Field of the invention

The present invention relates to aerated food products that include hydrophobins.

Background to the invention

A wide variety of food products contain introduced gas, such as air, nitrogen and/or carbon dioxide. Such foods include frozen and chilled food products, for example ice cream and mousses. Two key considerations arise in the production and storage of aerated food products, namely the ability to incorporate gas into the product during manufacture (foamability) and the subsequent stability of the gas bubbles during storage (foam stability). A number of additives are included in aerated food products to assist in the creation and maintenance of foam. These include proteins such as sodium caseinate and whey, which are highly foamable, and biopolymers, such as carrageenans, guar gum, locust bean gum, pectins, alginates, xanthan, gellan, gelatin and mixtures thereof, which are good stabilisers. However, although stabilisers used in the art can often maintain the total foam volume, they are poor at inhibiting the coarsening of the foam microstructure, i.e. increase in gas bubble size by processes such as disproportionation and coalescence. Further, many of the ingredients used to stabilise the gas phase in aerated food products need to be added at fairly high levels which can have deleterious textural and/or calorific consequences.

Summary of the invention

We have found that a class of proteins found in fungi, termed hydrophobins, combine high foamability and good foam stabilisation properties. In particular, hydrophobins have been found to provide both excellent foam volume stability and inhibition of coarsening. Further, the levels of hydrophobin required to achieve excellent product stability are relatively low. It will therefore be possible to replace some or all of the conventional ingredients used to form and stabilise aerated food products with smaller amounts of hydrophobin. Accordingly, the present invention provides an aerated food product comprising a hydrophobic In a related aspect, the present invention provides an aerated food product in which the air phase is at least partially stabilised with hydrophobin. In another related aspect, the present invention provides an aerated food product comprising hydrophobin in which the hydrophobin is associated with the air phase.

Preferably the hydrophobin is a class Il hydrophobin.

In a preferred embodiment, the hydrophobin is present in an amount of at least 0.001 wt%, more preferably at least 0.01 wt%.

In a related aspect, the present invention provides a composition for producing an aerated food product of the invention, which composition comprises hydrophobin, preferably hydrophobin in isolated form, together with at least one of the remaining ingredients of the food product. Preferably the composition comprises all the remaining ingredients of the food product.

In a related embodiment, the present invention provides a dry composition for producing an aerated food product of the invention, which composition comprises hydrophobin, preferably hydrophobin in isolated form, together with at least one of the remaining non-liquid ingredients of the food product. Preferably the composition comprises all the remaining non-liquid ingredients of the food product.

The present invention further provides the use of a hydrophobin in a method of inhibiting bubble coarsening in an aerated food product.

In a related aspect the present invention provides a method of inhibiting bubble coarsening in an aerated food product which method comprises adding hydrophobin to the food product prior to and/or during aeration of the product. The present invention also provides the use of a hydrophobin in a method of stabilising a foam in an aerated food product.

In a related aspect the present invention also provides a method of stabilising a foam in an aerated food product which method comprises adding hydrophobin to the food product prior to and/or during aeration of the product.

The present invention further provides the use of a hydrophobin in a method of improving shape retention and/or rigidity in an aerated food product.

In a related aspect the present invention provides a method of improving shape retention and/or rigidity in an aerated food product which method comprises adding hydrophobin to the food product prior to and/or during aeration of the product.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in chilled confectionery/frozen confectionery manufacture, chemistry and biotechnology). Definitions and descriptions of various terms and techniques used in chilled/frozen confectionery manufacture are found in Ice Cream, 4th Edition, Arbuckle (1986), Van Nostrand Reinhold Company, New York, NY. Standard techniques used for molecular and biochemical methods can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. - and the full version entitled Current Protocols in Molecular Biology).

Hydrophobins

Hydrophobins are a well-defined class of proteins (Wessels, 1997, Adv. Microb. Physio. 38: 1-45; Wosten, 2001 , Annu Rev. Microbiol. 55: 625-646) capable of self-assembly at a hydrophobic/hydrophilic interface, and having a conserved sequence:

Xn-C-X5-9-C-C-Xi i-ag-C-Xs^-C-Xs-g-C-C-Xe-iβ-C-Xm (SEQ ID No. 1)

where X represents any amino acid, and n and m independently represent an integer. Typically, a hydrophobin has a length of up to 125 amino acids. The cysteine residues (C) in the conserved sequence are part of disulphide bridges. In the context of the present invention, the term hydrophobin has a wider meaning to include functionally equivalent proteins still displaying the characteristic of self- assembly at a hydrophobic-hydrophilic interface resulting in a protein film, such as proteins comprising the sequence:

