PROTEIN-BASED PROBIOTIC ENCAPSULATES

TECHNICAL FIELD OF THE INVENTION

The present invention relates to protein-based probiotic encapsulates, more particularly to encapsulates that comprise a protein-based encapsulation matrix holding one or more probiotics. The probiotics contained in the encapsulates of the present invention are protected very effectively against gastric conditions.

BACKGROUND OF THE INVENTION Probiotic bacterial cultures are intended to assist the body's naturally occurring gut flora to reestablish themselves. They are sometimes recommended by doctors, and, more frequently, by nutritionists, after a course of antibiotics, or as part of the treatment for gut-related candidiasis. However, when probiotics are ingested (e.g. enteraly or by inhalation), these first have to pass and survive the extreme conditions existing in the stomach, before they reach the intestines.

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, food-grade 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 describes a method for microencapsulation of a volatile or a non- volatile 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. The resulting encapsulates were said to protect the core against deterioration by oxygen or from detrimental of other compounds or materials, to limit the evaporation or losses of volatile core materials and to release the core upon full hydration reconstitution. One example describes

encapsulation of anhydrous milk fat in whey protein isolate that has been heated at 80 ⁰ C for 30 minutes. This treatment results in denaturation of whey proteins. The publication is silent on probiotics.

It is an object of the invention to provide protein-based probiotic encapsulates that are acid resistant, thus exhibiting a high stomach survival rate, and that can be produced via a robust and relatively simple manufacturing process.

SUMMARY OF THE INVENTION

The inventors have discovered that the aforementioned objective can be realized by encapsulating a Gram-positive probiotic bacterial strain, in a matrix of protein that has been cross-linked by means of disulfide cross-links. It was found that these disulfide cross-linkages make the encapsulates acid resistant, i.e. the particles remain intact in the stomach and the sensitive probiotic core components, only to be released when contacted with enzymes secreted into the lower intestinal tract, such as pancreatic enzymes. In particular if a protein is utilized that is capable of forming a plurality of disulfide cross-links per molecule, it is possible to produce a cross-linked protein-based matrix that is virtually insoluble. Protein matrices that exhibit a high degree of disulfide cross-linking are further characterized by a very poor water solubility. Thus, encapsulates containing a disulfide cross-linked protein- based matrix in accordance with the present invention continue to effectively protect the probiotic component if exposed to moisture, in particular acid.

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 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.

The disulfide cross-linked protein-based matrix in the present encapsulates can, for example, be formed by a process that involves the following steps:

• providing an aqueous solution of a protein that is capable of forming disulfide cross-links;

• subjecting the aqueous solution to a heat treatment to produce an aqueous suspension of activated protein aggregates; and

• drying said aqueous suspension whilst supplying sufficient heat to cross-link the activated aggregates by means of disulfide links.

The activation step in the aforementioned process is a special form of protein denaturation and is crucial for the formation of disulphide cross-links between activated protein aggregates during the drying step. 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 preparation of the present encapsulates 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).

The protein-based probiotic encapsulates of the present invention can be produced through different manufacturing processes that all have in common that first a suspension is formed of activated protein aggregates. The suspension is then sprayed onto the probiotic component, thus forming a shell layer of protein around the probiotic. The activated protein aggregates are cross-linked in such a way that the probiotics becomes trapped within the cross-linked protein-based matrix. This 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.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention relates to an encapsulate comprising a protein-based encapsulation matrix that envelops one or more probiotic bacteria, preferably Gram-positive probiotic bacterial strains; said encapsulate having a mass weighted average diameter in the range of 0.5-5000 μm, wherein the protein-based encapsulation matrix contains at least 10 wt. % of a protein that has been cross-linked by means disulfide cross-links, said protein-based matrix further being characterized in that: i. less than 75 wt.%, preferably less than 40 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 5 ⁰ C 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 1.3 higher when in the procedure under i) the distilled water is replaced by an aqueous solution of 2 wt.% dithiothreitol (DTT).

