WO1999033341A1

WO1999033341A1 - Substantially water-insoluble matrix containing bioactive substances for slow release

Info

Publication number: WO1999033341A1
Application number: PCT/NZ1998/000193
Authority: WO
Inventors: Patrick Joseph Silcock; Karen Rachel Chenery; Jean-Pierre Ghislain Dufour
Original assignee: Kiwitech Limited
Priority date: 1997-12-24
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: AU1894099A; AU756668B2

Abstract

The invention provides a matrix which is substantially water-insoluble and is capable of effecting a slow release of an encapsulated bioactive substance. The matrix comprises a protein or protein-containing material cross-linked by a cross-linking agent to urea to form a 3-dimensional structure throughout which the bioactive substance is substantially uniformly dispersed. In preferred embodiments, the protein or protein-containing material is acid casein, and the bioactive substance is a trace element such as selenium or cobalt. Also provided is a process of preparing a matrix according to the invention.

Description

SUBSTANTIALLY WATER-INSOLUBLE MATRIX CONTAINING BIOACTIVE SUBSTANCES FOR SLOW RELEASE

TECHNICAL FIELD

This invention relates to the release of bioactive substances and in particular to matrices for effecting such release over an extended period.

BACKGROUND

In many industries, there is a need to be able to effect a controlled release of bioactive substances. In particular, in the agricultural industry, controlled release of veterinary medicaments and nutrients for both plants and animals is often desirable. Specific examples include the release of nutrients such as trace elements, including selenium and cobalt.

Selenium is an essential trace element for both livestock and humans. Unfortunately, the distribution of selenium on earth is uneven, leaving some regions with very high or alternatively very low levels in the soil, with New Zealand and regions of Australia particularly deficient. The selenate ion is the most biologically active form of selenium and is commonly used for livestock supplementation (usually as sodium selenate).

Livestock can be supplemented either directly (through drenching or injection), or indirectly through application to the soil to be taken up by plants to ultimately be ingested by the animal. However with drenching, due to the high toxicity of selenium, only small doses may be used and this increases the frequency with which it must be applied. Frequent re-application is both expensive and inconvenient.

Application of selenium to the soil is an efficient means to supplement the animal. The trace element is applied to the soil, taken up by the plant root system and in turn is taken up by the animal. However, this approach also has problems, which relate mainly to the fact that the selenate ion is very soluble. The selenate ion will either solubilise too quickly to be taken up by the plant and wash into the water table or will be taken up by the plant at high levels boosting an animal's short term levels but failing to provide sufficient levels long term. Sustained, or slow, release of the selenate ion is therefore desirable. This has been attempted through the use of the sparingly soluble barium salt. However, while effective in slow release terms, this approach has the disadvantage that barium is relatively toxic.

It is therefore an object of the invention to provide a slow release formulation which is suitable for use to release bioactive substances such as trace elements (including selenium) which at least provides the public with a useful choice.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the invention provides a matrix which is substantially water-insoluble and which is capable of effecting a slow release of an encapsulated bioactive substance, said matrix comprising a protein or protein-containing material cross-linked by a cross-linking agent to urea to form a 3-dimensional structure throughout which said bioactive substance is substantially uniformly dispersed.

Preferably, the protein or protein-containing material is or contains casein.

More preferably, the protein or protein-containing material is or contains acid casein.

Alternatively, the protein or protein-containing material is or contains rennet casein.

Conveniently, the cross-linking agent is an aldehyde such as formaldehyde, glutaraldehyde or glyceraldehyde. More conveniently, the cross-linking agent is formaldehyde. Alternatively, the cross-linking agent may be a compound which has an aldehydic function, such as a reducing sugar, or a dialdehyde such as malonaldehyde .

Preferably, the bioactive substance is a trace element or medicament. Most preferably, the bioactive substance is a trace element or a mixture of trace elements, most preferably a source of one or more of selenium, zinc, cobalt or copper-

In a preferred embodiment, the matrix can further include a particulate inert filler or bulking agent. It is presently preferred that the inert filler be selected from lime, silica or talc, with milled lime being particularly preferred-

In a further aspect, the invention provides a process for preparing a matrix as defined above comprising the steps of:

(a) forming a reaction mixture in aqueous solution of a protein or protein-containing material, urea, a cross-linking agent capable of cross-linking the protein and urea and a bioactive substance; and

(b) allowing sufficient time for the cross-linking of said protein and urea to occur.

