WO2007073188A1

WO2007073188A1 - Mixed gel system and method for preparation thereof

Info

Publication number: WO2007073188A1
Application number: PCT/NL2006/050324
Authority: WO
Inventors: Freddie Van De Velde; Saskia De Jong - Kok
Original assignee: Stichting Top Institute Food And Nutrition
Priority date: 2005-12-23
Filing date: 2006-12-21
Publication date: 2007-06-28
Also published as: RU2008150380A; ZA200810346B; RU2412607C2; EP2015643A1

Abstract

The present invention relates to a process of preparing a mixed gel system, containing at least one globular protein, at least one polysaccharide and at least 50 wt. % water, and comprising at least two distinct aqueous phases that are in direct contact, including a proteinaceous gelled phase and a non-proteinaceous aqueous phase, said process comprising: a. providing a suspension of protein aggregates of a globular protein that is capable of cold gelation, which aggregates have a hydrodynamic radius in the range of 10-500 nanometers, said suspension further containing one or more polysaccharides; and b. producing the mixed gel system by inducing gelation of the suspension of protein aggregates containing the one or more polysaccharides by reducing the pH of the mixed suspension. Other aspects of the invention concern a rehydratable mixed gel system, a suspension of protein particles and a free flowing powder comprising protein particles, each comprising a proteinaceous gel of globular protein.

Description

MIXED GEL SYSTEM AND METHOD FOR PREPARATION THEREOF

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for the preparation of a mixed gel system containing at least one globular protein, at least one polysaccharide and at least 50 wt.% water, which mixed gel system comprises at least two distinct aqueous phases that are in direct contact, including a proteinaceous gelled phase and a non- proteinaceous aqueous phase. In accordance with the present invention, the proteinaceous gelled phase is prepared by means of cold gelation of a globular protein, such as whey protein, egg protein or soy protein.

The present invention also relates to a rehydratable mixed gel system containing a proteinaceous gelled phase and a non-proteinaceous phase, as well as to a collection of protein particles comprising covalently cross-linked globular protein.

BACKGROUND OF THE INVENTION

Mixed gel systems containing two distinct aqueous phases, including at least one gelled aqueous phase, have been described in the prior art.

EP-A 0 298 561 (Unilever NV) describes edible plastic dispersions comprising at least two condensed aqueous phases, at least one of which is continuous, which dispersion comprises an aggregate-forming gelling agent and another gelling agent. Preferred combinations include a gelling agent (a) selected from gelatine, kappa- carrageenan, iota-carrageenan, alginate, agar, gellan, pectin or mixtures thereof and a gelling agent (b) selected from gelling starch, denatured whey protein, denatured bovine serum protein, denatured soy protein, microcrystalline cellulose and mixtures thereof. EP-A 1 466 630 (Wisconsin Alumni Research Foundation) discloses a hybrid protein-polysaccharide superabsorbing hydrogel comprising an acylated, cross-linked protein matrix; and an anionic polysaccharide matrix interpenetrating with the acylated, cross-linked protein matrix. This European patent teaches to produce the cross-linked protein matrix by adding carboxyl moieties to the lysyl residues in the protein matrix to yield an acylated protein matrix, and then cross-linking the acylated protein matrix with a bifunctional cross-linking reagent. The carboxyl moieties are preferably added to the lysyl residues by treating the protein matrix with ethylenediaminetetraacetic acid dianhydride (EDTAD). The preferred cross-linking agent is glutaraldehyde.

In an article by Bryant et al. (Food Hydrocolloids 14 (2000) 383-390) the results of a study into the influence of xanthan gum on the physical characteristics of heat- denatured whey protein solutions and gels is reported. It is described how a series of heat-denatured whey protein solutions containing different xanthan gum concentrations were prepared by adding varying proportions of 2 wt.% xanthan gum solution and distilled water to 10 wt.% heat denatured whey protein solutions. These solutions were stirred for 10 minutes to dissolve the xanthan gum and then salt solution (4M NaCl) was added. The addition of the salt caused cold gelation of the denatured whey protein. The final composition of the solutions was 8.5 wt.% protein, 0-0.2 wt.% xanthan gum and 200 mM NaCl. The authors conclude that the thermodynamic incompatibility of xanthan gum and heat denatured whey protein leads to phase separation and the formation of a water- in- water emulsion.

SUMMARY OF THE INVENTION

The inventors have discovered that particularly useful mixed gel systems may be prepared by employing a combination of globular protein and one or more polysaccharides, and by inducing cold gelation of the globular protein, using pH reduction. More particularly, the present process comprises the steps of: a. providing a suspension of protein aggregates of globular protein that is capable of cold gelation, said suspension further containing one or more polysaccharides; and; b. producing a mixed gel system by inducing gelation of the suspension of protein aggregates by reducing the pH of the mixed suspension. The present method enables the preparation of mixed gel systems in a particularly reproducible and controlled way. In the present method the one or more polysaccharides can be dispersed and be allowed to hydrate, without significant interference by the globular proteins. Once the one or more polysaccharides are fully dispersed and hydrated, gelation of the globular protein can be induced. Thus, the complex gelation/thickening dynamics of such a mixed protein/polysaccharide system can be controlled very effectively.