In the above mentioned solubility tests and the solubility tests described elsewhere in this document 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 poor solubility of the cross-linked protein-based matrix in distilled water is indicative for the high level of cross-linking of the present probiotic encapsulates. Without the disulfide cross-links the protein-based matrix of the present encapsulate would exhibit a much higher water solubility. This can be demonstrated by repeating the solubility test i) using an aqueous dithiothreitol (DTT) solution instead of distilled water. Since DTT reduces disulfide bonds and maintains the monothiols in a reduced state, the difference in solubility observed in the solubility tests with the DTT solution and distilled water is indicative of the level of disulfide cross-linking. The water-insoluble character improves the ability of the probiotic encapsulates to withstand the gastric conditions.

The term "encapsulate" as used herein refers to a particulate material. 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 "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.

Probiotics

Of all probiotics - including probiotic yeasts - acknowledged in the prior art, the invention pertains to encapsulated probiotic bacteria, and methods for obtaining such encapsulates. The probiotic bacteria of the invention are preferably Gram-positive probiotic bacteria.

The term "Gram positive probiotic" as used herein encompasses any lactic acid bacteria (LAB). The term "lactic acid bacteria" is used herein to refer to bacteria, which produce lactic acid as a product of fermentation, including e.g. bacteria of the genus Lactobacillus, Streptococcus, Lactococcus, Oenococcus, Leuconostoc, Pediococcus, Carnobacterium, Propionibacterium, Enterococcus and Bifidobacterium. The lactic acid bacterial strains of the invention are "probiotics" or "probiotic strains", which term herein refers to a strain of live bacteria, which have a beneficial effect on the host when ingested (e.g. enterally or by inhalation) by a subject. A "subject" refers herein to a human or non- human animal, in particular a vertebrate.

The preferred LAB are Lactobacillus and Streptococcus, particularly Lactobacillus. Most preferred are Lacobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius. A suitable example of a Streptococcus strain is Streptococcus thermophilus. Preferrred Bifidobacterium are Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium infantis and Bifidobacterium longum. A suitable Enterococcus is Enterococcus faecium. A preferred Lactococcus is Lactococcus lactis. PCT/NL2007/050233, which was not published prior to the filing date of the present application, contains an example in which a probiotic powder in general is encapsulated by spray drying a pre-emulsion containing reactive whey protein aggregates onto extruded mixture of polymer and a probiotic powder. No specific probiotic strains are disclosed therein.

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 present invention enables the preparation of encapsulates containing substantial levels of probiotics. Preferably, the probiotic materials in the encapsulate represent at least 1 wt.%, more preferably at least 5 wt.%, even more preferably at least 10 wt.% and most preferably at least 20 wt.% of the encapsulate. Typically, the probiotic materials represent not more than 90 wt.%, preferably less than 70 wt% of the encapsulate. Here the weight percentage of probiotic materials is calculated on the encapsulate exclusive any coating layers that may have been applied onto the (core) encapsulate. The high payload of probiotic materials that can advantageously be achieved in the encapsulates of the present invention is reflected in a high probiotic to protein ratio. Typically, the probiotics in the probiotic materials and the protein in the protein-based encapsulation matrix are contained in the encapsulate in a weight ratio probiotic : protein of 1:8 to 200:1, preferably in a weight ratio of 1:4 to 10:1. The protein-based encapsulation matrix, besides cross-linked protein may suitably contain other matrix components, such as 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 non cross-linked protein (e.g. gelatin). Preferably, the protein-based encapsulation matrix contains at least 20 wt.%, even more preferably at least 40 wt.% and most preferably at least 60 wt.% of a protein that has been cross-linked by means disulfide cross-links.

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 the protein that has been cross- linked by disulfide cross-links in its native form comprises at least three cystein residues per molecule, even more preferably at least 4 and 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 that has been cross-linked by disulfide cross-links in native form 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 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 that has been cross-linked by disulfide cross-links 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 protein based matrix 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, 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 protein in the protein-based matrix that has been cross-linked by disulfide cross- links is preferably 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, egg proteins, soy proteins and combinations thereof. Most preferably, the protein is a whey protein or a combination of whey proteins. The encapsulates of the present invention contain a protein-based matrix that is made up of macromolecules consisting of a hundreds or thousands of protein molecules that have been cross-linked by disulfide bonds. According to a particularly preferred 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.

