WO2015124447A1 - Stabilized aerated confection containing hydrophobin - Google Patents

Abstract

A chill or ambient aerated confection is disclosed whose microstructure is stable to temperature abuse during storage for the chill or ambient product.A synergistic stabilization effect regarding the combination of hydrophobin, a secondary protein and a co-surfactant is described that results in the observed stabilization.

Description

STABILIZED AERATED CONFECTION CONTAINING HYDROPHOBIN FIELD OF THE INVENTION

The present invention relates to the production of an aerated chilled or ambient confectionery comprising of hydrophobin, a secondary protein, and a co-surfactant (CSF).

BACKGROUND OF THE INVENTION

It is desirable to make small size bubbles in aerated frozen confections that are stable to temperature abuse in order to improve texture, palatability, and reduce calorific content. It is well understood that frozen confections such as sorbets, sherbets and ice cream experience temperature fluctuations in the distribution chain and in consumers' home freezers. This results in bubble growth and a degradation of the product quality and the palatability on consumption. It is preferable to produce an ice cream with stable bubbles.

It is also desirable to make small size, stable bubbles in aerated chill and ambient confections that are stable to long term storage in order to improve texture, palatability, and reduce caloric content. It is well understood that bubble growth, loss of air and separation of the aerated chill and ambient product cause a degradation of the product quality and the palatability on consumption. It is preferable to produce an aerated chill and ambient product with stable bubbles.

US 2006/0024417 A1 discloses aerated products comprising hydrophobin where the hydrophobin is used to inhibit bubble coarsening.

US 2008/0213453 A1 discloses aerated food products and methods producing them, where in the product comprises hydrophobin and a surfactant.

EP 1 800 543 A1 discloses aerated compositions containing hydrophobins and an additional surfactant.

WO 2007/039065 A1 discloses aerated compositions containing hydrophobins and having a pH below 5.5.

EP 2 052 628 A1 discloses aerated fat-continuous products containing hydrophobin.

We have unexpectedly found that when the combination of hydrophobin, at least one Secondary protein and at least one Co-surfactant (as those terms are defined below) are used to make a chill or ambient confectionery product then: The microstructure of the freshly produced product is preferable since the air bubbles sizes are generally smaller augmenting the beneficial qualities described above.

The microstructure of the product after storage and temperature abuse of the product is preferable since the air bubbles sizes are more stable and do not grow (coarsen) to the same extent as comparative cases.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect of the invention is a chill or ambient aerated food composition including but not limited to:

a. at least 0.01 wt.% of total hydrophobin(s) selected from the group consisting of HFBII, HFBI or Cerato Ulmin or blends thereof added in isolated form to the food composition;

b. one or more Co-surfactants in the total concentration range of about 0.001 to less than about 0.3 wt.% (preferably less than about 0.1 or 0.2 wt.%), and the Co- surfactant(s) to total hydrophobin wt. ratio is in the range of about 0.02 to less than 1.0. (Preferably the Co-surfactant to hydrophobin wt. ratio is at least about 0.05, more preferably at least about 0.3 and preferably at most about 0.75); and

c. one or more Secondary protein(s) that are different from hydrophobin(s);

wherein said Secondary protein(s) are present in a total concentration range of about 0.25 to less than 6.0 wt.% (Preferably the total Secondary protein(s) are at least 0.5 or 1.0 wt.% and at most 4 or 5 wt.%) .

In another aspect of the invention is a process of making an aerated food composition including but not limited to the steps of:

a. Blending the food composition ingredients together and mixing;

b. Adding hydrophobin and a Co-surfactant or Co-surfactants to the chilled mix of step (a);

c. Aerating the mix of step (b) to produce the aerated product; and

d. wherein pasteurisation may be accomplished after step (a) and/or step (b).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of Elastic modulus for Example 1 , samples A) HFBII (Δ), (B) HFBII+SMP (□) and (C) HFBII+SMP+CSF (TWN20 (Tween 20)) (o) at 5°C as a function of time. Figure 2: is a graphical representation of Elastic modulus for HFBI class II (Δ), HFBI class ll+SMP (□), and HFBI class ll+SMP+CSF (TWN20 (Tween 20)) (o) at 5°C for indicated concentrations as a function of time.

Figure 3: is a graphical representation of Elastic modulus for CU class II (Δ), CU class ll+SMP (□), and CU class ll+SMP+CSF (TWN20 (Tween 20)) (o) at 5°C for indicated concentrations as a function of time. Figure 4 shows a schematic depiction of a micrograph illustrating the guard frame concept for bubble size measurement.

Figure 5a depicts macroscope images of the fresh products D (0.02% Tween 20), E (0.02% Tween 60), and F ( 0.02% Panodan) described in Example 4. Also shown is an image for the comparative control sample (labelled Cont)

Figure 5b depicts macroscope images of the chill stored products D, E, and F described in Example 4. Also shown is an image for the comparative control sample (labelled Cont).

Figure 6 depicts the variation of dilational elastic modulus with time for HFBII (Δ), HFBII+ SMP (D),HFBII + SMP + co-surfactant (TWN20 (Tween 20)) (o, inventive), and comparative HFBII + SMP + co-surfactant (TWN20 (Tween 20)) (0, negative control), mixtures of Example5

Figure 7 depicts a photograph of the aerated product prepared using a level of Co- surfactant that is outside of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention is a chill or ambient aerated food composition including but not limited to:

a. at least 0.01 wt.% of total hydrophobin(s) selected from the group consisting of HFBII, HFBI or Cerato Ulmin or blends thereof added in isolated form to the food composition; b. one or more Co-surfactants in the total concentration range of about 0.001 to less than about 0.2 wt.%(preferably less than about 0.1 %), and the Co-surfactant(s) to total hydrophobin wt. ratio is in the range of about 0.02 to about 1.0. (Preferably the Co-surfactant to hydrophobin wt. ratio is at least about 0.05, more preferably at least about 0.3 and preferably at most about 0.75); and

c. one or more Secondary protein(s) that are different from hydrophobin(s);

wherein said Secondary protein(s) are present in a total concentration range of about 0.25 to less than 6.0 wt.% (Preferably the total Secondary protein(s) are at least 0.5 or 1.0 wt.% and at most 4 or 5 wt.%) .