The invention also relates to a rehydratable mixed gel system comprising at least two distinct aqueous phases, including a proteinaceous gelled phase and a non- proteinaceous phase, said mixed gel system containing: a. gelled globular protein selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof; b. polysaccharide selected from the group consisting of galactomannans, carrageenans, agar, pectins, pectin derivatives, cellulose derivatives, dextran, dextran derivatives, gellan gum, xanthan gum, alginate, starch, starch derivatives, inulin, inulin derivatives, exopolysaccharides and combinations thereof; wherein the pH of the mixed gel system is between 1 unit above and 1.5 units below the iso-electric point of the globular protein and wherein the mixed gel system has a rehydration value of at least 2.

Furthermore, the present invention provides an aqueous suspension of protein particles comprising at least 50% globular protein by weight of dry matter, at least 50 wt. % of said globular protein being covalently cross-linked, said globular protein being selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof, said particles having a volume weighted diameter in the range of 1-100 μm and being characterised in that the particles with a diameter in excess of 1 μm have an average surface roughness of less 1.2.

The present invention also relates to a free flowing powder comprising dry protein particles which can be hydrated to yield a suspension of protein particles as defined herein before.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a process of preparing a mixed gel system, containing at least one globular protein, at least one polysaccharide and at least 50 wt.% water, and comprising at least two distinct aqueous phases that are in direct contact, including a proteinaceous gelled phase and a non-proteinaceous aqueous phase, said process comprising: b. providing a suspension of protein aggregates of globular protein that is capable of cold gelation, which aggregates have a hydrodynamic radius in the range of 10-500 nanometers, preferably of 15-200 nanometers, said suspension further containing one or more polysaccharides; and c. producing the mixed gel system by inducing gelation of the suspension of protein aggregates containing the one or more polysaccharides by reducing the pH of the mixed suspension. The term "proteinaceous gelled phase" as used herein refers to an aqueous phase that is gelled by a three-dimensional protein network.

The term "non-proteinaceous aqueous phase" as used herein refers to an aqueous phase that does not contain a protein gel in the form of a three-dimensional protein network. Preferably, said non-proteinaceous aqueous phase contains not more than a limited amount of protein, e.g. less than 1 wt.%, more preferably less than 0.5 wt.%, most preferably less than 0.2 wt.% of protein.

The suspension of aggregates of globular protein containing one or more polysaccharides may suitably be produced by: i. preparing an aqueous solution or dispersion containing at least 1% of the globular protein by weight of water; ii. if necessary, adjusting the pH of the solution to at least 1 unit above the iso-electric point of the globular protein; iii. heating and/or pressurising said aqueous solution so as to obtain the suspension of aggregates of the globular protein; and wherein the one or more polysaccharides are admixed to the aqueous solution or dispersion and/or to the suspension of protein aggregates.

Alternatively, the suspension of globular protein may be prepared by dispersing a powder or slurry of previously prepared globular protein aggregates in water. The one or more polysaccharides may be added simultaneously, before or after the globular protein aggregates.

In case the present process encompasses the preparation of the suspension of aggregates of globular protein by heating and/or pressurising, such globular protein should be essentially undenatured before it is subjected to heating and/or high pressures to obtain a suspension of protein aggregates. If a denatured globular protein is employed in the present method, it is not feasible to obtain a suspension of aggregated globular protein that can be cross-linked to form a gel by means of pH reduction. Naturally, the present method may employ a mixture of native and denatured protein, provided the amount of native protein is sufficient to enable the preparation of a proteinaceous gelled phase.

In order to ensure that a solution or dispersion of essentially undenatured globular protein is converted in a cold gelling suspension of protein aggregates, the aqueous solution or dispersion is heated to a temperature of at least 60°C and less than 159°C. Typically, at least 70 wt.% of the globular protein present in the aqueous solution or dispersion is aggregated after the heat and/or pressure treatment. Preferably, at least 90 wt.%, more preferably at least 95 wt.% of the globular protein is aggregated after said treatment.

The mixed gel system of the present invention comprises at least two distinct aqueous phases that are in direct contact, including a proteinaceous gelled phase and a non-proteinaceous aqueous phase. Thus, the mixed gel system may, for instance, essentially consist of a continuous proteinaceous gelled phase and a dispersed non- proteinaceous aqueous phase. The dispersed non-proteinaceous aqueous phase may be thickened or gelled by the one or more polysaccharides, or it may be virtually free of polysaccharides and globular protein. Alternatively, the mixed gel system may essentially consist of a dispersed proteinaceous gelled phase and a continuous non- proteinaceous aqueous phase, which non-proteinaceous phase may be gelled or thickened by the one or more polysaccharides, or it may be virtually free of polysaccharides and globular protein. Another embodiment of the present mixed gel system essentially consist of a continuous proteinaceous gelled phase and a continuous non-proteinaceous aqueous phase, which again may be gelled or thickened by the one or more polysaccharides, or which may be virtually free of polysaccharides and globular protein.