The encapsulates of the present invention typically have a mass weighted average diameter in the range of 1-2000 μm. Even more preferably the mass weighted average diameter of the present encapsulates is in the range of 5-1000 μm, most preferably in the range of 5-500 μm. The present encapsulate can be prepared in the form of a granulate with a relatively large diameter by e.g. extrusion or alternatively by agglomerating an encapsulate with a smaller particle distribution. Encapsulates with a relatively small particle size can be obtained by means of e.g. spray drying. The particle 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.

The present invention also encompasses 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 use of gluteraldehyde as cross-linking agent and the use of transglutaminase. Preferably, however, the protein in the present encapsulate has not been cross-linked in any other way than by disulfide cross-links.

The poor water solubility of the protein-based matrix of the present encapsulate is associated with the high level of disulfide cross-linking within said matrix. Without these cross-links the protein-based matrix normally has a much higher water solubility. Thus, according to a very preferred embodiment, the protein-based matrix is characterized in that more than 60 wt.%, more preferably more than 80 wt.% and most preferably at least 90 wt.% of the protein contained in the protein-based matrix dissolves in an aqueous solution of 2 wt.% DTT having a temperature of 25 ⁰ C and a pH in the range of 3.0-7.0.

As mentioned herein before, the protein-based probiotic 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. 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 ⁰ C 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.% 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 ⁰ C 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 ⁰ C 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 ⁰ C 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 present encapsulate the number of probiotic celss per gram of encapsulate can vary widely, e.g. in the range from 10 ⁶ - 10 ¹² cfu/g (colony forming units per gram encapsulate), preferably in the range from 10 ⁷ - 10 ¹¹ cfu/g, more preferably in the range of 10 ⁸ - 10 ¹⁰ cfu/g.

The encapsulate of the present invention advantageously is substantially water-free. Typically, the encapsulate contains less than 20 wt.% of water. Even more preferably the water content does not exceed 10 wt.%. The present invention also encompasses an embodiment in which the encapsulate comprising the probiotic materials and the protein-based matrix is coated with one or more protective layers. The use of such coating layers may even further improve the survival rate of the probiotics-containing encapsulate. Such coatings may also be applied in order to make the encapsulate less sensitive to conditions of shear. According to a particularly preferred embodiment the present encapsulate contains 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.

The present invention also provides a method of producing an encapsulate as defined herein before, said method comprising:

• providing an aqueous solution of a protein that is capable of forming disulfide cross-links;

• submitting said aqueous solution 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; and

• spraying said aqueous suspension onto core particles containing one or more of the aforementioned probiotics, to produce microcapsules having activated protein aggregates contained in the interface layer; or

• providing a concentrated suspension of probiotic cells to said suspension of protein aggregate; followed by spraying of said suspension containing probiotic cells and protein aggregates; and

• forming disulfide cross-links between the activated protein aggregates contained in the interface layer. Optionally further layers are added around the microcapsules obtained by said method.

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.

Activation treatment

In the present method, the aqueous protein solution (which optionally further comprises additives) is submitted to a protein activation treatment. The nature of this treatment is not essential, as long as 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 heat treatment, 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 90 ⁰ C in a water bath without stirring resulted in a reactivity of more than 15 μmol per gram of protein.

The activation treatment preferably comprises heating the aqueous solution to a temperature of at least 60 ⁰ C and less than 200 ⁰ C for at least a period of time equal to t, which period of heating t is governed by the following formula: t = (500/(T-59))-4 wherein: t = duration of heating (in seconds) and

T = heating temperature (in ⁰ C). More preferably the heating conditions complied are governed by the following formula: t = (90000/(T-59))-900.

For any given type of protein and protein-comprising solution, 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.

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. The absorbance is measured at 20-25 ⁰ C. 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.

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.

In the present method the aqueous suspension containing the activated protein aggregates typically contains from 0.1-25 wt.% of the protein capable of forming disulfide links. More preferably, said suspension contains from 0.5-15 wt.% of the protein capable of forming disulfide links.