Advantageously the total hydrophobin(s) concentration is at most 1.5 wt.%. Preferably the Co-surfactants is or are water soluble non-ionic surfactant(s). More preferably the Co-surfactant(s) are selected from Polysorbates, polyglycerol esters of alkyl or alkenyl fatty acids, diacetyl tartartic acid esters of mono-/di- glycerides, sucrose esters with an HLB > about 8 or blends thereof. Most preferably the Co-surfactant(s) has a minimum effective HLB value of about 8. Effective HLB value is here defined as the arithmetic mean of the HLB values of a blend of Co-surfactants. Advantageously the Co- surfactant(s) is selected from Tweens 20, 60 or 80, PGE-O-80; Panodan-Visco Lo 2000 and blends thereof.

Preferably the average bubble diameter (d3,2) is at least 10 % smaller after the standard temperature abuse protocol described below than the same product prepared the same way but absent either hydrophobin(s), Co-surfactant(s) or if both are present then outside the total Co-surfactant(s) to hydrophobin(s) ratio range of about 0.02 to less than 1.0. Preferably the average bubble size is at least 20, 30, 40 or 50 % smaller.

Preferably the average bubble diameter d(3,2) of the freshly prepared product is at least 10% smaller, preferably 15% smaller, and more preferably 20% smaller than the same product prepared the same way but absent either hydrophobin(s), Co- surfactant(s) or if both are present then outside the total Co-surfactant(s) to

hydrophobin(s) ratio range of about 0.02 to less than 1 .0.

In the case of the chill or ambient embodiment, preferably after 6 weeks storage at 5°C no more than 10 bubbles, preferably no more than 7 bubbles, more preferably no more than 5 bubbles and most preferably no more than 3 bubbles of a size greater than 6 mm in diameter are observed in a sample size of 9437 mm squared area where the sample is in a 10 mm deep container and had an initial overrun between 30 and 200% overrun, more preferably between 50 and 150% overrun. In another aspect of the invention is a process of making an aerated food composition including but not limited to the steps of:

a. Blending the food composition ingredients together and mixing;

b. adding hydrophobin and a Co-surfactant or Co-surfactants to the chilled mix of step (a);

c. Aerating the mix of step (b) to produce the aerated and frozen product; and d. Optionally further ingredients could be added after aeration

e. wherein pasteurisation may be accomplished after step (a) and/or step (b).

All percentages, unless otherwise stated, refer to the percentage by weight, with the exception of percentages cited in relation to the overrun.

Confections

The term "chill or ambient confection" means an edible confection made by a mix of ingredients which includes water. Such confections typically contain fat, non-fat milk solids and sugars, together with other minor ingredients such as stabilisers, emulsifiers, colours and flavourings. Chill or ambient confections include mousses, desserts, yoghurts, milk shakes and the like.

Aeration

The term aeration means that gas has been incorporated into a product to form air cells. The gas can be any gas but is preferably, particularly in the context of food products, a food-grade gas such as air, nitrogen or carbon dioxide or a mixture of the aforementioned. The extent of the aeration can be measured in terms of the volume of the aerated product. The stability of the aeration can be assessed by monitoring the volume of the aerated product over time and or the bubble size change over time.

Microstructure

The microstructure of chill or ambient confections is critical to their organoleptic properties. The air cells incorporated into confections are preferably small in size which ensures that the confections do not have a coarse texture and also ensures that they deliver a smooth creamy mouth-feel. In typical aerated products, the air bubbles coarsen over time (through distribution and storage) leading to a degradation in quality.

Chill product

A chill product is one that is typically distributed and stored at temperatures between 0°C and 10°C

Ambient product

An ambient product is one that is typically distributed and stored at temperatures between 15°C and 40°C, and more preferable at temperatures between 15°C and 30°C

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-39-C-X₈-23-C-X₅-9-C-C-X6-i8-C-X_m (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:

X_n-C-Xi-5o-C-Xo-5-C-Xi-ioo-C-Xi-ioo-C-Xi-5o-C-Xo-5-C-Xi-5o-C-X_m (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, a-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+n <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 hydrophobilic interfaces into amphipathic films. Assemblages of class I hydrophobins are relatively insoluble whereas those of class II 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 conserved 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

W096/41882 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 hydrophobin. 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 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.

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).

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 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 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". 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. The hydrophobin used in the present invention can be a Class I or a Class II hydrophobin. Preferably, the hydrophobin used is a Class II hydrophobin. Most preferably, the hydrophobin used is HFBI, HFBII, or CU (cerato ulmin). The

hydrophobin used can also be a mixture of hydrophobins, e.g. Class II hydrophobins HFBI and HFBII.

The product should comprise at least 0.01 wt.% hydrophobin, more preferably at least 0.025 wt.% hydrophobin and most preferably at least 0.05 wt.%. Preferably the hydrophobin is present in an amount of 1.5 wt.% maximum and more preferably 0.5 wt.% maximum and most preferably 0.2 wt.% maximum.

Overrun

The extent of aeration of a product is measured in terms of "overrun", which is defined as:

weight of mix— weight of aerated product

% Overrun = - - - x 100

weight of aerated product

Where the weights refer to a fixed volume of mix or product. Overrun is measured at atmospheric pressure.

