°C during 2 hours in a water bath. The solution was further cooled down in ice and then brought to room temperature. The reactivity of the particles was determined using the DTNB-method as described before. The reactivity was about 17 μmol thiol groups per gram protein.

A pre-emulsion was prepared by mixing 128 g of demineralized water and 400 g of a solution of 9% of reactive protein aggregates and 72 g of fish oil (batch 116kl837 Sigma Aldrich) using an Ultra-turrax (2 min at power 9.5). The mixture was then homogenized using a two-stage high pressure homogenizer (Model NSlOOlL - PANDA, Niro Soavia S.P.A., Italy; flow 10 L/h) (400/40 bar). The suspension of fish oil capsules with activated protein aggregates was subsequently heated for at 9O⁰C during 30 minutes hours in a water bath. The emulsion was further cooled down in ice and then brought to room temperature.

Example 4: Measurement of oxidation products of fish oil capsules The aqueous suspension of fish oil capsules was prepared as described in example 3. The aqueous suspension of fish oil capsules was stored in an open container at ambient conditions during 5 weeks. An open container containing the fish oil used for the preparation of the suspension of the fish oil capsules was used a reference and stored on the conditions described for the capsules. The amounts of oxidation products of fish oil were measured in the capsules and the free fish oil sample using gas chromatography. The amount of the oxidation products measured for the aqueous suspension of fish oil capsules were expressed as the percentage of the amount of oxidation products measured for the free fish oil (Table 1).

As shown in Table 1, the aqueous solution of fish oil capsules prepared with reactive protein aggregates was much more resistant against oxidation compared to the unprotected fish oil.

Table 1: Relative amount of fish oil oxidation products measured after 5 weeks of ambient storage.

Claims

1. A method of manufacturing microcapsules containing a hydrophobic core and a protein-based shell, said method comprising the steps of: a) preparing a dispersion of hydrophobic material in a continuous aqueous phase containing activated protein aggregates, said preparation comprising: al) providing an aqueous solution or suspension of a protein that is capable of forming disulfide cross-links; a2) submitting said aqueous solution or suspension to a protein activation treatment to produce an aqueous suspension of activated protein aggregates, said suspension having a reactivity of at least 5.0 μmol thiol groups per gram of protein as determined in the Ellman's assay; wherein the aqueous solution or suspension provided in step al) contains a dispersed phase of hydrophobic material and/or wherein hydrophobic material is dispersed throughout the suspension of activated protein aggregates in or after step a2) in any event prior to step b); b) allowing the activated protein aggregates to form a layer at the interface of the dispersed phase of hydrophobic material and the aqueous phase; and c) producing the microcapsules by forming disulfide cross-links between the activated protein aggregates contained in the interface layer by: cl) heating to a temperature in excess of 40 ⁰C; c2) pressurization to a pressure in excess of 50 MPa; and/or c3) contacting the activated protein aggregates with an oxidizing agent.

2. The method according to claim 1, wherein said hydrophobic material is dispersed throughout the suspension of activated protein aggregates in a step a3).

3. The method according to claim 1 or 2, wherein said protein activation treatment involves heating to a temperature of 60 ⁰C - 200 ⁰C, for a period of time equal to t, governed by the following formula: t = (500/(T-59)) - 4 in which t is the duration of heating (in seconds) and T is the heating temperature (in ⁰Q.

4. The method according to any one of the preceding claims, wherein the hydrophobic material comprises at least 60 wt% of one or more components selected from the group consisting of oils, waxes, oil- soluble vitamins, fatty acids, lipophilic drugs, active pharmaceutical ingredients (APIs), oil-soluble flavors, oil- soluble colorants, hydrophobic peptides and combinations thereof.

5. The method according to claim 4, wherein the hydrophobic material comprises at least 60 wt% of an oil.

6. The method according to any one of the preceding claims, wherein the dispersed hydrophobic material submitted to step b) contains at least 3 wt% of polyunsaturated fatty acids (PUFAs) selected from the group consisting OfCi₈-C₂₄ ω3-fatty acids, Ci₈-C₂₄ ω6- fatty acids and combinations thereof.

7. The method according to claim 4 or 5, wherein the dispersed hydrophobic material comprises at least 3 wt.% of PUFAs selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid, conjugated linoleic acid (CLA) and gamma-linoleic acid.

8. The method according to any one of the preceding claims, wherein the protein that is capable of forming disulfide cross-links comprises at least three cystein residues per molecule.

9. The method according to claim 8, wherein the protein that is capable of forming disulfide cross- links is selected from one or more of the group consisting of whey proteins, egg proteins, soy proteins, lupine proteins, rice proteins, pea proteins, wheat proteins and combinations thereof, preferably a whey protein.

10. The method according to any one of the preceding claims, wherein the dispersion of hydrophobic material submitted to step b) contains 0.1 - 50 wt% of the protein that is capable of forming disulfide cross- links, based on the weight of the aqueous phase.

11. The method according to any one of the preceding claims, wherein the dispersion submitted to step b) contains 1-50 wt% of the dispersed hydrophobic material.

12. The method according to any one of the preceding claims, wherein the weight ratio of hydrophobic material to protein in the aqueous dispersion submitted to step b) is comprised between 1 :8 to 200: 1.

13. The method according to any one of claims 1-11, wherein c3) involves an oxidizing agent selected from the group consisting of salts, oxides or ligands of transition metals, reactive oxygen compounds and oxidizing enzymes, and combinations thereof.

14. The method according to claim 13, wherein the transition metals are selected from the group consisting of copper, iron, manganese, zinc, ruthenium, cobalt and combinations thereof.

15. The method according to claim 13, wherein the oxidizing enzyme is selected from the group consisting of oxidoreductases, preferably oxidases, peroxidases, laccases and combinations thereof.

16. The method according to claim 15, wherein the oxidizing enzyme is selected from the group consisting of glutathione peroxidase, horseradish peroxidase, microperoxidase, coprinus cinereus oxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase and combinations thereof.

17. The method according to any one of claims 1 - 16, comprising a further step d) of removing water, to yield a free flowing powder.

18. A dispersion containing microcapsules obtainable by a method according to any one of claims 1-17.

19. Microcapsules obtainable by the method according to claim 18.

20. The dispersion according to claim 18 or microcapsules according to claim 19, wherein a hydrophobic dispersed phase is enclosed by a protein-based layer containing at least 10 wt.% of a protein that has been cross- linked by means of disulfide cross-links, and wherein at least 50 wt% of this cross-linked protein dissolves when dithiothreitol (DTT) is incorporated in the aqueous phase in a concentration of 2% by weight of water.

21. The method according to any one of claims 1 - 17, wherein the microcapsules obtained in step c) or d) are combined with one or more other edible ingredients to produce a food, a beverage, a nutritional supplement or animal feed that is subsequently packaged.

22. A foodstuff, a beverage, a nutritional supplement or animal feed containing from 0.5-98 wt.% of the dispersion according to claim 18 or from 0.3-80 wt.% of the microcapsules according to claim 19.

23. A method of preparing a foodstuff, a beverage, a nutritional supplement or animal feed, said method comprising incorporating from 0.5-98 wt.% of the dispersion according to claim 18 or from 0.3-80 wt.% of the microcapsules according to claim 19.

24. Use of the dispersion according to claim 18 or microcapsules according to claim 19 in cosmetic and pharmaceutical applications.