Xn-C-Xi-so-C-Xo-δ-C-Xi-ioo'C-Xi-ioo-C-Xi-so-C-Xo-δ-C-Xi -so-C-Xm (SEQ ID No. 2) or parts thereof still displaying the characteristic of self-assembly at a hydrophobic-hydrophilic interface resulting in a protein film. In accordance with the definition of the present invention, self-assembly can be detected by adsorbing the protein to Teflon and using Circular Dichroism to establish the presence of a secondary structure (in general, α-helix) (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

The formation of a film can be established by incubating a Teflon sheet in the protein solution followed by at least three washes with water or buffer (Wosten et al., 1994, Embo. J. 13: 5848-54). The protein film can be visualised by any suitable method, such as labeling with a fluorescent marker or by the use of fluorescent antibodies, as is well established in the art. m and n typically have values ranging from 0 to 2000, but more usually m and n in total are less than 100 or 200. The definition of hydrophobin in the context of the present invention includes fusion proteins of a hydrophobin and another polypeptide as well as conjugates of hydrophobin and other molecules such as polysaccharides.

Hydrophobins identified to date are generally classed as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at hydrophobic interfaces into amphipathic films. Assemblages of class I hydrophobins are relatively insoluble whereas those of class Il hydrophobins readily dissolve in a variety of solvents.

Hydrophobin-like proteins have also been identified in filamentous bacteria, such as Actinomycete and Steptomyces sp. (WO01 /74864). These bacterial proteins, by contrast to fungal hydrophobins, form only up to one disulphide bridge since they have only two cysteine residues. Such proteins are an example of functional equivalents to hydrophobins having the consensus sequences shown in SEQ ID Nos. 1 and 2, and are within the scope of the present invention.

The hydrophobins can be obtained by extraction from native sources, such as filamentous fungi, by any suitable process. For example, hydrophobins can be obtained by culturing filamentous fungi that secrete the hydrophobin into the growth medium or by extraction from fungal mycelia with 60% ethanol. It is particularly preferred to isolate hydrophobins from host organisms that naturally secrete hydrophobins. Preferred hosts are hyphomycetes (e.g. Trichoderma), basidiomycetes and ascomycetes. Particularly preferred hosts are food grade organisms, such as Cryphonectria parasitica which secretes a hydrophobin termed cryparin (MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65: 5431-5435).

Alternatively, hydrophobins can be obtained by the use of recombinant technology. For example host cells, typically micro-organisms, may be modified to express hydrophobins and the hydrophobins can then be isolated and used in accordance with the present invention. Techniques for introducing nucleic acid constructs encoding hydrophobins into host cells are well known in the art. More than 34 genes coding for hydrophobins have been cloned, from over 16 fungal species (see for example WO96/41882 which gives the sequence of hydrophobins identified in Agaricus bisporus; and Wosten, 2001 , Annu Rev. Microbiol. 55: 625-646). Recombinant technology can also be used to modify hydrophobin sequences or synthesise novel hydrophobins having desired/improved properties. Typically, an appropriate host cell or organism is transformed by a nucleic acid construct that encodes the desired hydrophobic The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector encoding the necessary elements for transcription and translation and in such a manner that 5. they will be expressed under appropriate conditions (e.g. in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.

o A number of expression systems may be used to express the polypeptide coding sequence. These include, but are not limited to, bacteria, fungi (including yeast), insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors. Preferred hosts are those that are considered food grade - 'generally regarded as safe' (GRAS). 5 Suitable fungal species, include yeasts such as (but not limited to) those of the genera Saccharomyces, Kluyveromyces, Pichia, Hansenula, Candida, Schizo saccharomyces and the like, and filamentous species such as (but not limited to) those of the genera Aspergillus, Trichoderma, Mucor, Neurospora, Fusarium and 0 the like.

The sequences encoding the hydrophobins are preferably at least 80% identical at the amino acid level to a hydrophobin identified in nature, more preferably at least 95% or 100% identical. However, persons skilled in the art may make 5 conservative substitutions or other amino acid changes that do not reduce the biological activity of the hydrophobin. For the purpose of the invention these hydrophobins possessing this high level of identity to a hydrophobin that naturally occurs are also embraced within the term "hydrophobins".

0 Hydrophobins can be purified from culture media or cellular extracts by, for example, the procedure described in WO01 /57076 which involves adsorbing the hydrophobin present in a hydrophobin-containing solution to surface and then contacting the surface with a surfactant, such as Tween 20, to elute the hydrophobin from the surface. See also Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50; Calonje et al., 2002, Can. J. Microbiol. 48: 1030-4; Askolin et al., 2001, Appl Microbiol Biotechnol. 57: 124-30; and De Vries et al., 1999, Eur J Biochem. 262: 377-85.