Naturally, the mixed gel systems according to the present invention may contain more than two distinct phases. Thus, the mixed gel system may contain three or more distinct aqueous phases. Furthermore, the mixed system may also contain a fat phase, said phase preferably being a dispersed fat phase. The term "distinct" as used herein, e.g. in relation to the proteinaceous gelled phase and the non-proteinaceous aqueous phase, means that phases are clearly separated in such a way that a clear interface between those phases can be shown to exist. The occurrence of the two distinct phases is suitably determined by means of confocal scanning laser microscopy, whereby the proteinaceous gelled phase is stained with an appropriate dye, e.g. Rhodamine B (see Example 9).

The terminology "two distinct aqueous phases that are in direct contact" is used to make it clear that these two distinct aqueous phases are not separated from one another by an intermediate phase, as is the case, for instance, in a water-in-oil-in-water emulsion.

The present invention offers the advantage that non-modified natural proteins can be employed. Thus, in a preferred embodiment, the globular protein is not acylated or cross-linked by means of a chemical cross-linking reagent, e.g. a bifunctional cross- linking reagent. The gel- forming globular protein employed in the present mixed gel system is advantageously selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof. More preferably, said globular protein is selected from whey proteins, egg proteins and combinations thereof. Most preferably, the globular protein is whey protein. The term "whey protein" covers individual whey proteins, e.g. β-lactoglobulin and α-lactalbumin. Whey proteins may also suitably be employed e.g. in the form of whey powder, whey protein concentrates or whey protein isolates.

The one or more polysaccharides applied in the mixed gel system are advantageously selected from the group consisting of galactomannans, carrageenans, agar, pectins, pectin derivatives, cellulose derivatives, dextran, dextran derivatives, gellan gum, xanthan gum, alginate, starch, starch derivatives, inulin, inulin derivatives, exopolysaccharides and combinations thereof. Particularly preferred polysaccharides include galactomannans, carrageenans, gellan gum, xanthan gum, pectins and combinations thereof. The term "derivatives" as used in relation to polysaccharides refers to polysaccharides that have been chemically modified, e.g. by esterification, carboxymethylation or hydrolysis. The one or more polysaccharides are typically employed in a concentration of 0.001 wt.% to 10 wt.%, preferably between 0.005 wt.% and 8 wt.%, more preferably between 0.01 wt.% and 5 wt.%.

By reducing the pH of the one or more polysaccharides containing suspension of aggregates, cross-linking between the aggregates can be induced. Initially, mainly physical crosslinks are formed. However, at a later stage, in particular in case of whey protein, chemical covalent cross-links may also be formed. Typically, the pH of the one or more polysaccharides containing suspension of aggregates is reduced by at least 0.5 pH unit, preferably by at least 1.0 pH unit, more preferably by at least 1.5 pH unit in order to initiate sufficient cross-linking to obtain a proteinaceous gel. Cross-linking will occur when the pH is reduced to a level that is close to the electric point of the globular protein. Preferably, the pH is reduced to between 1 unit above and 1.5 units below the iso-electric point of the globular protein. More preferably , the pH is reduced to between 0.8 unit above and 1 unit below the iso-electric point of the globular protein. In a preferred embodiment, most of the globular protein present is cross-linked into a proteinaceous gel. Accordingly, more than 50 wt.% of the globular protein is covalently bound in the proteinaceous gelled phase. Even more preferably at least 70 wt.%, most preferably at least 80 wt.% of the globular protein is covalently bound in the proteinaceous gelled phase. The one or more polysaccharides employed in accordance with the present invention may be concentrated in the non-proteinaceous aqueous phase or in the proteinaceous gelled phase, or alternatively these polysaccharides may be distributed across these two phases. It is feasible to produce a mixed gel system comprising a non- proteinaceous aqueous phase that contains virtually no polysaccharides by applying a negatively charged (anionic) polysaccharide in combination with a gelled proteineous phase at a pH below the isoelectric point of the protein.

If there are no negatively charged polysaccharides at the pH at which the mixed gel system is produced, the non-proteinaceous aqueous phase of the mixed gel system typically contains a substantial amount, e.g. at least 30 wt.%, more particularly at least 50 wt.%, even more particularly at least 70 wt.% of the one or more polysaccharides is contained in the non-proteinaceous aqueous phase

According to a particularly advantageous embodiment of the invention the non- proteinaceous aqueous phase is thickened or gelled by the one or more polysaccharides. According to one preferred embodiment of the invention, the mixed gel system obtained after gelation of the globular protein contains a dispersed proteinaceous gelled phase and a continuous non-proteinaceous aqueous phase. Even more preferably, said continuous non-proteinaceous aqueous phase is non-gelled. By removing the continuous non-proteinaceous aqueous phase, the dispersed proteinaceous phase may be recovered in the form of gelled protein particles. These gelled protein particles may advantageously be employed as fat replacer, flavour or drug delivery systems, etc. Thus, in an advantageous embodiment, the dispersed proteinaceous gelled phase is isolated from the mixed gel system by removing the non- proteinaceous aqueous phase and collecting the isolated proteinaceous gelled particles. Even more preferably, the dispersed proteinaceous gelled phase is isolated from the mixed gel system by removing the non-proteinaceous aqueous phase through hydrocyclonation, filtration, centrifugation, sedimentation, and/or washing. Even more preferably, the dispersed proteinaceous gelled phase is isolated form the mixed gel system by removing the non-proteinaceous aqueous phase through washing. The collected particles may suitably be dried to obtain a rehydratable powder.