Preferably the overrun of the product is between 10 and 400% overrun, more preferably between 10 and 300% overrun, and most preferably between 20 and 250% overrun. Preferably the measurements are taken immediately after aeration is ended. Dilational Interfacial Rheology

Interfacial or surface rheology, defines the functional relationship between stress, deformation and rate of deformation at an interface in terms of coefficients of elasticity, and viscosity, arising from relaxation processes. The technique is referred to as dilational interfacial rheology when the experimentally imposed interfacial deformation arises from variation of area at constant shape. The investigation of the dilational rheology of adsorbed layers is useful to access the macroscopic viscoelastic properties of interfaces and can be used to predict the stability of a foam once formed. In the dilational deformation mode, the use of the interfacial tension (γ) response to relative area variation (ΔΑ/Α) provides for the definition of the dilational viscoelasticity. Assuming harmonic area perturbations of small amplitude of frequency v, the dilational viscoelasticity can be written using a linear approximation approach, as d in A where the viscoelastic modulus E can be further split into its elastic (E_e) and viscous (Ev) components (see e.g. R. Miller, L. Liggieri, Interfacial Rheology, Brill, Leiden, 2009, Ch.5, 138). One criterion to reduce bubble coarsening (e.g. coalescence and/or disproportionation) is to confer adequate interfacial properties (particularly elasticity) to the air/water surface, i.e. the bubble surface. Experimental data show that high interfacial elasticity (for completely "elastic" interfaces) is able to slow down the rate of disproportionation (see e.g. W. Kloek, T. van Vliet, M. Meinders, J Colloi Interf Sci, 2001 , 237, 158), and consequently this bubble/foam coarsening.

This criterion has been used here to predict the stability of fully formulated aerated confections stored under controlled thermal conditions. Verification of the stability of the ice cream was assessed using SEM (Scanning Electron Microscopy) and observations compared with predictions from the dilational interfacial rheology experiments.

Co -surfactants

A Co-surfactant (CSF) is defined as:

An ingredient which, when mixed in an aqueous solution containing: 0.001 wt.% hydrophobin

and between 0.0015 and 0.2 wt.% of at least one non-hydrophobin (Secondary) protein at a concentration effective to confer an air/water surface dilatational elasticity that is at least 30% of that of 0.001 wt.% pure hydrophobin (absent the Co-surfactant), more preferably at least 50%, more preferably at least 55%, more preferably at least 65% and most preferably at least 70%, measured between 600 and 7200^" s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05 Hz using the procedure provided below. The effective concentration will depend on the identity of the Co-surfactant. Preferably the effective concentration will be in the range of 0.001 to less than 0.2 wt.% based on the product; more preferably in the range of 0.005 to 0.2 wt.% and most preferably in the range of 0.01 to 0.1 wt.%.

Preferably the Co-surfactant is chosen as one of more of the following:

- E.g. Polysorbates, including Polysorbate 20, 60 and/or 80 also known as Tween

20, 60, and 80 or Polyoxyethylene (20) sorbitan monolaurate, Polyoxyethylene (60) sorbitan monolaurate and Polyoxyethylene (80) sorbitan monolaurate respectively.

Polyglycerol esters of fatty acids, particularly PGE-O-80 as supplied by Danisco - Diacetyl tartartic acid esters of mono-/di- glycerides, particularly Panodan

Visco-Lo 2000as supplied by Danisco

- Sucrose esters of HLB > 8 or 12 (HLBs of SP70 and SE1670 are 15 and 16, respectively) Secondary protein(s)

Secondary protein(s) are defined as non-hydrophobin proteins that when mixed with 0.001 wt.% hydrophobin in aqueous solution at a concentration of 0.04 wt.% results in an air/water surface dilatational elasticity that is at least 35% less than that of hydrophobin alone, more preferably at least 40% less, more preferably at least 50% less, where the dilatational measurement is made between 600 and 4000s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05Hz. Secondary proteins, are advantageously chosen from food proteins such as: skim milk protein (SMP), whey protein, soy protein, or mixtures thereof and the like. It may be noted that many Secondary proteins such as skim milk powder are not typically received and used as pure proteins, since they consist of other ingredients such as lactose and other non-protein materials. Therefore the protein content must be taken into account during formulation.

Other Product Ingredients

Further additional ingredients are typically added to make the inventive confectionary product(s). These include and are not restricted to: Sugars, e.g. sucrose, fructose, dextrose, corn syrups and sugar alcohols and the like. Fats, e.g. coconut oil, butter oil, palm oil and the like. Preferably the fat content of the product is less than 5 wt.%, more preferably less than 3 wt.%, more preferably less than 2 wt.%, most preferably less than 1.5 wt.%, 1.0, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.01 , 0.001 wt.% or zero.

Emulsifiers, e.g. mono/di glycerides of fatty acids and the like other than Co- surfactants.

Stabilisers or thickeners, e.g. locust bean gum, guar gum, tara gum, carrageenans, alginates, pectins, citrus fibres, xanthan, gelatine and the like.

Flavours and colours, e.g. vanilla, fruit purees, chocolate, mint and the like.

The invention will now be described in greater detail by way of the following non- limiting examples. The examples are for illustrative purposes only and not intended to limit the invention in any way. Physical test methods are described below:

Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts or ratios of materials or conditions or reaction, physical properties of materials and/or use are to be understood as modified by the word "about".

Where used in the specification, the term "comprising" is intended to include the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more features, integers, steps, components or groups thereof.

All percentages in the specification and examples are intended to be by weight unless stated otherwise

EXAMPLES:

Example 1 : Surface dilatational rheology measurements to demonstrate the effect of one inventive Co-surfactant

Surface dilatational rheology measurements were taken using formulations A, B, and C shown in Table 1.