Aerated Food Products Aerated food products of the invention typically fall into one of four groups - hot, ambient, chilled or frozen. The term "food" includes beverages. Hot food products include beverages such as cappuccino coffee. Ambient aerated food products include whipped cream, marshmallows and bakery products, e.g. bread. Chilled aerated food products include whipped cream, mousses and beverages (, such as beer, milk shakes and smoothies. Frozen aerated food products include frozen confections such as ice cream, milk ice, frozen yoghurt, sherbet, slushes, frozen custard, water ice, sorbet, granitas and frozen purees.

Preferably the aerated food product is an aerated confectionery product.

The term "aerated" means that gas has been intentionally incorporated into the product, such as by mechanical means. The gas can be any food-grade gas such as air, nitrogen or carbon dioxide. The extent of aeration is typically defined in terms of "overrun". In the context of the present invention, %overrun is defined in volume terms as:

((volume of the final aerated product - volume of the mix) / volume of the mix) X 100

The amount of overrun present in the product will vary depending on the desired product characteristics. For example, the level of overrun in ice cream is typically from about 70 to 100%, and in confectionery such as mousses the overrun can be as high as 200 to 250 wt%, whereas the overrun in water ices is from 25 to 30%. The level of overrun in some chilled products, ambient products and hot products can be lower, but generally over 10%, e.g. the level of overrun in milkshakes is typically from 10 to 40 wt%. The amount of hydrophobin present in the product will generally vary depending on the product formulation and volume of the air phase. Typically, the product will contain at least 0.001 wt%, hydrophobin, more preferably at least 0.005 or 0.01 wt%. Typically the product will contain less than 1 wt% hydrophobin. The hydrophobin may be from a single source or a plurality of sources e.g. the hydrophobin can a mixture of two or more different hydrophobin polypeptides.

Preferably the hydrophobin is a class Il hydrophobin.

The present invention also encompasses compositions for producing- an aerated food product of the invention, which composition comprises a hydrophobin. Such compositions include liquid premixes, for example premixes used in the production of frozen confectionery products, and dry mixes, for example powders, to which an aqueous liquid, such as milk or water, is added prior to or during aeration.

Such compositions include liquid premixes, for example premixes used in the production of frozen confectionery products, and dry mixes, for example powders, to which an aqueous liquid, such as milk or water, is added prior to or during aeration.

The compositions for producing a frozen food product of the invention, will comprise other ingredients, in addition to the hydrophobin, which are normally included in the food product, e.g. sugar, fat, emulsifiers, flavourings etc. The compositions may include all of the remaining ingredients required to make the food product such that the composition is ready to be processed, i.e. aerated, to form an aerated food product of the invention.

Dry compositions for producing an aerated food product of the invention will also comprise other ingredients, in addition to the hydrophobin, which are normally included in the food product, e.g. sugar, fat, emulsifiers, flavourings etc. The compositions may include all of the remaining non-liquid ingredients required to make the food product such that all that the user need only add an aqueous liquid, such as water or milk, and the composition is ready to be processed to form an aerated food product of the invention. These dry compositions, examples of which include powders and granules, can be designed for both industrial and retail use, and benefit from reduced bulk and longer shelf life.

The hydrophobin is added in a form and in an amount such that it is available to stabilise the air phase. By the term "added", we mean that the hydrophobin is deliberately introduced into the food product for the purpose of taking advantage of its foam stabilising properties. Consequently, where food ingredients are present or added that contain fungal contaminants, which may contain hydrophobin polypeptides, this does not constitute adding hydrophobin within the context of the present invention.

Typically, the hydrophobin is added to the food product in a form such it is capable of self-assembly at an air-liquid surface.

Typically, the hydrophobin is added to the food product or compositions of the invention in an isolated form, typically at least partially purified, such as at least 10% pure, based on weight of solids. By "added in isolated form", we mean that the hydrophobin is not added as part of a naturally-occurring organism, such as a mushroom, which naturally expresses hydrophobins. Instead, the hydrophobin will typically either have been extracted from a naturally-occurring source or obtained by recombinant expression in a host organism.