The mixed gel systems according to the present invention may suitably contain additional components besides water, globular protein and polysaccharides. Examples of additives that may suitably be incorporated in the present mixed gel system include: colourings, flavourings, preservatives, vitamins, minerals, pharmaceutically active substances, non-gelling proteins, sugars, fat, emulsifiers etc. The aforementioned additives can be added at different stages of the present process, e.g. during preparation of the aqueous solution or dispersion containing the globular protein. Alternatively, additives may be added to the suspension of aggregates. In particular if additives are sensitive to the heat and/or pressure conditions employed to obtain the suspension of protein aggregates, it may be advantageous to incorporate additives to the suspension of protein aggregates before gelation is induced.

The present mixed gel system may suitably contain dispersed lipid material, e.g. fat. Typically, the present mixed gel system contains between 60 and 98 wt.% of water and between 0 and 38 wt.% of dispersed lipids, and wherein the water and the optional lipids together constitute at least 90 wt.% of the mixed gel system. Examples of dispersed lipids include fat, emulsifiers, flavonoids, sterols, etc. Another advantageous embodiment of the present invention relates to a process wherein the mixed gel system obtained after gelation of the globular protein contains a continuous proteinaceous gelled phase and a non-proteinaceous aqueous phase. The non-proteinaceous aqueous phase can be a continuous phase or it can be a dispersed phase, the former being preferred. A bi-continuous mixed gel system offers the advantage that the non-proteinaceous phase can be removed relatively easily by means of pressing, whereas the continuous proteinaceous phase provides the "sponge structure". In order to facilitate the removal of the non-proteinaceous aqueous phase, said phase preferably is non-gelled. It was found by the inventors that these types of mixed gel systems can be dehydrated to yield a product that rehydrates extremely well. Although the inventors do not wish to be bound by theory, it is believed that the aforementioned mixed gel systems act as sponges, meaning that they can be compressed and dried to produce a materials that is capable of absorbing a large quantity of water relative to its own weight. Thus, these dehydrated product may be advantageously be employed as absorbing tissue in packaging, as moisture barrier, etc. In accordance with this advantageous embodiment, the process comprises the step of dehydrating the mixed gel system by removing at least 50 wt.% of the water contained therein by means of pressing, centrifuging and/or drying. Even more preferably, at least 70 wt.%, most preferably at least 80 wt.% of the water contained in the mixed gel system is removed. In an advantageous embodiment the water is removed by first pressing the mixed gel system to remove at least 20 wt.% of the water, followed by drying.

In case the present process include dehydration of the mixed gel system, said mixed gel system advantageously comprises two continuous phases, one being a proteinaceous gelled phase. This type of mixed gel may suitably be dehydrated to produce a product that rehydrates very easily and effectively. According to a preferred embodiment, the non-proteinaceous aqueous phase is a thickened phase, wherein the one or more polysaccharides provide the viscosity.

In a particularly preferred embodiment of the process involving dehydration, the one or more polysaccharides largely consist of anionic polysaccharides, such as alginates, pectins, carrageenans, gellan gum, xanthan gum and carboxymethyl celluloses. Even more preferably at least 60 wt.% of the polysaccharides, more preferably at least 80 wt.% of the one or more polysaccharides are anionic polysaccharides. Anionic polysaccharides offer the advantages that they can bind positively charged proteins, especially at pH-values below the isoelectric pH of the globular protein. Thus, mixed gel systems can be produced that contain anionic polysaccharides that are complexed to positively charged gelled globular protein. The water content of these mixed gel systems can be reduced by pressing without loosing a substantial fraction of the anionic polysaccharides, even if said polysaccharides are not present in the form of a polysaccharide gel.

Another aspect of the invention relates to a rehydratable mixed gel system comprising at least two distinct phases, including a proteinaceous gelled phase and a non-proteinaceous phase, said mixed gel system containing 0.01-40 wt.% water, and by weight of dry matter: a. 50-99.5 wt.% of gelled globular protein selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof; b. 0.1-50 wt.% of polysaccharide selected from the group consisting of galactomannans, carrageenans, agar, pectins, pectin derivatives, cellulose derivatives, dextran, dextran derivatives, gellan gum, xanthan gum, alginate, starch, starch derivatives, inulin, inulin derivatives, exopolysaccharides and combinations thereof; wherein the pH of the mixed gel system is between 1 unit above and 1.5 units below the iso-electric point of the globular protein and wherein the mixed gel system has a rehydration value of at least 2. Even more preferably, the pH of the mixed gel system is between 0.8 unit above and 1 unit below said iso-electric point.