Table 1 : Formulations used for Example 1 in wt.%

* Concentration for SMP (skim milk powder) is that stated for the total powder. The amount of protein in SMP was stated by the manufacturer as 35 wt.%.

The elastic modulus data are summarised in Figure 1 . We can appreciate the unexpectedly different effects of adding SMP or SMP + Co-surfactant (Tween20) to HFBII at the indicated concentrations. Addition of SMP (squares, Example 1 B) clearly reduces the elastic interfacial modulus of HFBII ( triangles, Example 1A). Addition of Co-surfactant ( circles Example 1 C) to the SMP + HFBII mix promotes a recovery of the interfacial elasticity that is substantially different than the comparative case i.e. HFBII + SMP.

This recovery is preferred since it will lead to a more stable foam structure in an aerated confectionery stored at chill temperatures where the ratio of HFBII : Co- surfactant : SMP is similar to that described in this example, although the absolute concentrations will be greater in order to make the food product. Example 2: Surface dilatational modulus for a range of hydrophobin, skim milk protein, and Co-surfactant comparative and inventive cases.

Evaluating the extent of the recovery (if any) of the elastic modulus (E_ei) in presence of a Co-surfactant over the comparative hydrophobin + SMP case (e.g. Example 1 B) is a method that was found useful to predict the quality of the ice cream microstructure after the chill storage test.

The elasticity of the a/w (air/water) interface in presence or absence of a Co-surfactant can be listed at set time points (e.g. 1200s, 1800s, 2400s, 3600s) and compared with the elasticity (absolute and percentage) of reference hydrophobin example alone (measured at 3600s).

Elasticity of HFBII (0.001 wt.%) measured after 3600s is 212.9±19.4 mN/m (average of five repetitions) - referring to solution in Example 1A, and illustrated in Figure 1 .

Table 2: Elastic modulus (mN/m) compared at defined time points for HFBII

(0.001 wt.%) + SMP(0.041 1 wt.%) + either inventive Co-surfactant or a comparative surfactant (at indicated concentration). Parenthetical expressions indicate percentage (%) of HFBII(0.001 wt.%) elastic modulus (219 mN/m = 100%) measured at 3600s.

Inventive Co-surfactant or comparative Repli¬

1200s 1800s 2400s 3600s Notes surfactant cates

(wt. %)

NONE (Comp.) 84.3 87.0 90.2 93.6 2

(39.6%) (40.08%) (42.3%) (43.9%)

Tween 20 (Inv.) 76.8 86.9 104.6 138.8 3

(0.00005 wt.%) (36.0%) (40.8%) (49.1 %) (65.2%)

Tween 20 (Inv.) 72.2 97.4 127.4 139.0 4

(0.0001 wt.%) (33.9%) (45.7%) (59.8%) (65.2%)

Tween 91.2 89.3 1 13.6 146.5 2

20(lnv.) (0.00015 (42.8%) (41.9%) (53.3%) (68.8%)

wt.%)

Tween 60(Comp.) 63.5 71.2 78.1 90.2 1

(0.0001 wt.%) (29.8%) (33.4%) (36.6%) (42.3%) Inventive Co-surfactant or comparative Repli¬

1200s 1800s 2400s 3600s Notes surfactant cates

(wt. %)

Tween 60 (Comp.) 75.3 92.0 99.6 1 14.9 2

(0.0002 wt.%) (35.4%) (43.2%) (46.8%) (54%)

Tween 60 (Inv.) 82.1 108.2 123.2 2 exp. (0.0003%) (38.5%) (50.8%) (57.8%) term.

at

2400s

Tween 68.9 80.1 98.7 124.1 3

80 (Inv.) (0.0001 %) (32.3%) (37.6%) (46.3%) (58.3%)

PGE-O-80 (Inv.) ^•1 19.5 1 exp. (0.0001 wt.%) (56.1 %) term. Poly-glycerol Ester at ^*

900s

PGE-O-80 (Inv.) 1 14.5 126.3 121 .1 1 exp. (0.00005 wt.%) (53.8%) (59.3%) (56.9%) term. Poly-glycerol Ester at

2400s

Panodan Visco Lo 25.7 56.5 95.8 148.7 1

2000 (Inv.) (0.0002 (14.31 %) (31.47%) (53.36%) (82.8%)

wt.%)

Sucrose Ester 60.5 109.1 129.2 152.4 1

SE1670 (Inv.) (28.4%) (51.2%) (60.7%) (71.6%)

(0.0002 wt.%)

Sucrose Ester 59.7 71.3 83 (39%) 124.7 2

SE1670 (Inv.) (28.0%) (33.5%) (58.6%)

(0.0001 wt.%)

Sucrose Ester SP70 57.9 62.7 72.8 102.4 2

(Comp.) (0.0001 (27.2%) (29.5%) (34.2%) (48.1 %)

wt.%)

Erythritol (Comp.) 80.5 85.7 89.2 94.9 1

(0.0001 wt.%) (37.8%) (40.2%) (41.8%) (44.5%) Inventive Co-surfactant or comparative Repli¬

1200s 1800s 2400s 3600s Notes surfactant cates

(wt. %)

PGE55 (Comp.) 61.1 65.5 67.3 75.8 2

(0.0001 wt.%) (28.7%) (30.7%) (31.6%) (35.6%)

Hygel (Comp.) 74.8 79.0 79.9 81.1 1

(0.00015 wt.%) (35.1 %) (37.1 %) (37.5%) (38.0%)

Example 3: Use of a Co-surfactant with Class II hydrophobin HFBI and CU in the presence of milk protein

In the following section we show that the recovery of interfacial elastic modulus promoted by CSF is a common feature to different variants of class II hydrophobins. In this case, using either HFBI or CU, both of which are class II hydrophobins. Surface dilational rheology experiments were carried out in the same manner as in the previous examples, except in these cases ΔΑ/Α=2.5%.