In one embodiment, the hydrophobin is added to the food product in monomeric, dimeric and/or oligomeric (i.e. consisting of 10 monomeric units or fewer) form. Preferably at least 50 wt% of the added hydrophobin is in at least one of these forms, more preferably at least 75, 80, 85 or 90 wt%. Once added, the hydrophobin will typically undergo assembly at the air/liquid interface and therefore the amount of monomer, dimer and oligomer would be expected to decrease. In one embodiment, the hydrophobin is added to the aerated food product or compositions of the invention in an isolated form, typically at least partially purified.

The added hydrophobin can be used to stabilise the air phase in an aerated food product, generally by inhibiting bubble coarsening, i.e. hydrophobins have been found not only to stabilise foam volume but also the size of the bubbles within the foam.

The present invention will now be described further with reference to the following examples which are illustrative only and non-limiting.

Description of the figures

Figure 1 is a graph showing overrun as a function of protein concentration of hydrophobin, sodium caseinate and skimmed milk powder in water

Figure 2 is a graph showing the foam stability of 0.1 wt% Hydrophobin expressed as overrun. Foam stability is shown for hydrophobin in (1) water (2) a 20 wt% sucrose solution and (3) a solution of 20 wt% sucrose and 1 wt% guar gum.

Figure 3a is a graph comparing the foam stability of 0.1 wt% Hydrophobin in water with aqueous solutions of 2 wt% sodium caseinate, 2.86 wt% skimmed milk powder (equivalent to about 1 wt% protein) and 6.67 wt% whey protein (equivalent to about 2 wt% protein). The foams produced using hydrophobin are considerably more stable than those from conventional proteins.

Figure 3b is a graph comparing the foam stability of 0.1 wt% Hydrophobin and 2 wt% sodium caseinate in 20 wt % sucrose solution. The foam produced using hydrophobin is considerably more stable than that from 2% sodium caseinate.

Figure 3c is a graph comparing the foam stability of 0.1 wt% Hydrophobin and 2 wt% sodium caseinate in a solution of 20 wt % sucrose and 1 wt% guar gum. The foam produced using hydrophobin is considerably more stable than that from 2% sodium caseinate.

Figure 4 is a scanning electron micrograph of an aerated food product of the invention after (A) 1 day and (B) 2 weeks at chill temperature.

Figure 5 is a graph showing the interfacial rheological properies (G' and G") of the air/water interface in the presence of hydrophobin. It should be noted that the values increase to such an extent that they go beyond the capability of the instrument.

Figure 6 is a graph showing the interfacial elasticity (G') at the air/water interface of 0.00035 wt% hydrophobin in comparison with 0.0007 wt% sodium caseinate and whey protein. Although the hydrophobin reading goes off scale, the result show that the interfacial elasticity of hydrophobin is significantly higher than those formed by convention proteins.

Figure 7 is a diagram showing shear regimes for the aerated frozen products.

Figure 8 is a scanning electron micrograph of product microstructures - fresh and after abuse (Magnification x100)

Figure 9 is a scanning electron micrograph of product microstructures - fresh and after abuse (Magnification x300)

Example 1 - Foamability

(a) Sodium Caseinate, Skimmed Milk Protein or Hydrophobin in water

The foamability of Trichoderma reesei hydrophobin Il (HFB II) was compared to that of the widely used, foamable, dairy protein sodium caseinate (DMV International, the Netherlands. 88-90% protein, i .5% fat and 6% moisture) and skimmed milk (United Milk, UK. 33-36% protein, 0.8% fat, 3.7% moisture). HFBII was obtained from VTT Biotechnology, Finland (purified from Trichoderma reesei essentially as described in WO00/58342 and Linder et al., 2001 , Biomacromolecules 2: 511-517).

The table below shows the concentrations of the protein solutions that were prepared.

Table 1 - Solutions prepared

The dairy protein solutions were prepared using a magnetic stirrer and the protein was sprinkled into the water at room temperature. The solution was then heated to 600C, held for 5 minutes and then cooled to 5°C. The HFB Il solutions were prepared by using a Sonicor ultrasonic bath model SC-42 (Sonicor Instrument Corp). The HFB Il was added either as an aliquot or dry powder which was sonicated for between 30 seconds to 1 minute at room temperature until all of the HFB was dispersed and a clear liquid obtained. This solution was also cooled to 5° C before aeration.

Foams were produced by shearing each solution for up to a maximum of 10 minutes in a cooled (2°C) cylindrical, vertically mounted, jacketed stainless steel vessel with internal proportions of 105 mm height and diameter 72 mm. The lid of the vessel fills 54% of the internal volume leaving 46% (180 ml) for the sample. The rotor used to shear the sample consists of a rectangular impeller of the correct proportions to scrape the surface edge of the container as it rotates (dimensions 72 mm x 41.5mm). Also attached to the rotor are two semi-circular (60 mm diameter) high-shear blades positioned at a 45° angle to the rectangular attachment.