Preferably, once formed, the water-insoluble matrix is then dried.

Preferably, the protein is casein, more preferably acid casein.

Optionally, the reaction mixture can further include an anti-foam agent and/ or a dispersing agent.

Preferably, the anti-foam agent is polydimethylsiloxane, octamethyl cyclotetrasiloxane or a combination of these.

Preferably, the dispersing agent is maltodextrin.

Preferably, the cross-linking agent is formaldehyde, glutaraldehyde or glyceraldehyde with formaldehyde being particularly preferred-

Preferably, the bioactive substance is a trace element or medicament. Most preferably, the bioactive substance is a trace element which is selected from a selenium source, a zinc source, a cobalt source and a copper source, with a selenium source being presently preferred.

Conveniently, said protein and urea are added to the reaction mixture as a water- soluble matrix comprising the protein and urea in aqueous solution.

Optionally, said water-soluble matrix further includes the bioactive substance.

Conveniently, said cross-linking agent is added to the reaction mixture alone or in aqueous solution.

Optionally, said cross-linking agent is added in aqueous solution and said bioactive substance is dispersed in said solution.

Where the slow release matrix is to include an inert particulate filler, it is preferred that said filler be added to the reaction mixture together with the cross-linking agent. Most preferably, the inert filler is added to the reaction mixture as part of an aqueous solution containing the cross-linking agent and the bioactive substance.

Where the final matrix is to include an inert filler, it is preferred that the filler be lime, more preferably milled lime.

Preferably, the pH of the reaction mixture is approximately neutral (pH 7.0). More preferably, the pH is 7.1.

Preferably, the slow release matrix containing the bioactive substance is shaped, eg granulated or pelletised.

It is preferred that the drying step is performed after the shaping step.

Preferably, the reaction mixture will be heated to accelerate the cross-linking between the protein and the urea.

Preferably, the soluble matrix comprises acid casein, urea, maltodextrin, anti-foam agent and alkali in an aqueous solution at a pH of approximately 7. 1. These components will conveniently be present, on a dry weight basis, in the following proportions:

acid casein 20-50% urea 40-70% maltodextrin (DE10) 0-7% ammonia (25% NH₃) 0.5-4% antifoam 0-0.2%

The amount of water added to the soluble matrix is preferably in the range of from 0-233g per lOOg of the soluble matrix ingred ents listed above.

In its presently most preferred form, the soluble matrix comprises, on a wet weight basis:

38% acid casein; 45% urea;

5% maltodextrin (D.E. 10); 2.2% ammonia solution (25% NH₃); 0.2% anti-foaming agent; and 10% water.

On a dry weight basis, this preferred soluble matrix therefore comprises:

42.3% acid casein; 49.6% urea;

5.5% maltodextrin (D.E. 10); 2.4% ammonia solution (25% NH₃); and 0.2% anti-foaming agent.

In a further aspect, the invention provides a slow release substantially water- insoluble matrix obtainable by a process as defined above.

In still a further aspect, the invention provides a water-soluble matrix suitable for use in the preparation of a slow release matrix by a process as defined above comprising, in aqueous solution, casein and urea. Preferably, the soluble matrix will further include a dispersing agent, an anti-foam agent and an alkali.

Most preferably, the soluble matrix will comprise:

38% w/w acid casein; 45% w/w urea;

5% w/w maltodextrin (D.E. 10); 2.2% w/w ammonia solution (25% NH₃); 0.2% w/w anti-foam agent; and 10% w/w water.

The water-soluble matrix may optionally further include a bioactive substance, preferably a trace element, most preferably a selenium source, a zinc source, a cobalt source or a copper source.

DESCRIPTION OF THE DRAWING

While the present invention is broadly as defined above, it will be appreciated that it is not limited thereto and that it further includes embodiments of which the following description provides examples. In particular, a better understanding of the invention will be gained by reference to the accompanying drawings in which:

Figure 1 is a graph showing the cumulative results for elution of selenium from a slow release matrix of the invention.