Figure 2 shows the elastic interfacial modulus for pure Class II hydrophobin HFBI in water (top, triangles) at a concentration of HFBI = 0.2/200 wt.%. The addition of SMP to such a solution at a concentration of 8.22/200 wt.% (bottom, squares) clearly reduces the elastic interfacial modulus of compared Class II hydrophobin HFBI alone. Addition of a CSF (middle, circles) to the SMP + Class II hydrophobin HFBI mix at a concentration of 0.02/200 wt.% promotes a recovery of the interfacial elasticity. The same pattern is seen in Figure 3 for CU, the other class II hydrophobin, although CU is active (top, triangles) at a higher concentration (1.5/200 wt.%) than the other hydrophobins. The addition of SMP (8.22/200 wt.%) clearly reduces the elastic interfacial modulus (bottom, squares) in comparison to value measured for Class II hydrophobin CU alone (triangles). Addition of a CSF (middle, circles) to the SMP + Class II hydrophobin CU mix at a concentration of 0.02/200 wt.% promotes a recovery of the interfacial elasticity These data demonstrate that the use of a Co-surfactant works with other class II hydrophobins.

Example 4: Stability of foams at chill temperature in the presence and absence of a preferred level of co-surfactant

Chilled, aerated confections (D), (E), and (F) were made using formulations

summarised in Table 3. A control formulation with no Co-surfactant (Cont) was also prepared. The relative amounts of hydrophobin, SMP, and Co-surfactant are selected from the examples measured in Examples 1 and 2.

Table 3: Formulations of chilled aerated confections for macroscope images shown in Figure 5.

The macrographs of the fresh and stored products D, E, and F, are shown in Figure 5. Also shown are the images for the control samples (labelled Cont). Figure 5a shows macroscope images of the foams prior to storage and Figure 5b shows images of the foams after 4 weeks storage at chill temperature. From the images presented it can be seen that: Products D, E and F have smaller air bubbles initially than the control sample (with no co-surfactant), and after storage, the differences between the test samples and the control are even greater. In particular, we note that after storage, Products D, E, and F, show a much smaller proportion of bubbles > about 5mm in diameter in comparison to the control. Table 4 shows the changes in product volume with time for the inventive samples and the comparative example function of storage time. From Table 4 it can be seen that the samples containing a Co-surfactant at a preferred level have greater stability to overrun loss than the comparative example.

Table 4: Changes in product volume with storage time at chill temperature for the inventive samples D, E and F and the comparative control sample. The product volume decreases as overrun is lost.

It was found that the recovery of the elastic modulus (E_e) for the inventive Co- surfactant containing solutions (shown in Examples 1 and 2) was associated with smaller foam bubble sizes initially and increased stability of the foam during storage in our inventive samples D, E and F.

Example 5: Surface dilatational modulus for a range of hydrophobin, skim milk protein, and Co-surfactant within the inventive range and level outside of the inventive range

Figure 6 shows how the dilational elastic surface modulus of a bubble varies with each composition using the same dilational measurement technique as described in the Methods' section. The formulations used are given in Table 5. Table 5: Formulations used for Example 5 ^* Concentration for SMP (skim milk powder) is that stated for the total powder. The amount of protein in SMP was stated by the manufacturer as 35 wt.%.

Ingredient A B C Negative Test

(reference) (comparative) (inventive)

HFBII 0.001 % 0.001 % 0.001 % 0.001 %

SMP ^* - 0.041 % 0.041 % 0.041 %

Co-surfactant 0.0001 % 0.0015%

Tween 20

HFBII to Co- - - 0.1 1 .5 surfactant ratio

Addition of SMP reduced elasticity by over half. Addition of a co-surfactant at a level that lies within our claimed HFBI I/co-surfactant ratio was found to unexpectedly improve the surface elasticity of H FBI I in an HFBI I/SMP mixture. However, if we use a higher level of Co-surfactant we are unable to produce a bubble that was stable enough to measure within the time window (1 hour) of the experiments. Table 6 shows the interfacial surface elasticity (E_ei) for a sample within the preferred Co-surfactant range, sample C (Inventive), and a sample outside the preferred range (Negative Test). From the Table it may be seen that in the Negative Test sample case the bubble produced had a low elastic modulus and was very unstable

Table 6 : Interfacial Elastic modulus (E_e) (mN/m) compared at defined time points for cases shown in Example 5

Therefore, we conclude that the selection of a Co-surfactant is not obvious and is not taught in the prior art. This is because Co-surfactants have to be used within our inventive ranges e.g. Co-surfactant Tween 20 in the Co-surfactant to HFBI I ratio of 0.1 (inventive) provides a high Interfacial Elastic modulus of 140.2 mN/m but the same Co- surfactant in the Co-surfactant to HFBI I ratio of 1 .5 ( the Negative Test sample) produced a low Interfacial Elastic modulus of 48.3 mN/m after 1200 seconds and the bubble becomes totally unstable after 1800 seconds.

Example 6: Stability of foams at chill temperature in the presence of a Co- surfactant used at a level that is outside of the inventive range.

Chilled, aerated confections were made using formulations described in Table 7. A control formulation with no co-surfactant (Cont) was also prepared. The relative amounts of hydrophobin HFBI I , SMP are the same as those selected for Example 5. However, the co-surfactant level was selected to be outside of the preferred range for the Negative Test 2 sample.