80 ml of solution was poured into the vessel, enough to cover half the rotor, and the lid secured. The solution was then sheared at 1250 rpm for the aforementioned period (table 1). The aerated solution was then immediately poured into a measuring cylinder, thus giving a measure of overrun by volume. Foamability refers to the volume of foam is stated in terms of percentage "overrun", and based on the definition by Arbuckle (ibid).

Overrun % = 100 x (volume of foam - volume of unaerated solution) / (volume unaerated solution)

Figure 1 shows the overruns obtained for the sodium caseinate, SMP and HFB II.

These results show that hydrophobin is at least as foamable as sodium caseinate and SMP, with a lower concentration needed to generate a similar overrun.

(b) Sodium caseinate and HFB in the presence of other ingredients

Sodium caseinate and HFB Il were also aerated in the presence of 20% sucrose (Tate and LyIe) and 20% sucrose + 0.5% guar gum (Willy Benecke, Germany. 78% gum, 14% moisture, 7% protein, 4% acid insoluble residue, 1% fat and 1 % ash). In the case of sodium caseinate with sucrose the dry powders were combined and then slowly added to the water at room temperature that was being mixed on a magnetic stirrer. The solution was then heated to 600C, held for 5 minutes and then cooled to 5°C. When guar gum was present the guar was added to the solution first with half of the sucrose at room temperature. This solution was then heated to 800C and held for 5 minutes before being cooled to 600C. At this point the sodium caseinate was added with the rest of the sucrose. Stirring was continued at the temperature for 30 minutes before cooling to 5°C. In the case of HFB II, it was added separately to a cooled sucrose or sucrose guar solution either as an aliquot or a dry powder. Initial mixing was carried out on a magnetic stirrer followed by 30 seconds in the ultrasonic bath. Table 2 shows the solutions prepared.

These solutions were aerated for 10 minutes as described in section (a) and the overrun obtained by volume in a measuring cylinder. Table 2 shows the overrun obtained for each sample.

Table 2

These results show that hydrophobin has similar foamability to sodium caseinate in a more complex system including sugar, and optionally guar.

Example 2 - Foam stability

The stability of an HFB Il foam was compared to some commonly used dairy proteins: whey, skimmed milk powder and sodium caseinate. After production, foams were poured into a measuring cylinder to assess their stability in terms of foam volume as a function of time. The volume of foam is stated in terms of percentage "overrun", and based on the definition by Arbuckle (ibid).

Overrun % = 100 x (volume of foam - volume of unaerated solution) / (volume unaerated solution) The stability of these foams was measured by monitoring samples contained in 250 ml measuring cylinders and recording serum level and foam height over time at room temperature. The liquid in the foams drain over time, leading to two separate and distinct layers: a foam on top, and aqueous solution below. This is because the aqueous phase does not contain a significant amount of, or any, viscosifiers. However, it is the stability of the foam phase that is the point of interest here. For the calculation of overrun, the volume of foam is taken as the entire volume of the system, i.e. both air (foam) phase and liquid phase irrespective of whether they have separated into two distinct layers. The value of overrun therefore gives a quantitative indication of the stability of the foam to typical break down mechanisms such as coalescence (with themselves and the atmosphere) and disproportionation.

Proteins were dispersed in water alone and in the presence of both 20% sucrose and 20% sucrose + 1% guar gum. Table 3 shows the samples that were prepared. Whey powder (Avonol 600 - 30 wt% protein, 3.5 wt% moisture, 2.5 wt% fat, 7 wt% ash and 53 wt% lactose) was obtained from Glanvia, Ireland.

Table 3

The dairy protein solutions were prepared using a magnetic stirrer and the protein was sprinkled into the water at room temperature. The solution was then heated to 6O0C, held for 5 minutes and then cooled to 5°C. The HFB Il solutions were prepared by using a Sonicor ultrasonic bath model SC-42 (Sonicor Instrument Corp). The HFB Il was added either as an aliquot or dry powder which was sonicated for between 30 seconds to 1 minute at room temperature until all of the HFB was dispersed and a clear liquid obtained. This solution was also cooled to 5°C before aeration.