The activated protein aggregates in the suspension preferably 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.

Spraying

In one embodiment, the aqueous suspension comprising the reactive protein aggregates (and optionally other additives) is advantageously sprayed onto a core material to form protein- coated core particles, preferably having a volume weighted average diameter in the range of 0.5-5000 μm, more preferably in the range of 1-2000 μm, most preferably 5 - 1000 μm, particularly 5-500 μm. The spraying may be performed in air or in a gas. Preferably the gas has low or zero reactivity towards the thiol groups contained in the activated protein aggregates. Preferred examples of gases that may be used in accordance with the invention include nitrogen, carbon dioxide, air, argon, helium and combinations thereof.

The suspension of activated protein aggregates is typically sprayed in air or gas by means of a nozzle. Preferably, spraying of the activated protein aggregate suspension onto the core particles and subsequent drying may be performed 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).

Spraying advantageously involves a temperature in excess of 30 ⁰ C, even more preferably in excess of 40 ⁰ C. By subjecting the suspension to a substantial temperature increase initial cross-linking of the protein aggregates can be instigated. By partially cross- linking the protein aggregates in the suspension droplets the stability of these droplets is enhanced, which makes it easier to oxidatively cross-link the protein aggregates in a next step.

In a preferred embodiment the aqueous suspension is dispensed into a hot gas to remove water and to convert the droplets into partially cross-linked protein-based particles which are subsequently contacted with the oxidizing agent. Particularly good results are obtained if the dispensed suspension is contacted with the hot gas in countercurrent fashion. The present method preferably comprises spraying the suspension of protein aggregates onto core particles, e.g. in a fluidized bed, said core particles containing the probiotic to be encapsulated. Thus, the suspension droplets are deposited on the surfaces of the core particles.

In accordance with a preferred embodiment, the core particles containing said one or more probiotics comprise at least 5 wt.%, more preferably 5-98 wt%, most preferably 10-50 wt% of a bulk ingredient. Various bulk ingredients may be used in accordance with the invention. For example, the bulk ingredient may comprise or consist of hydrocolloids (e.g. carboxymethylcellulose, starch, maltodextrin) and/or fats and/or waxes and/or carbohydrates (e.g. sugars) and/or proteins. Preferably said core particles further comprise one or more of the components selected from the group consisting of fibres, vitamins, minerals, peptides, polyphenols, fatty acids, oils, drugs, bioactive components, flavours, colourants, gas and combinations thereof.

Suitably, the probiotic can be entrapped within the core particle made by e.g. extrusion or other technique, such as spheronization. Hence, the particles referred to in the above method are particles containing one ore more probiotics.

The core material may contain one or more additional (sensitive) additives, such as an enzyme , a prebiotic, a vitamin, a polyunsaturated fatty acid (PUFA), a flavour (e.g. a bitter component, a salty component, an acid components, 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, any part of the intestinal tract environment (e.g. mouth / saliva, stomach acids, intestine, etc.) etc. may be used.

The core particles are preferably spherical. Suitable core particles include particles of at least 50 μm. Preferably the core particles have a diameter of at least 100 μm even more preferably of at least 200 μm and most preferably of at least 300 μm. Typically, the diameter of the core particles does not exceed 5000 μm.

Alternatively, probiotics are provided as a concentrated suspension of cells to the aqueous suspension of activated protein aggregates. Hence, the invention also pertains to a method of producing a probiotic encapsulate, said method comprising the aforementioned

steps a) and b),and further comprising a step c) of providing a concentrated suspension of probiotic cells, preferably containing at least 10 ⁸ cfu/g of one or more probiotics, to said suspension of protein aggregate, and spraying said suspension; and d) forming disulfide cross-links between the activated protein aggregates. The aforementioned spraying conditions may also be applied in the present case.

In the present method one or more (food-grade) additives may be added to the protein aggregates, 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, e.g. the additives do not react with free thiol groups as this would interfere with the cross-linking of the protein. The exception to this concerns cross- linkers which will assist in cross-linking the activated protein aggregates.