DESCRIPTION OF THE INVENTION

As defined above, the invention has a number of aspects. In its first aspect, it provides a slow release matrix. This matrix is capable of effecting release of a bioactive substance it contains over an extended period.

In principle, the matrix can be used to effect a sustained release of any bioactive substance or combination of these. It is however preferred that the bioactive substance be a trace element or medicament- In particular, a matrix which is able to effect a slow release of trace elements such as selenium sources, zinc sources, cobalt sources and copper sources is a particular focus of this invention.

The slow release matrix of the invention is effectively a 3-dimensional structure containing cross-linked protein and urea. The bioactive substance intended for controlled release is substantially uniformly dispersed throughout the 3- dimensional matrix structure.

The preferred protein or protein- containing material included in the matrix is casein. Further, while rennet casein is not excluded, it is particularly preferred that the protein be acid casein.

Casein is the preferred protein due to its open structure, relatively large proportion of lysine residues and relatively pure natural state. These properties mean that casein will readily polymerise with little fat or carbohydrate to interfere in the reaction. The result is a highly cross-linked structure.

The preference for urea as the component to which the protein (preferably casein) is cross-linked is that urea allows a high solids level to be attained in the final product while retaining a workable viscosity. Urea is also able to participate in the cross-linking (polymerisation) reaction, particularly where the preferred aldehyde cross-linking agents are used. Through its amine group, urea reacts with the aldehyde cross-linking agent to form aldehyde-urea-chains between the protein (casein) monomers. In addition, urea is preferred as it is a relatively low cost reagent.

As indicated above, the urea and protein (casein) components are cross-linked by a cross-linking agent, which is preferably an aldehyde-based agent. Examples of suitable cross-linking agents include glyceraldehyde and glutaraldehyde. Other examples of cross-linking agents which may be used are compounds which contain an aldehydic function, such as reducing sugars or dialdehydes such as malonaldehyde. It is however preferred that the cross-linking agent be formaldehyde. This preference is due to the ability of formaldehyde to react rapidly with both the preferred protein (casein) and urea to form an insoluble matrix. In addition to the protein, urea, cross-linking agent and bioactive substance, it will be usual for the matrix to include an inert filler or bulking agent. This filler will be particulate, with convenient examples including lime (preferably milled lime) or talc.

The inert filler can be added to further reduce the cost of the matrix, or to supplement the bioactivity of the matrix (eg as a fertiliser) .

The slow release matrix of the invention can be prepared by taking a number of approaches. Essentially, what is involved is the formation of a reaction mixture in aqueous solution of the protein (preferably casein), the urea, the bioactive substance and the cross-linking agent, and then allowing sufficient time for the cross-linking reaction to proceed to completion.

In one preferred approach, the protein and urea components are added to the reaction mixture in aqueous solution together. In this form, the protein and urea constitute a soluble matrix.

The soluble matrix can optionally include a number of additional components. These include dispersing agents (such as maltodextrin) and anti-foam agents. Desirably, an alkali such as ammonia is also included to maintain the pH of the soluble matrix at approximately 7. 1.

In the presently preferred embodiment, the soluble matrix contains acid casein, urea, dispersing agent (maltodextrin D.E. 10), an anti-foam agent, an alkali (ammonia) and added water. Preferably, these components are included in the soluble matrix in the following proportions, on a dry weight basis:

acid casein 20%- 50% urea 40%-70% maltodextrin (D.E. 10) 0%-7% ammonia solution (25% NH₃) 0.5%-4% anti-foam 0%-0.2%

Water may also be included in the soluble matrix. It is preferred that water is present in the soluble matrix in a proportion ranging from 0 to about 233g of water per lOOg of total weight of the remaining ingredients of the soluble matrix. Most preferably, the water soluble matrix is made up of the following components in the following percentages: wet weight basis dry weight basis acid casein 38% 42.2% urea 45% 49.6% maltodextrin (D.E. 10) 5% 5.5% ammonia solution (25% NH₃) 2.2% 2.4% antifoam 0.2% 0.2% water 10.0%

The water soluble matrix will conveniently be prepared by first blending the dry ingredients (the casein, urea, maltodextrin and powdered antifoam) and then mixing the blended dry ingredients with the water and ammonia solution. The resulting mixture is then blended until free of lumps, and matrix allowed to form.