Table 7 Formulation of chilled aerated confections for stability test of a Negative Test 2 sample, prepared with a Co-surfactant level outside of the inventive range

Figure 7 shows a photograph of the aerated product prepared using a level of Co- surfactant that is outside of our preferred ranges. Inspection of the image clearly indicated that this product is totally unstable. Table 8 shows the change in product volume with storage time at chill temperature. Inspection of the data in the Table 8 suggests that the foam is much less stable than the control sample.

Table 8: Variation of sample volume with time for the Comparative Control sample (Cont) and the Negative Test 2 sample.

The product volume decreases as overrun is lost. The Negative Test 2 sample looses far more overrun on storage than the Comparative Control, the formulations for which are described in Table 7. This result confirms that Co-surfactants type and level can only be selected reliably by using our inventive method, and that was surprisingly discovered in the present invention. Test Methods and Material Sources:

1. Dilatational Surface Rheology Measurements

Materials

Preparation of the solutions for the experimental work was done using the same ingredients as in the preparation of fully formulated products except that tap water was used to prepare chilled and ambient products. All other solutions were prepared in de- ionised water (18.2 ΜΩ cm). Concentrations of ingredients are expressed as weight %.

Solutions were prepared using combinations of hydrophobin, Secondary protein e.g. a milk protein, and an added Co-surfactant.

In the interfacial rheology experiments the concentration of used ingredients was scaled down 200-times with respect to the levels used in ice cream manufacturing. The concentrations used for interfacial rheology experiments are (as an example and not restricted to these):

HFBII = 0.001 wt.% = 0.2/200 wt.%

SMP = 0.041 wt.% = 8.22/200 wt.%

CSF = 0.0001 wt.%=0.02/200 wt.%

2. Interfacial rheology method

Reported values of the viscoelastic modulus (E) were measured using the Drop Shape tensiometer PAT-1 (Sinterface, Germany). The measuring configuration is that of a bubble emerging from a J-shaped capillary positioned inside the cell containing the solution. The PAT-1 tensiometer implements a feature allowing for an accurate control of the bubble interfacial area with the possibility of varying it during the measurement according to predetermined patterns. This feature is utilised for the measurement of the dilational viscoelasticity. Purely harmonic oscillations of the bubble interfacial area with small amplitude and frequency are imposed (immediately after the bubble formation) while the surface tension response, γ (t), is measured. From the amplitude of the two signals, A (t) and γ (t), and the phase shift between them, the elastic and viscous components of E are calculated. Amplitude and phase of the measured A (t) and γ (t) oscillatory signals are extracted by standard Fourier analysis techniques.

In the experiments reported here an air bubble of area Ao = 18 mm² was formed at the tip of a J-shaped capillary in a glass cell containing about 27 ml of the solution. An area variation between 2.5 and 3.5% was imposed during oscillations at the frequency of 0.05 Hz and a temperature of 5^° C, unless otherwise stated. A gentle nitrogen stream was directed onto the cell glass walls (front and back) to prevent air humidity condensation which obscures the cell field of view

3. Scanning Electron Microscopy (SEM) Method

The microstructure of each product was visualised using Low Temperature Scanning Electron Microscopy (LTSEM). The sample was cooled to -80 °C 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 1 1 wt.%, Carbowax 5 wt.% and 85 wt.% 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 °C. 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 -1 10°C ending 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; JEOL LTD. Japan), fitted with an Oxford Instruments cold stage at a temperature of -160°C. The sample is examined and areas of interest captured via digital image acquisition software e.g. using the method described below.

4. Determination of bubble size distribution of Products

The gas bubble size (diameter) distribution as used herein is defined as the size distribution obtained from the two dimensional representation of the three dimensional microstructure, as visualized in the SEM micrograph, determined using the following methodology. Samples are imaged at 3 different magnifications (for reasons explained below), and the bubble size distribution of a sample is obtained from this set of micrographs in three steps:

1. Identification and sizing of the individual gas bubbles in the micrographs

2. Extraction of the size information from each micrograph

3. Combination of the data from the micrographs into a single size distribution

All of these steps, other than the initial identification of the gas bubbles, can

conveniently be performed automatically on a computer, for example by using software such as MATLAB R2006a (MathWorks, Inc) software.

Identification and sizing of the individual gas bubbles in the micrographs

Firstly, a trained operator (i.e. one familiar with the microstructures of aerated systems) traces the outlines of the gas bubbles in the digital SEM images using a graphical user interface.

Secondly, the size is calculated from the selected outline by measuring the maximum area as seen in the two dimensional cross-sectional view of the micrograph (A) as defined by the operator and multiplying this by a scaling factor defined by the microscope magnification. The bubble diameter is defined as the equivalent circular diameter d:

This is an exact definition of the diameter of the two-dimensional cross-section through a perfect sphere. Since most of the gas bubbles are approximately spherical, this is a good measure of the size.

Extraction of the size information from each micrograph

Gas bubbles which touch the border of a micrograph are only partially visible. Since it is not therefore possible to determine their area, they must be excluded. However, in doing so, systematic errors are introduced: (i) the number of gas bubbles per unit area is underestimated; and (ii) large gas bubbles are rejected relatively more often since they are more likely to touch the border, thus skewing the size distribution. To avoid these errors, a guard frame is introduced (as described in John C. Russ, "The Image Processing Handbook", second edition, CRC Press, 1995). The guard frame concept uses a virtual border to define an inner zone inside the micrograph. The inner zone forms the measurement area from which unbiased size information is obtained, as illustrated in Figure 3 (a schematic depiction of a micrograph, in which gas bubbles that touch the outer border of the micrograph have been drawn in full, even though in reality only the part falling within the actual micrograph would be observed.)