The rehydratable mixed gel system preferably has a water content of less than 30 wt.%, more preferably of less than 20 wt.% and most preferably of less than 15 wt.%. Typically, the rehydratable mixed gel system contains at least 0.01 wt.% of water.

The non-proteinaceous phase in the rehydratable mixed gel system preferably is a continuous phase. In another preferred embodiment, the proteinaceous gelled phase is a continuous phase. Most preferably, both the non-proteinaceous phase and the proteinaceous gelled phase are continuous.

As explained herein before it is advantageous to employ anionic polysaccharides as these enable easy production of rehydratable mixed gel systems without substantial losses of polysaccharide during pressing. Consequently, the polysaccharide of the rehydratable gel system is advantageously selected from the group consisting of alginates, pectins, carrageenans, gellan gum, xanthan gum, carboxymethyl celluloses and combinations thereof.

According to a particularly preferred embodiment, the rehydratable mixed gel system has a rehydration value of at least 2.5, preferably of at least 3, most preferably of at least 3.5. The rehydration value is indicative of the amount of water that a given material is capable of absorbing. The rehydration value is calculated from the weight of the starting material and the weight of wet material obtained after sufficient time in excess water to reach maximum absorption, using the following formula: Rehydration value = (wet weight - dry weight)/dry weight.

The rehydratable mixed gel system of the present invention may suitably take the form of a powder, an agglomerate or a film. The rehydratable mixed gel system may also take the form of a shaped article, similar to rehydratable pasta products (macaroni, spaghetti, etc.). Most preferably, the rehydratable mixed gel system is a free flowing powder.

The amount of globular protein contained in the rehydratable mixed gel system preferably is within the range of 70-99% by weight of dry matter. Typically, at least 50 wt. % of the globular protein is cross-linked. Preferably at least 70 wt.%, more preferably at least 80 wt.% of the globular protein contained in the mixed gel system is cross-linked.

As described above, mixed gel systems obtained by the present method and comprising a dispersed proteinaceous gelled phase and a continuous non-proteinaceous aqueous phase can advantageously be used to produce gelled protein particles. Thus, a further aspect of the invention relates to an aqueous suspension of protein particles comprising at least 50% globular protein by weight of dry matter, at least 50 wt.% of said globular protein being covalently cross-linked, said globular protein being selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof, said particles having a volume weighted diameter in the range of 1-100 μm and being characterised in that the particles with a diameter in excess of 1 μm have an average surface roughness of less than 1.2, preferably of less than 1.15.

The surface roughness is indicative of the irregularity of the surface of the protein particles. The surface roughness is calculated from (confocal scanning laser) microscopic images, using quantitative image analysis software (see Example 9). The surface roughness is given by the following formula:

Surface roughness = measured perimeter / perimeter of an ellipse with equal dimensions. The present suspension of protein particles typically contains from 1-60 wt.% of globular protein, preferably from 3-40 wt.% of globular protein. The water content of said suspension typically is within the range of 99-60 wt.%, preferably of 97-60 wt.%. The continuous aqueous phase of the present invention preferably is non-gelled. As explained herein before these gelled particles can advantageously be employed as fat replacer, flavour or drug delivery systems, etc. The present suspension exhibits a texture that can be described as oily or ointment-like. The pleasant texture of the suspension makes it suitable for use as a product base in e.g. dressings, mayonnaise, desserts, spreads and ointments. Typically in these product applications the present suspension can advantageously applied as a product base in a concentration in excess of 70 wt.%, preferably in excess of 80 wt.%.

Fat replacers based on aqueous suspension of protein particles, especially whey protein particles are commercially available under the tradename Simplesse®. The protein particles of Simplesse® differ from the present particles in that they are composed of small agglomerated protein particles and are irregular in shape. Typically, the Simplesse® agglomerates are composed of smaller particles with a diameter that typically lies well below 1 μm. In contrast, the present particles are spheroidal granules with a diameter that usually is well above 1 μm. Furthermore, the present gelled protein particles exhibit a very smooth surface as exemplified by the CSLM images contained in this document. The inventors have conducted experiments in which a 1 wt.% aqueous suspension of a commercially available Simplesse® product (Simplesse® 100) was produced by dispersing said product in reverse osmosis water. The particle size distribution of the suspension was determined using a Mastersizer® 2000 laser diffractometer. Next, the suspension was subjected to 2 hours sonication in a sonication bath (Branson™, model 2510). Again the particle size distribution was measured. It was found that as a result of the sonication treatment, the volume fraction of particles with a diameter of less than 1 μm had increased from about 40% to approximately 90 vol.%, which serves to demonstrate that the agglomerates break up during sonication. Applying the same treatment to an aqueous suspension of gelled protein particles according to the present invention does not result in the formation of large volume fraction of particles with a diameter of less than 1 μm. Accordingly, the present invention also encompasses an aqueous suspension of protein particles comprising at least 50% globular protein by weight of dry matter, at least 50 wt.% of said globular protein being covalently cross- linked, said globular protein being selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof, said particles having a volume weighted diameter in the range of 1-100 μm and being characterised in that after 2 hours of sonication at least 60 vol.%, preferably at least 80 vol.%, more preferably at least 90 vol.% of the gelled protein particles have a diameter of more than 1 μm. According to another preferred embodiment, after 2 hours of sonication, at least 60 vol.%, preferably at least 80 vol.% of the particles have diameter of more than 3 μm, most preferably of more than 5 μm.