When 20% sucrose and 20% sucrose + 0.5% guar were present the preparation was slightly different. In the case of sodium caseinate with sucrose the dry powders were combined and then sprinkled into the water at room temperature which was being mixed on a magnetic stirrer. The solution was then heated to 600C, held for 5 minutes and then cooled to 5°C. When guar gum was present the guar was added to the solution first with half of the sucrose at room temperature. This solution was then heated to 800C and held for 5 minutes before being cooled to 6O0C. At this point the sodium caseinate was added with the rest of the sucrose, stirring was continued at the temperature for 30 minutes before cooling to 5°C. In the case of HFB II, it was added separately to a cooled sucrose or sucrose guar solution either as an aliquot or a dry powder. Initial mixing was carried out on a magnetic stirrer followed by 30 seconds in the ultrasonic bath. Foams were produced as described in Example 1 , except that different shear terms were used so as to generate in each case about 100% overrun.

The microstructure of the hydrophobin foam was visualised by Low Temperature Scanning Electron Microscopy (LTSEM). The foam sample was first cut at +50C and plunged into liquid nitrogen. The sample was left at -8O0C on dry ice prior SEM sample preparation. A sample section was cut carefully because of its very fragile structure. This section, approximately 6mmx6mmx10mm in size, was mounted on a sample holder using a compound: OCT ™ on the point of freezing (supplied by Agar Scientific). The sample including the holder is plunged into liquid nitrogen slush and transferred to a low temperature preparation chamber: Oxford Inst. CT1500HF. The chamber is under vacuum, approximately 10"4 - 10"5 mbar. The sample is kept at a temperature below -11O0C on a cold stage. The sample is fractured inside the chamber using a scalpel blade and coated with gold using argon plasma This process also takes place under vacuum with an applied pressure of 10"1 millibars and current of 5 milliamps for 30 seconds. The sample is then transferred to a conventional Scanning Electron Microscope (JSM 5600), fitted with an Oxford Instruments cold stage at a temperature of -15O0C. The sample is examined and areas of interest captured via digital image acquisition software.

Results - Foam stability of foam created using hydrophobin

Foam produced using hydrophobin remained stable over a long time period in all three systems tested (water, + sucrose, + sucrose and guar) - see Figure 2.

Results - Comparison of foam stability of proteins in water.

Foams produced from sodium caseinate, skimmed milk powder, and whey protein are all very unstable compared to foam produced using hydrophobin (see Figure 3A). Further, higher concentrations of skimmed milk powder and whey protein solutions are required to attain an initial overrun of 100% than the concentration needed for hydrophobin.

Results - Comparison of foam stability of hydrophobin and sodium caseinate in the presence of sucrose/guar gum.

Foams produced using hydrophobin remaining very stable for a considerable period of time (2 weeks) whereas foams produced using sodium caseinate were stable for under 20 mins in the presence of sucrose (Figure 3b) and under about 2 hours in the presence of the sucrose and guar gum (Figure 3c).

Therefore, hydrophobin can be used at a low concentration to create significant amounts of foam which remain very stable relative to other commonly used proteins.

In summary, the data show that the foam that is created with 0.1 % HFB Il is more stable that those produced by the other proteins tested. All the foams drain over time (which can be reduced by the addition of thickeners), but the bubbles for the hydrophobin foams still remain stable, i.e. the foam system still retains the air (overrun). In addition, we have found that the bubbles present in foams made with hydrophobin remain stable to bubble coarsening at chill temperatures for at least 2 weeks (see Figure 4 which shows SEM micrographs demonstrating that bubble size is substantially unchanged after 2 weeks). Hence, hydrophobin improves the stability of foams in terms of both foam volume and bubble size. It should be noted that the fractures observed on the surface of the bubbles are believed to be artefacts of the SEM preparation procedure.

Example 3 - Measurement of surface viscosity and elasticity using surface rheometry

A Camtel ClR-100 interfacial rheometer (Camtel Instruments Limited, Royston, Herts, UK), was used to measure the surface viscosity and elasticity. Such data give an indication of how well an adsorbed molecule will stabilise a foam.

The instrument was used in the normalised resonance mode, using a 13 mm diameter du Nouy ring at the surface of the liquid in a 46 mm diameter sample dish. The ring oscillates on the sample surface, and a high-resolution displacement sensor is used to monitor strain amplitude over the range +/- 1°.

Each run was carried out using the same experimental conditions. The runs were time sweeps, with the starting frequency at 3 Hz, and starting amplitude at 10,000 //Radians, and measurements taken at room temperature. The test duration was set at 36,000 seconds, with 240 data points gathered during that time. The physical parameters of interest are G' (storage modulus) and G" (loss modulus) as a function of time, which give an indication of the viscoelasticity of the adsorbed surface layer.

Protein samples were diluted with water to the required concentration. The surface rheology measurements are made relative to pure water, which was measured prior to measurement of the protein solutions.