When it is desired to form the substantially insoluble matrix, the water-soluble matrix is combined with the cross-linking agent. This will usually be formaldehyde.

The formaldehyde will conveniently be added to the reaction mix in aqueous solution. A 37% -40% formaldehyde aqueous solution has been found suitable. The amount of the formaldehyde aqueous solution to be added can vary depending on the desired properties of the finished matrix. However, in a preferred embodiment, an amount of formaldehyde corresponding to approximately 26% by weight of the finished matrix of 40% formaldehyde solution will be added.

The bioactive substance to be dispersed throughout the final matrix can be included as part of the water-soluble matrix. However, it is more convenient for the bioactive substance to be included with the aqueous solution of the cross-linking agent (usually formaldehyde) and added to the reaction mixture at the same time as the cross-linking agent. This has been found to reduce problems with ensuring adequate mixing of the bioactive substance.

The proportion of bioactive substance included will vary depending on the nature of the bioactive substance and the end use to which the matrix is to be put and in particular the desired release rate of the bioactive substance in the finished matrix.

Purely by way of example, in particularly preferred embodiments of the invention in which the bioactive substance is selenium (in the form of sodium selenate) or cobalt

(in the form of cobalt hydroxide) , the sodium selenate may conveniently be present in an amount of up to 2.4% of dry weight of the finished matrix, and the cobalt hydroxide may conveniently be present in an amount of up to 1 1% by weight of the finished matrix.

Where the inert filler is also to be included in the final insoluble matrix, this is also conveniently added to the reaction mixture at the same time as the cross-linking agent and bioactive substance. Most conveniently, the cross-linking agent, the bioactive substance and the inert filler will be mixed in aqueous solution for addition to the reaction mixture.

The amount of the inert filler can vary. For example, where the inert filler is milled lime, it can be added to the reaction mixture in an amount such that the filler constitutes up to 74% of the finished matrix (on a dry weight basis). More preferably, the lime will be added in an amount to be from 29% to 59% by dry weight of the final matrix, more preferably approximately 39% by dry weight of the final matrix.

Once all of the components of the reaction mixture are present, the reaction is permitted to proceed to form the substantially insoluble matrix. A suitable reaction period is approximately 7 hours at room temperature. However, it will be appreciated that the reaction period can be substantively reduced if desired through heating of the reaction mixture. For example, the insoluble matrix will be formed in as little as 10 minutes by heating the reaction mixture to a temperature of between 60°C and 70°C.

Following formation of the substantially insoluble matrix, the matrix is preferably dried. For example, the matrix can be formed into a relatively thin layer or sheet and then subjected to heat for a defined period. A period of 24 to 72 hours at a temperature of approximately 65°C has been found to be suitable.

The drying step serves a number of functions: it reduces the moisture content of the matrix; it allows the polymerisation reaction to go to completion; and it removes any residual formaldehyde. It is further preferred that the matrix be shaped, eg granulated or pelletised, in its final form. The process of production can therefore include a shaping eg granulation or pelleting step. It is preferred that the shaping step be performed prior to drying.

Prior to drying, the texture of the insoluble matrix is such that it can be readily formed into small pellets or balls. These are preferably formed and then placed or dropped onto a bed of granulated material such as starch. Proceeding in this way allows the granules or balls of matrix to avoid being stuck together. It also ensures that the granules or balls of matrix have smooth surface characteristics.

In one preferred embodiment, the granulation step involves forming small cylindrical pellets by extrusion of the insoluble matrix, conveniently using a die in a hydraulic press.

As indicated above, this shaping step is preferred. The granules or balls of matrix have an average weight of approximately 0.01 to 0. 1 g and an average diameter of 1.5 to 3 mm.

The particularly preferred matrix formulations which include sodium selenate or cobalt hydroxide as the bioactive substance to be released therefore consist of the following, on a dry weight basis:

(a) Matrix containing sodium selenate:

water soluble matrix 32.3% formaldehyde solution 26.5% sodium selenate 1.9% milled lime 39.2%.