The aforementioned gelled proteinaceous particles may suitably be dried to produce a free flowing powder with a water content of less than 40 wt.%. Accordingly, the present invention also relates to a free flowing powder with a water content of less than 40 wt.%, preferably of less than 30 wt.% and most preferably of less than 20 wt.%, which powder can be hydrated to yield a suspension of protein particles as defined herein before. In order for the free flowing powder to have good handling properties, it is preferred that the protein particles are present as agglomerates. These agglomerates are composed of individual protein particles that are loosely bound together, such that they will detach easily when dispersed in water, especially under stirring. The agglomerates of the gelled protein particles can be produced by suitable agglomeration techniques known in the art. Preferably, the free flowing powder has a volume weighted average particle size of at least 30 μm, more preferably of at least 50 μm and most preferably of at least 200 μm. Typically, the free flowing powder has a volume weighted average particle size of not more than 2000 μm, preferably of nor more than 1000 μm..

The amount of globular protein contained in the aforementioned protein particles, whether contained in a suspension or a free flowing powder, preferably exceeds 60%, more preferably it exceeds 75% and most preferably it exceeds 90% by weight of dry matter. Typically, at least 50 wt.% of the globular protein present in the protein particles is cross-linked. Preferably at least 70 wt.%, more preferably at least 80 wt.% of the globular protein contained in the mixed gel system is cross-linked. The invention is further illustrated by means of the following examples.

EXAMPLES

Example 1 Whey protein isolate (WPI; Bipro™ from Davisco Foods International Inc.; La

Sueur, MN, USA) was dissolved in water under continuous stirring (2 h at room temperature). Soluble WPI aggregates were prepared by incubating the WPI solution with an initial concentration of 9 wt.% in portions of 400 mL at 68.5 ⁰C in a water bath for 2.5 h. These heat treatments resulted in over 95% aggregation of the proteins. WPI aggregate solutions were cooled to room temperature using running tap water (30 min). The WPI aggregate solutions were diluted to 3 wt.% concentration by adding a locust bean gum solution. The stock solution of locust bean gum (lbg; C- 130 from CP Kelco Inc.; Lille Skensved, Denmark) had previously been prepared by hydrating the powder over night at 4 ⁰C, followed by heating to 80 ⁰C for 30 minutes under continuously stirring. The final composition of the mixed protein aggregates and polysaccharide suspension was 3.0 wt.% WPI aggregates and 0.175 wt.% locust bean gum.

Gelation was induced by the addition of glucono-δ-lactone (GDL; Gluconal™ from Purac Biochem; Gorinchem, The Netherlands). An amount of 0.25 wt.% was added to reach a pH value of approximately 4.8 after 20 h incubation at 25 ⁰C. All analyses were carried out after incubation of the samples during 20 h at 25°C.

Example 2

The mixed gel system prepared in Example 1 was compressed to 20% of its initial height using an Instron universal testing system (model 5543, Instron Corp.) to remove part of the liquid from the gel. The compressed gel piece (3.7 gram) was dried at 60⁰C for 5h. The dehydrated gel piece (0.3 gram) was brought into an excess water and allowed to rehydrate for at least 2h. The mass of the rehydrated material was 1.5 gram. Thus the rehydration value of the dried gel piece is ((1.5 - 0.3) / 0.3) = 4.

Example 3

Example 1 was repeated except that the concentration of locust bean gum was adjusted. The final composition of the mixed gel system was 3.0 wt.% WPI and 0.40 wt.% locust bean gum. The mixed gel system was dilute with an equal volume of water and centrifuged for 15 minutes at 400Ox g. The obtained pellet was resuspended in water to obtain the original volume and centrifuged for a second time. The protein pellet so obtained was resuspended with water to the original volume. The size distribution of the protein particles was measured using a Malvern MasterSizer X (Malvern Instruments, Spring Lane South) and the mean particle size is reported as the volume-to-surface diameter:

where n, is the number of particles with diameter d, and N is the total number of paarticles. The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 6.8μm.

Example 4

Example 3 was repeated except that, instead of locust bean gum, pectin was added as the polysaccharide during the dilution step. The final composition of the mixed gel system was 3.0 wt.% WPI and 0.40 wt.% pectin (HM pectin, H-6 from CP Kelco Inc.; Lille Skensved, Denmark). The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 12.3μm.

Example 5

Example 3 was repeated except that, instead of locust bean gum, gellan gum was added as the polysaccharide during the dilution step. The final composition of the mixed gel system was 3.0 wt.% WPI and 0.10 wt.% gellan gum (Kelcogel™ F from CP Kelco Inc.; Lille Skensved, Denmark). The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 20.3μm.