The surface rheology data is shown in Figures 5 and 6. For the hydrophobin protein, G' and G" increase gradually over time, before a rapid increase in both is observed. In the examples shown, the values increase to such an extent that they go beyond the measuring capabilities of the experimental set up. Even at very low concentrations (less than 0.001 wt%), the values for G' and G" reach values far in excess of the proteins used as comparisons: whey protein and sodium caseinate.

It can be concluded from these data that hydrophobin stabilises foams effectively by forming very strong viscoelastic surface layers around the bubbles. These lead to good stability against typical foam destabilising mechanisms such as coalescence and disproportionation. We believe that whey protein and sodium caseinate foams are both less stable that hydrophobin foams, since the surface layers do not exhibit G' and G" values as high as hydrophobin at comparable solution concentrations.

Accordingly, hydrophobins can be used to inhibit bubble coarsening in an aerated food product, for example by inhibiting or reducing disproportionation and/or coalescence. Similarly, hydrophobins can be used to stabilise foams in an aerated food product. Further, given that hydrophobins inhibit bubble coarsening, it will be possible to improve shape retention and rigidity of aerated products.

Example 4 - Aerated Frozen Products

Aerated frozen products were prepared using 3 types of protein:

A: Sodium Caseinate (Na Gas) B: Skimmed Milk Powder (SMP) C: Hydrophobin (HFBII) from Trichoderma Reesei

Microstructural and physical properties of the products were compared, both before and after temperature abuse regimes. Materials

Details of the materials used are summarised in Table 4 and the formulations

from which each of the aerated frozen products was prepared are shown in

Table 5.

Table 4. Materials used

Table 5. Formulations used 2

Preparation of the Aerated Frozen Products

Mix (Emulsion) preparation

All mixes were made in 10O g batches. For Mixes A and B (containing sodium caseinate and skimmed milk powder, respectively), the protein was combined with the sucrose and dispersed into cold water using a magnetic stirrer. The solution was then heat to 6O0C with stirring and held for 5 minutes before being cooled to 4O0C. Molten coconut fat was then added and the aqueous mix immediately sonicated (Branson Sonifer with 6.4 mm tapered tip) for 3 minutes at 70% amplitude with the tip well immersed in the solution. The emulsion was then cooled rapidly in a -100C water bath until the solution temperature was 5°C, to crystallise the fat droplets. The mixes were stored at 5°C until further use.

For Mix C (containing HFB II), the sucrose was first dispersed into cold water with stirring. Then, half of the required concentration of HFB Il was added to this as an aliquot. The solution was then gently sonicated in a sonic bath for 30 seconds to fully disperse the HFB II. This solution was then stirred on a magnetic stirrer and heated to 400C. Before the molten fat was added the solution was again sonicated in a sonic bath for 30 seconds. The molten fat was then added and the mix was emulsified and cooled as described for Mixes A and B. A further aliquot of HFB Il was then added to this cold solution to bring the HFB Il concentration up to 0.2%. The first 0.1% of HFB Il was for emulsifying and stabilising the fat. The second addition of HFB Il would provide adequate excess HFBII to provide good aeration and foam stability.

Particle size analysis on the chill emulsions was performed using a Malvern Mastersizer 2000.

Analysis of Emulsions Following this methodology, we were able to make emulsions with small fat droplets. A summary of oil droplet sizes measured are shown in Table 6.

Table 6. Emulsion particle size as measured using the Malvern Mastersizer 2000

Shear Freezing Process 80 ml of mix was sheared and frozen simultaneously in the vessel described in Example 1. In essence an aerated and frozen prototype is produced as follows: The mix inside the enclosed container is mixed with an impeller at a high shear rate in order to incorporate air. Simultaneously, the coolant flows around the container jacket to cool and freeze the mix. The impeller also scrapes the inside wall, removing the ice that forms there and incorporating this into the rest of the mix. High shear is used to initially aerated the mix, and then the shear rate is slowed in order to allow better cooling and freezing. The shear regimes used for each mix are graphically presented in Figure 7.

For the freezing and aeration step with Mixes A and B (containing sodium caseinate and skimmed milk powder, respectively) the coolant (set at -18°C) was set to circulate from Time = 0 minutes. The relatively slow stirring at the start for Mixes A and B allowed for cooling of the mix and generation of some viscosity (via ice formation and incorporation) prior to aeration using higher shear. A short time at high speed incorporated the air and then the speed was stepped down to allow the samples to reach at least -5°C.