(b) Matrix containing cobalt hydroxide:

water soluble matrix 32.3% formaldehyde solution 26.5% cobalt hydroxide 8.9% milled lime ^" 32.2%. The invention, in its particularly preferred but still illustrative aspects as a slow- release matrix for selenium or cobalt ions, will now be described in the foUowing examples.

Example 1: Formation of Soluble Casein Matrix

The soluble casein matrix consisted of a dry blend of acid casein (46g), urea (54g), maltodextrin (6g) and antifoam (0.2g), which were mixed with water ( 1.2g) and lastly ammonia solution (2.6g, 25% ammonia). The mixture was stirred until free of lumps and left to stand overnight-

Example 2: Formation of a Selenium Fertiliser

Powdered lime (97g) was dry blended with powdered sodium selenate (4.8g, 99% purity) and mixed under low shear with formaldehyde (32.75g, 40% w/v formaldehyde) at room temperature. A soluble casein matrix formed as in Example 1 (71.8g, 55.7% soluble soUds) was added to the above blend and mixed under low shear until homogenous. The wet mixture was left to stand at room temperature for eight hours with intermittent mixing. Upon standing, the mixture became thick and elastic, prior to being roUed out onto a greased tray. Spherical chips are cut from the mass and dried at 60°C for 48 hours.

Example 3: Assessment of Selenium Release from Fertiliser

The selenium release characteristics of 4 separate samples of fertiliser granules made by the general preparative method described in Example 1 were assessed. The granules contained the following, on a dry weight basis: Sample 1 2 3 4 soluble casein matrix ("glue") 30.0% 40.0% 50.0% 40.0% formaldehyde 9.0% 12.3% 18.2% 12.3% sodium selenate 2.4% 2.4% 2.4% 2.4% lime 58.6% 45.3% 29.4% 45.3%

Samples 1 and 2 were ball-like in shape whereas samples 3 and 4 were irregular shaped-chips.

For each fertiUser sample assessed, 5g (approximately 50mg selenium) was mixed with 35g of silica sand (acid purified) and 35g of glass beads (acid steriUsed). The fertiliser-sand-glass bead mixture was poured into a verticaUy mounted column (2.5cm diameter x 18cm long) between two 2cm layers of glass wool. After opening the column tap, 50ml of distilled water was gently added to the top of the column to moisten the contents. The tap was then closed and the column was left to stand for one hour.

Using a 250ml plastic bottle, extended with a 5cm length of tube, 200ml of distiUed water was added to the column by inverting the bottle and clamping it in place. After letting the column stand for an hour, the tap was opened and the first 50ml of filtrate was coUected in a small flask. The flow rate was just less then one drop per second. The column was run continuously for approximately eight hours a day and specific volumes coUected; the first 50ml, the foUowing 200ml, the foUowing 11 and the foUowing five Utres in 11 volumes. The plastic bottle was refilled when it became empty to enable a continuous flow of fresh distiUed water.

The results are summarised in the foUowing Table and in Figure 1 : SELENIUM ANALYSIS RESULTS (SAMPLES 1-4)

Expressed as mg of selenium lost in waste water (total in 5g sample = 50mg)

The results suggest the mode of release is related to the matrices very low solubiUty. As the matrix slowly absorbs water the selenium (as selenate) is able to be released.

A spherical product that is very dense with a smooth surface would appear to provide the best release properties.

Example 4: Preparation of Selenium Fertiliser

Formulation

Soluble casein matrix Fertiliser (wet basis) % weight/weight %weight / weight Ingredient Urea 44.67 14.45

Acid casein 38.05 12.31 Maltodextrin 4.96 1.60 Ammonia (25%) 2.15 0.70 Antifoam 0.16 0.05 Water 10.00 3.23

Powdered lime 39.22 Sodium selenate 1.94 Formaldehyde solution (40%) 26.48

The urea, casein, maltodextrin and antifoam were blended together; and the water and ammonia also blended together. The water and ammonia were added to a