Forming disulfide links Although the spraying step already initiates the formation of disulfide cross-links, the protein aggregates deposited on the surface of the core particles may get further cross-linked as soon as they have been deposited onto the core particles, e.g. by applying heat treatment or by applying core particles that contain a suitable oxidizing agent.

Preferably, in this particular embodiment, the formation of the disulfide cross-links is brought about by subjecting the dispensed droplets to heat. A convenient way of subjecting the dispensed droplets to a heat treatment is to dispense the aqueous suspension into a hot gas and/or by spraying the aqueous suspension onto hot core particles.

Alternatively or in addition, cross-linking may be established by pressurization to a pressure in excess of 50 MPa, more preferably at least 100 MPa. Yet another method of establishing further cross-linking of the activated protein aggregates 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 International 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 crosslink 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 are not expected to change significantly during cross-linking.

Another aspect of the present invention relates to a foodstuff, a beverage, a nutritional supplement or animal feed containing from 0.05-60 wt.% of an encapsulate as defined herein.

Yet another aspect of the invention relates to a method of preparing a foodstuff, a beverage, a nutritional supplement or animal feed, said method comprising incorporating from 0.05-60 wt.% of an encapsulate according to the present invention.

Another aspect of the present invention relates to a nutritional supplement or a pharmaceutical product containing from 50-100 wt.% of an encapsulate as defined herein, and suited for oral administration. Yet another aspect of the invention relates to a method of preparing a nutritional supplement or a pharmaceutical product, said method comprising incorporating from 50-100 wt.% of an encapsulate according to the present invention.

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

EXAMPLES

Example 1: Preparation of encapsulated probiotics

Protein solutions were prepared by mixing 70 g of whey protein isolate (BiPRO™; Davisco, USA) in 700 g of demineralized water at room temperature (stirred for 2 h).

Reactive protein aggregates were prepared by heating the whey protein isolate solution at 90°C during 30 min 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 20 μmol thiol groups per gram of protein.

A mixture of a probiotic powder (70 wt.%) and native corn starch mixture (30 wt.%)

(probiotic powder was containing a lactic acid bacteria) was brought in a free flowing spouted bed (Procell technology). Particles were made spherical by agglomeration/erosion in the spouted bed with an aqueous solution containing 1 wt.% methylcellulose and 5 wt.% trehalose as binder. The material thus obtained is called core material.

The activated protein mixture prepared as described above is then sprayed using a fluidized bed coater (Glatt, Germany) onto 281 g spherical core material comprising the lactic acid bacteria.

The encapsulates were then tested under stomach conditions and the survival of the pribiotic cells in the encapsulates was higher than that of the non encapsulated probiotic cells.

Example 2: Solubility assay of the encapsulates containing the lactic acid bacteria

The solubility of the encapsulation matrix of the probiotica-containing encapsulates of example 1 was tested at pH 7 at 20 ⁰ C in distilled water and in an aqueous solution of 2 wt.%

dithiothreitol (DTT). To this end, 75 mg of the encapsulate was dispersed in 50 ml distilled water and 75 mg of the encapsulate was dispersed in 50 ml of an aqueous solution of 2 wt.% dithiothreitol (DTT). The capsules were gently stirred overnight. The supernatant was filtered and the soluble proteins were quantified by spectrophotometer reading at 280 nm.

As shown in Table 1, the encapsulate prepared with reactive protein aggregates (Example 1) were almost insoluble in water at pH 7, whereas they were soluble in the aqueous solution of 2 wt.% dithiothreitol (DTT).

Table 1: Solubility of encapsulates

Example 3: Activity assay of the encapsulates containing the lactic acid bacteria The activity of the lactic acid bacteria encapsulated in the core material and the probiotica- containing encapsulates of example 1 was tested in a MRS agar (Merck) medium after incubation for 3 days at 37°C.

Table 2 showed that the probiotica-containing encapsulates of Examples 1 had an activity of 1.20 10 ¹⁰ cfu/g (colony forming units per gram material). This showed that the probiotica-containing encapsulates had a sufficient high activity to be applied in food applications.

Table 2: Activity of encapsulates