For Mix C (containing HFB II) the pot was chilled to about 50C and the sample added and the high shear for aerated started. The coolant (set at -18°C) was not switched to circulate on until 9 minutes due to the increased time required to generate 100% overrun. Once the coolant was switched on to circulate (at 9 minutes), the same shear-cooling pattern as previous (for A and B) was adopted.

At the end of the process, overrun was measured and samples (approximately 15 g) were placed into small pots. Each product was cooled further for 10 minutes in a freezer set at -8O0C before being stored at -200C.

Analysis of Aerated Frozen Products

All aerated frozen products were stored under two temperature regimes: (a) -200C. Subsequent analysis was made within one week of production and we deem this as "fresh" product. (b) Temperature abused samples were subject to storage at -1O0C for 1 or 2 weeks, and then subsequently stored at -20°C before analysis.

Table 8. Process details and product overrun for products prepared from Mixes A, B, and C.

Final products were analysed as follows:

Overrun of freshly made product SEM analysis on fresh and temperature abused product Melting behaviour on fresh and temperature abused product Overrun

The overrun for each of the products is summarised in Table 8. All of the mixes were aeratable and incorporated significant amounts of air.

Microstructural Stability: Methodology

Scanning Electron Microscopy (SEM) The microstructure of each products was visualised using Low Temperature Scanning Electron Microscopy (LTSEM). The sample was cooled to -80 0C on dry ice and a sample section cut. This section, approximately 5mmx5mmx10mm in size, was mounted on a sample holder using a Tissue Tek : OCT ™ compound (PVA 11%, Carbowax 5% and 85% non-reactive components). The sample including the holder was plunged into liquid nitrogen slush and transferred to a low temperature preparation chamber: Oxford Instrument CT1500HF . The chamber is under vacuum, approximately 10"4 bar, and the sample is warmed up to -90 0C. Ice is slowly etched to reveal surface details not caused by the ice itself, so water is removed at this temperature under constant vacuum for 60 to 90 seconds. Once etched, the sample is cooled to -11O0C ending up the sublimation, and coated with gold using argon plasma. This process also takes place under vacuum with an applied pressure of 10"1 millibars and current of 6 milliamps for 45 seconds. The sample is then transferred to a conventional Scanning Electron Microscope (JSM 5600), fitted with an Oxford Instruments cold stage at a temperature of -16O0C. The sample is examined and areas of interest captured via digital image acquisition software.

Microstructural Analysis: Results Scanning Electron Microscopy (SEM) was used to examine the microstructure of the fresh and temperature abused frozen products. Representative images can be seen in Figures 8 and 9 at different magnifications.

After temperature abuse the SEM images clearly show that the HFB Il containing product (from Mix C) has retained its original microstructure, i.e. there is little apparent air bubble coarsening. This is the case after 1 and 2 weeks storage at -10°C. However, the prototypes containing Na Cas and SMP (from Mix A and B, respectively) show very severe coarsening of the gas structure under temperature abused at -100C after just one week.

Overall, it is clear that the frozen product made containing HFBII shows much greater stability to temperature abuse than the frozen product made using sodium caseinate or skim milk powder. HFBII has an influence on air bubble stability.

Melting Behaviour: Methodology

Samples of frozen product were placed on a metal grid at room temperature (200C). Differences in the way the products melted, notably shape retention and foam stability, were observed as a function of time.

Melting Behaviour: Results

These microstructural differences (stable foam and stable ice) had some impact on the melting behavior of the frozen product. The aerated frozen sample made from Mix C (containing HFBII) retained its shape better on melting, compared to the product made with sodium caseinate or skimmed milk powder (i.e. Mixes A and B, respectively).

As the ice melted and formed water, it flowed through the melting grid. However, for the product with HFBII, much of the foam also remained on the grid with some stable drops of foam observed beneath - neither of these characteristics was observed with the conventional proteins (sodium caseinate and skimmed milk powder). This illustrates the differences in the foam stability between each of the proteins used.

Textural Differences between Products A, B, and C

Clear differences in texture between the three samples could also be observed after one week storage at -1O0C (i.e. temperature abused samples). On handling the product made using sodium caseinate (A) and skimmed milk powder (B), these were noticed to have a very soft and very flaky texture, which was difficult to cleanly remove from the silicon paper used to line the sample pot. The product made using HFBII (C), on the other hand, was very firm and released from the silicon paper lining the sample pot very cleanly. In other words, the product prepared using HFBII shows much greater stability to temperature abuse on both a microscopic and macroscopic scale than product prepared using sodium caseinate or skim milk powder.

The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and products of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the relevant fields are intended to be within the scope of the following claims.