Brabender Plasticorder with W50 mixer set at 50 °C and 50RPM; the blended urea, casein, maltodextrin and antifoam were then added immediately- Mixing was continued for 3 minutes. The powdered Ume, sodium selenate and formaldehyde were blended together and added to the mixer. The mass was then mixed for between 14-24 minutes (or untU the mass was sufficiently cured to enable extrusion). The resulting mixture (a rubbery dough) was removed from the mixer and transferred to an extrusion die. The die was placed in a hydrauUc press and sufficient pressure appUed to extrude a 3mm diameter "spaghetti". The spaghetti was cut into 3mm long cylinders then dried to finish curing for 3 days at 50 °C. Example 5: Preparation of a cobalt fertiliser

Formulation

Soluble casein matrix FertiUser (wet basis) % weight/ weight %weight / weight

Ingredient

Urea 44.67 14.45

Acid casein 38.05 12.31

Maltodextrin 4.96 1.60

Ammonia (25%) 2.15 0.70

Antifoam 0.16 0.05

Water 10.00 3.23

Powdered lime 32.20

Cobalt hydroxide 8.94 Formaldehyde solution (40%) 26.48

A cobalt fertiUser was prepared using the same method as that described above for Example 4, with the variation that the mixing time for the resulting mass was reduced to between 6-14 minutes.

Example 6: Preparation of a cobalt fertiliser

Formulation

Soluble casein matrix Fertiliser (wet basis) %weight/weight %weight / weight

Ingredient

Urea 27.00 11.91

Acid casein 23.00 10.14

Maltodextrin 3.00 1.32

Ammonia (25%) 1.30 0.57

Antifoam 0.10 0.04

Water 45.60 20.1 1

Powdered lime 26.60 Cobalt hydroxide 7.40 Formaldehyde solution (40%) 22.00 The urea, casein, maltodextrin and antifoam were blended together; the ammonia and water were also separately blended together. The water and ammonia were then added to the urea, casein, maltodextrin and antifoam and the mixture mixed until it was free of lumps. The powdered Ume, cobalt hydroxide and formaldehyde were blended together then added to the mixture. The resulting mass was left to cure for 1 to 2 hours at room temperature (or until the mass could be roUed into baUs). The mass was then roUed into Uttle balls and the baUs dropped onto a bed of starch. After 3 hours the baUs were removed from the starch by sieving and cured until hard (48 hours at 70 ^* C).

Example 7: Assessment of cobalt release from fertiliser

The cobalt release characteristics of fertiUser granules made by the preparative method described in Example 6 were assessed, using the same method as that described in Example 3. The results are summarised in the foUowing Table:

COBALT ANALYSIS RESULTS

(Fertiliser of Example 6) Expressed as mg of Cobalt lost in waste water (total in 5g sample = 350 mg) mg in mg in mg in mg in mg in mg in mg in

50ml 200ml 1^st L 2^«i 3 L 4* L 5* L

0.26 0.15 0.41 0.20 0.21 0.18 0.22

INDUSTRIAL APPLICATION

The water-insoluble matrix of the invention has a number of appUcations. These will be dependent primarily upon the bioactive substance dispersed within, and to be released from, the matrix.

The principal description has been of casein-urea matrices which contain a selenium or cobalt source- Such matrices can be used to supply selenium or cobalt to Uvestock, usuaUy indirectly through appUcation to the soil for uptake by pasture plants. This is especiaUy the case where the matrix includes milled lime as an inert filler, which renders the product suitable for use as a selenium- or cobalt- containing fertiUser.

In other appUcations, the selenium-or cobalt-containing matrix can be used to coat seeds to ultimately increase the selenium or cobalt content of products such as cereals.

This can be achieved by either dipping or spraying the seeds with the matrix prior to drying, or by coating the seed with a powdered dried matrix.

It wiU of course be appreciated that the matrix can be used to effect the slow release of other trace elements such as zinc and copper in the same manner as for selenium and cobalt.

Other appUcations wiU involve the use of the matrix to release medicaments, particularly veterinary medicaments. For such an appUcation, the matrix containing the medicament would normally form part of a bolus for administration to the animal.

StiU other appUcations of the matrix, and of its variations, wiU be apparent to those persons skilled in this art.