Example 6

Ovalbumin (Albumin from chicken egg white, grade III from Sigma-Aldrich, Zwijndrecht, the Netherlands) was dissolved in water under continuous stirring (2 h at room temperature). Soluble protein aggregates were prepared by incubating the ovalbumin solution with an initial concentration of 5 wt.% in portions of 400 mL at 78 ⁰C in a water bath for 22 h. The resulting ovalbumin aggregate solutions were cooled to room temperature using running tap water (30 min). The protein aggregate solutions were diluted by adding a pectin solution. The final composition of the mixed protein aggregates and polysaccharide suspension was 2.0 wt.% ovalbumin and 0.40 wt.% pectin.

Gelation was induced by the addition of glucono-δ-lactone (GDL). An amount of 0.16 wt.% was added to reach a pH value of approximately 4.8 after 20 h incubation at 25 ⁰C. All analyses were carried out after incubation of the samples during 20 h at 25°C. The mixed gel system was diluted with an equal volume of water and centrifuged for 15 minutes at 400Ox g. The obtained pellet was resuspended in water to obtain the original volume and centrifuged for a second time. This protein pellet was resuspended with water to the original volume. The size distribution of the protein particles was measured according to example 3. The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 25.3μm.

Example 7

Example 6 was repeated except that, instead of pectin, kappa carrageenan was added as the polysaccharide during the dilution step. The final composition of the mixed gel system was 2.0 wt.% ovalbumin and 0.20 wt.% kappa-carrageenan (C-40 from CP Kelco Inc.; Lille Skensved, Denmark). The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 15.3μm.

Example 8 Example 6 was repeated except that, instead of pectin, gellan gum was added as the polysaccharide during the dilution step. The final composition of the mixed gel system was 2.0 wt.% ovalbumin and 0.20 wt.% gellan gum. The mean particle size (d_3j2) of the protein particles obtained from this mixed gel system was 10.7μm.

Example 9

The microstructures of the mixed gel systems prepared according example 1 was analysed by means of Confocal Scanning Laser Microscopy (CSLM). The mixed gel systems analysed contained 3.0 wt.% WPI and 0.1 wt.% gellan (Sample A), 3.0 wt.% WPI and 0.2 wt.% gellan (Sample B) and 3.0 wt.% WPI and 0.3 wt.% locust bean gum (Sample C). The mixed gel systems were diluted with an equal volume of water and centrifuged for 15 minutes at 400Ox g. The obtained pellets were resuspended in water to obtain the original volume and centrifuged for a second time. The protein pellets so obtained were resuspended with water to the original volume. Aliquots of the final suspension of protein particles were mixed with Rhodamine B solution (10 μL of a 0.2 wt.% solution per mL sample) and allowed to gel inside a special CSLM cuvette at 25°C.

In addition, a suspension containing 2.5 wt.% of Simplesse® 100 was prepared by mixing the powdered Simplesse® 100 with reversed osmosis water and stirring for at least 5 min with a magnetic stirrer. An aliquot of this sample was mixed with Rhodamine B solution as described above (Sample D).

CSLM-images (160 μm x 160 μm) of the 4 aforementioned samples were recorded at NIZO food research B.V. (Ede, The Netherlands) on a LEICA TCS SP Confocal Scanning Laser Microscope (Leica Microsystems CMS GmbH., Manheim, Germany), equipped with an inverted microscope (model Leica DM IRBE), used in the single photon mode with an Ar/Kr visible light laser. A Leica objective lens (63x/UV/1.25NA/water immersion/PL APO) was used. The excitation wavelength was set at 568 nm for Rhodamine B with an emission maximum at 625 nm. Digital image files were acquired in multiple .tif formats and in 1024x1024 pixel resolution.

The CSLM-images of samples A, B and C as well as of the Simplesse ® sample D are depicted in Figure 1 (Sample A), Figure 2 (Sample B), Figure 3 (Sample C) and Figure 4 (Sample D).

The protein particles prepared according the process of this invention have a spheroidal shape with a smooth surface. In contrast, the commercially available Simplesse® protein particles have an irregular shape with an rough surface. The roughness of the different protein particles was calculated by applying quantitative image analysis on images of Samples A, B, C and D. The Leica Q Win Pro (Leica, Microsystems Imaging Systems Ltd., Cambridge, UK) software package was used for this purpose. The Detect routine of this package was used to create a binary image.

Next the Measure Feature routine was used to calculate for each individual object in the binary image the following parameters (using a detection limit of 10 pixels): Area (μm ), Length (μm), Breadth (μm), Perimeter (μm), and Aspect Ration (-). The Length and Breadth are the long and short axis of the object, whereby the Aspect Ratio is the ratio between these two axis. The length (L) and breadth (B) were used to calculate the perimeter of the corresponding ellipse according to the following formula: P-ellipse = pi * square root (2((L/2)²+(B/2)²) - ((L/2) - (B/2))²/2.458338 The roughness of the objects in the images is defined as calculated Perimeter divided by the perimeter of the corresponding ellipse. Absolutely smooth spheres have a roughness equal to 1. The average roughness was calculated for the object with an aspect ratio smaller or equal to 1.25 to avoid artefacts that result from touching objects. The calculated roughness for the different protein particles is 1.09, 1.06, 1.09, and 1.35 for respectively Sample A, Sample B, Sample C, and Sample D.