Bubbles are classified into 5 classes depending on their size and position in the micrograph. Bubbles that fall fully within the inner zone (labelled class 1 ) are included. Bubbles that touch the border of the virtual micrograph (class 2) are also included

(since it is only a virtual border, there is fact full knowledge of these bubbles). Bubbles that touch the actual micrograph border (class 3) and / or fall within the outer zone (class 4) are excluded. The exclusion of the class 3 bubbles introduces a bias, but this is compensated for by including the bubbles in class 2, resulting in an unbiased estimate of the size distribution. Very large bubbles, i.e. those larger than the width of the outer zone (class 5), can straddle both the virtual (inner) border and the actual outer border and must therefore be excluded, again introducing bias. However, this bias only exists for bubbles that are wider than the outer zone, so it can be avoided by excluding all bubbles of at least this size (regardless of whether or not they cross the actual border). This effectively sets an upper limit to the gas bubble size that can be reliably measured in a particular micrograph. The width of the inner zone is chosen to be 10% of the vertical height of the micrograph as a trade-off between the largest bubble that can be sized (at the resolution of the particular micrograph) and the image area that is effectively thrown away (the outer zone).

There is also minimum size limit (at the resolution of the micrograph) below which the operator cannot reliably trace round gas bubbles. Therefore bubbles that are smaller than a diameter of 20 pixels are also ignored. Combination of the data from the micrographs into a single size distribution

As explained above, it is necessary to introduce maximum and minimum cut-off bubbles sizes. In order that these minimum and maximum sizes are sufficiently small and large respectively so as not to exclude a significant number of bubbles, some samples may need to be imaged at 3 different magnifications: e.g. 100x, 300x and 1000x. This occurs if there is a wide distribution in bubble sizes, and the skilled user can determine what magnifications are appropriate in order to capture the full size distribution: one magnification or more. As an example for the case of 3 different magnifications, each magnification yields size information in a different range, given in Table9.

Table 9

Thus bubbles as small as 2ΜΓΠ and as large as 83ΜΠΊ are counted. Visual inspection of the micrographs at high and low magnifications respectively confirmed that essentially all of the bubbles fell within this size range. The magnifications are chosen so that there is overlap between the size ranges of the different magnifications (e.g. gas bubbles with a size of 20-28 m are covered by both the 100x and 300x micrographs) to ensure that there are no gaps between the size ranges. In order to obtain robust data, at least 500 bubbles are sized; this can typically be achieved by analysing one micrograph at 10Ox, one or two at x300 and two to four at x1000 for each sample.

The size information from the micrographs at different magnifications is finally combined into a single size distribution histogram. Bubbles with a diameter between 20 Mm and 28 m are obtained from both the 100x and 300x micrographs, whereas the bubbles with a diameter greater than 28 m are extracted only from the 100x micrographs. Double counting of bubbles in the overlapping size ranges is avoided by taking account of the total area that was used to obtain the size information in each of the size ranges (which depends on the magnification), i.e. it is the number of bubbles of a certain size per unit area that is counted. This is expressed mathematically, using the following parameters:

N = total number of gas cells obtained in the micrographs

d_k = the k^th outlined gas cell with k≡ [1, N]

A, = the area of the inner zone in the I^th micrograph

R; = the range of diameters covered by the I^th micrograph (e.g. [20 ηΊ,83 ηι]) B(j) = the j^th bin covering the diameter range : [j W, (j + 1) W) The total area, S (d), used to count gas bubbles with diameter d is given by adding the areas of the inner zones (A,) in the micrographs for which d is within their size range

The final size distribution is obtained by constructing a histogram consisting of bins of width W μηη. B (j) is the number of bubbles per unit area in the j^th bin (i.e. in the diameter range j x W to (j+1 ) x W). B (j) is obtained by adding up all the individual contributions of the gas bubbles with a diameter in the diameter range j x W to (j+1 ) x W, with the appropriate weight, i.e. 1/S (d).

B{j ) =∑l / S{d_k )

k≡D where

Magnifications used are chosen by the skilled user in order to extract bubble size through the analysis software.

The bubble size distributions are conveniently described in terms of the normalised cumulative frequency, i.e. the total number of bubbles with diameter up to a given size, expressed as a percentage of the total number of bubbles measured.

Alternative expressions of bubble size distribution can also be used, e.g. d3,2. 5. Preparation of samples for Macroscope analysis

Sample preparation

A transparent square container was used to contain a sample of each foam under investigation. The plastic container consisted of a hinge on one side and a catch on the opposing side. The edges of the container (=1 -2 mm thick) were coated with a thin layer of vacuum grease (Dow Corning) using a syringe before addition of a foam sample. Sufficient quantity of foam was used such that the container when closed was overfilled meaning surplus foam was ejected. The closed unit was then sealed with clear nail varnish around the container edges and left to dry. Three containers were set up for each foam sample. The weight of the containers was monitored over time - Initial, 1 , 4 and 6 weeks storage. This allowed a reliable estimate of any foam escaping from the container over time. This was proved to be minimal. Characterisation of foams using a macroscope

The sample container was placed on the stage with the light used to illuminate the sample on full power. The image was obtained by appropriately focussing the lens and then subsequently adjusting the camera settings to obtain a high quality picture. The camera settings outlined blow allowed optimal imaging;

- Exposure: 50-70ms;

- Gain: 2. Ox;

Gamma: 2.00x;

Image type: Greyscale;

- Captured format: 1728 x 1296, Interlaced Medium High Quality.