FinaUy, it will be understood that the above description is exemplary only and is not to be construed as limiting the invention to the specific embodiments set forth.

Claims

1. A matrix which is substantiaUy water-insoluble and which is capable of effecting a slow release of an encapsulated bioactive substance, said matrix comprising a protein or protem-containing material cross-linked by a cross-Unking agent to urea to form a 3-dimensional structure throughout which said bioactive substance is substantiaUy uniformly dispersed.

2. A matrix as claimed in claim 1 wherein the protein or protein-containing material is or contains casein.

3. A matrix as claimed in claim 1 or 2 wherein the protein or protein- containing material is or contains acid casein.

4. A matrix as claimed in any one of claims 1 to 3 wherein the cross-Unking agent is selected from the group consisting of formaldehyde, glutaraldehyde and glyceraldehyde .

5. A matrix as claimed in any one of claims 1 to 4 wherein the cross-Unking agent is formaldehyde.

6. A matrix as claimed in any one of claims 1 to 5 wherein the bioactive substance is source of a trace element or a medicament.

7. A matrix as claimed in claim 6 wherein the bioactive substance is a source of one or more trace elements selected from the group consisting of selenium, cobalt, zinc and copper.

8. A matrix as claimed in claim 7 wherein the bioactive substance is a source of selenium.

9. A matrix as claimed in claim 7 wherein the bioactive substance is a source of cobalt.

10. A matrix as claimed in any one of claims 1 to 9 which further includes a particulate inert filler.

11. A matrix as claimed in claim 10 wherein the inert filler is selected from lime, silica or talc.

12. A matrix as claimed in claim 1 1 wherein the inert filler is milled lime.

13. A process for preparing a substantiaUy water- insoluble matrix as claimed in claim 1 comprising the steps of:

(a) forming a reaction mixture in aqueous solution of a protein or protein-containing material, urea, a bioactive substance and a cross-Unking agent capable of cross-Unking the protein and urea; and (b) aUowing sufficient time for the cross- nking of said protein and urea or occur.

14. A process as claimed in claim 13 wherein the substantiaUy water-insoluble matrix is dried once formed.

15. A process as claimed in claim 13 or 14 wherein the reaction mixture further includes an anti-foam agent.

16. A process as claimed in claim 15 wherein the anti-foam agent is selected from the group consisting of polydimethylsiloxane, octamethyl cyclotetrasUoxane and mixtures thereof.

17. A process as claimed in any one of claims 13 to 16 wherein the reaction mixture further includes a dispersing agent.

18. A process as claimed in claim 17 wherein the dispersing agent is maltodextrin.

19. A process as claimed in any one of claims 13 to 18 wherein the protein or protein-containing material is or contains casein.

20. A process as claimed in any one of claims 13 to 19 wherein the protein or protein- containing material is or contains acid casein.

21. A process as claimed in any one of claims 13 to 20 wherein the cross-

Unking agent is selected from the group consisting of formaldehyde, glutaraldehyde and glyceraldehyde.

22. A process as claimed in any one of claims 13 to 21 wherein the bioactive substance is a source of a trace element or a medicament.

23. A process as claimed in claim 22 wherein the bioactive substance is a source of one or more trace elements selected from the group consisting of selenium, zinc, cobalt and copper.

24. A process as claimed in any one of claims 13 to 23 wherein the protein and urea are added to the reaction mixture as a water-soluble matrix comprising the protein and urea in aqueous solution.

25. A process as claimed in claim 24 wherein the water-soluble matrix comprises acid casein, urea, maltodextrin, anti-foam agent and alkaU in an aqueous solution at a pH of approximately 7.1.

26. A process as claimed in any one of claims 13 to 24 wherein the reaction mixture formed in step (a) further includes an inert particulate filler and the f╧èUer is added to the reaction mixture as part of an aqueous solution containing the cross- Unking agent and the bioactive substance.

27. A process as claimed in any one of claims 14 to 26 wherein the substantially insoluble matrix is granulated or peUetised before the matrix is dried.

28. A substantiaUy insoluble matrix obtainable by a process as claimed in any one of claims 13 to 27.