Claims

1. A process of preparing a mixed gel system, containing at least one globular protein, at least one polysaccharide and at least 50 wt.% water, and comprising at least two distinct aqueous phases that are in direct contact, including a proteinaceous gelled phase and a non-proteinaceous aqueous phase, said process comprising: a. providing a suspension of protein aggregates of globular protein that is capable of cold gelation, which aggregates have a hydrodynamic radius in the range of 10-500 nanometers, preferably of 15-200 nanometers, said suspension further containing one or more polysaccharides; and; b. producing the mixed gel system by inducing gelation of the suspension of protein aggregates containing the one or more polysaccharides by reducing the pH of the mixed suspension.

2. Process according to claim 1, wherein the suspension of protein aggregates containing the one or more polysaccharides is produced by: i. preparing an aqueous solution or dispersion containing at least 1% of the globular protein by weight of water; ii. if necessary, adjusting the pH of the solution to at least 1 unit above the isoelectric point of the globular protein; iii. heating and/or pressurising said aqueous solution so as to obtain a suspension of aggregates of the globular protein; and wherein the one or more polysaccharides are admixed to the aqueous solution or dispersion and/or to the suspension of protein aggregates.

3. Process according to claim 1 or 2, wherein the gel- forming globular protein is selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof.

4. Process according to any one of the preceding claims, wherein the one or more polysaccharides are selected from the group consisting of galactomannans, carrageenans, agar, pectins, pectin derivatives, cellulose derivatives, dextran, dextran derivatives, gellan gum, xanthan gum, alginate, starch, starch derivatives, inulin, inulin derivatives, exopolysaccharides and combinations thereof.

5. Process according to any one of the preceding claims, wherein the aqueous solution is heated and/or pressurised until at least 70 wt.% of the globular protein is aggregated.

6. Process according to any one of the preceding claims, wherein the pH is reduced by at least 0.5 pH unit, preferably by at least 1.0 pH unit.

7. Process according to any one of the preceding claims, wherein the pH is reduced to between 1 unit above and 1.5 units below the iso-electric point of the globular protein.

8. Process according to any one of the preceding claims, wherein the mixed gel system obtained after gelation of the globular protein contains a dispersed proteinaceous gelled phase and a continuous non-proteinaceous aqueous phase.

9. Process according to claim 8, wherein the dispersed proteinaceous gelled phase is isolated from the mixed gel system by removing the non-proteinaceous aqueous phase and collecting the isolated proteinaceous gelled particles.

10. Process according to claim 9, wherein the non-proteinaceous aqueous phase is removed through hydrocyclonation, filtration, centrifugation, sedimentation, and/or washing.

11. Process according to claim 10, wherein the non-proteinaceous aqueous phase is removed through washing.

12. Process according to any one of claims 1-7, wherein the mixed gel system obtained after gelation of the globular protein contains a continuous proteinaceous gelled phase and a non-proteinaceous aqueous phase.

13. Process according to claim 12, wherein the mixed gel system is dehydrated by removing at least 50 wt.% of the water contained therein by means of pressing, centrifuging, drying or combinations thereof.

14. An aqueous suspension of protein particles comprising at least 50% globular protein by weight of dry matter, at least 50 wt.% of said globular protein being covalently cross-linked, said globular protein being selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof, wherein the particles with a diameter in excess of 1 μm have an average surface roughness of less than 1.2.

15. Aqueous suspension according to claim 14, wherein after 2 hours of sonication at least 60 vol.%, preferably at least 80 vol.% of the protein particles have a diameter of more than 1 μm.

16. A free flowing powder with a water content of less than 40 wt.%, which powder can be hydrated to yield an aqueous suspension according to claim 14 or 15.

17. A rehydratable mixed gel system comprising at least two distinct phases, including a proteinaceous gelled phase and a non-proteincaceous phase, said mixed gel system containing 0.01-40 wt.% water, and by weight of dry matter: a. 50-99.5 wt.% of gelled globular protein selected from the group consisting of whey proteins, egg proteins, soy proteins and combinations thereof; b. 0.1-50 wt.% of polysaccharide selected from the group consisting of galactomannans, carrageenans, agar, pectins, pectin derivatives, cellulose derivatives, dextran, dextran derivatives, gellan gum, xanthan gum, alginate, starch, starch derivatives, inulin, inulin derivatives, exopolysaccharides and combinations thereof; wherein the pH of the mixed gel system is between 1 unit above and 1.5 units below the iso-electric point of the globular protein and wherein the mixed gel system has a rehydration value of at least 2.

18. Rehydratable mixed gel system according to claim 17, comprising a continuous non-proteinaceous phase.

19. Rehydratable mixed gel system according to claim 17 or 18, comprising a continuous proteinaceous gelled phase.

20. Rehydratable mixed gel system according to any one of claims 17-19, wherein the polysaccharide is an anionic polysaccharide selected from the group consisting of alginates, pectins, carrageenans, carboxymethyl celluloses and combinations thereof.