Each of the images produced had 20mm scale bars on the top left hand corner of the image

6. Method for bubble size analysis from macroscope images

The gas bubble size (diameter) distribution as used herein is defined as the size distribution obtained from the two dimensional representation of the three dimensional microstructure, as visualized in the macrospopic images, determined using the following methodology. The bubble size distribution of a sample is obtained from this set of macrographs in three steps:

1. Identification and sizing of the individual gas bubbles in the micrographs

2. Extraction of the size information from each micrograph

3. Combination of the data from the micrographs into a single size distribution

All of these steps, other than the initial identification of the gas bubbles, can

conveniently be performed automatically on a computer, for example by using software such as MATLAB R2006a (MathWorks, Inc) software or by a trained microscopist using a suitably calibrated scale bar. 7. Measurement of overrun volume loss on storage at chill temperature

100cm³ of each foam was placed in a measuring cylinder. The measuring cylinder was then sealed using Parafilm® in order to prevent evaporative losses. The tubes were then stored at chill temperature. The height of the foam was recorded as a function of storage period.

8. Preparation of aerated chilled confections.

Preparation of co-surfactant solutions

The following Co-surfactant solutions were prepared in advance as 1 % solutions for the test samples or at 10% for the negative co-surfactant control sample. All dilutions were prepared using deionised water. The Tween 20 solutions were manually agitated in order to disperse (dissolve) the Co-surfactant. The Panodan solution once prepared was sonicated for 2-3 minutes at high power in a sonic bath to disperse, forming a milky solution. The Tween 60 solution was prepared by melting a sample of the stock solution and then adding the appropriate amount to D.I water, which had been heated to 70°C.

Preparation of stock SMP/Xanthan/Sucrose solution

Dry xanthan (0.41 %w/w) was mixed with sucrose (20.4 %w/w) to aid dispersion of the xanthan. The mix was dissolved in water at 80°C and stirred manually for 10 minutes. Having allowed the mix to cool to 70°C SMP (8.36 %w/w) was added gradually with stirring. The remaining water was then added to complete the formulation (to 1 kg) and the resulting solution sheared on a Silverson Mixer for 2-3 minutes. The solution was left to cool to room temperature and then stored at chill ready for use in foam preparation stage the following day.

Aeration of chilled product

4g of the Co surfactant solution was added to the hydrophobin (138mg/g, 0.7246g) and the mix stirred manually. The HFBII /co-surfactant mix was then combined with 200g of the SMP/Xanthan/Sucrose mix and the resulting mix stirred. Aeration was carried out using a laboratory scale scraped surface heat exchanger operating at 1 1000 rpm for 10 minutes. The equipment was cooled with circulating water at 5°C. 4 grams of a 5% phenoxethanol solution was then added as an antimicrobial and mixed into the foam at low speed (250 rpm for 30s). The foam produced was decanted into Sterlin® pots prior to setting up samples for analysis. The control foam was prepared by the same procedure except that deionised water was mixed with HFBII at the start (i.e. no co-surfacant added). The foam was decanted into Sterilin® pots prior to setting up samples for analysis.

All samples were prepared with an overrun of at least 100%,

9. Material Sources

Ingredient Source Comments

Hydrophobic HFBII Danisco, Denmark Class II hydrophobin

Hydrophobic HFBI VTT, Finland Class II hydrophobin

Hydrophobin, Cerato Unilever R&D Class II hydrophobin

Ulmin Vlaardingen

Skim Milk Protein (SMP) Dairy Crest 35 wt.% protein content

Xanthan Keltro F CP Kelco Cold water soluble

Corn syrup LF9 Brenntag

Panodan Visco Lo 2000 Danisco, Denmark A diacetyl tartaric acid ester of mono-diglycerides; Saponification value 435-465; Acid value 50-70; Iodine value ~ 75.

Tween 60: Sigma Chemicals A water soluble (HLB = 14.7), low

Polyoxyethylene (20) molecular weight, non-ionic sorbitan monostrearate surfactant

Tween 20 Sigma Chemicals A water soluble (HLB = 16.7), low

Polyoxyethylene (20) molecular weight, non-ionic sorbitan monolaurate surfactant

PGE-O-80/D Danisco, Denmark A polyglycerol ester; polyglycerol moiety is mainly di-, tri-, and tetraglycerol; Iodine value ~ 55; Saponification value 1 15-135.

Sucrose ester SE1670 Ryoto Sucrose Esters-

(Mitsubishi-Kagaku

Foods) Ingredient Source Comments

Sucrose ester SP70 Ryoto Sucrose Esters- Sucrose ester SP70

(Mitsubishi-Kagaku

Foods)

PGE55 Danisco, Denmark A polyglycerol ester; polyglycerol moiety is mainly di-, tri-, and tetraglycerol; Iodine level max. 2; Saponification value 130-145.

Hygel Kerry Foods Hydrolysed milk protein

Ice Structuring Protein Martek Ice Structuring Protein (ISPIII) (ISPIII)

IcePro Danisco

Claims

1. A non-frozen aerated chill or ambient composition comprising:

a. at least 0.01wt.% of total hydrophobin(s) selected from the group consisting of class I hydrophobin(s), class II hydrophobin(s) or blends thereof added in isolated form to the food composition;

b. one or more co-surfactant(s) in the total concentration range of 0.001 to less than 0.2 wt.%;

wherein the one or more co-surfactant(s) are ingredients which, when mixed in an aqueous solution containing 0.001 wt.% hydrophobin and between 0.0015 and 0.2 wt.% of at least one non-hydrophobin (secondary) protein at a concentration effective to confer an air/water surface dilatational elasticity that is at least 30% of that of 0.001 wt.% pure hydrophobin, absent the co- surfactant, measured between 600 and 7200^"s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05 Hz using the procedure as described in here; and

c. one or more secondary protein(s) that are different from hydrophobin(s); wherein said secondary protein(s) are present in a total concentration range of 0.25 to less than 6.0 wt.% and

wherein the one or more secondary protein(s) are defined as non- hydrophobin proteins that when mixed with 0.001 wt.% hydrophobin in aqueous solution at a concentration of 0.04 wt.% results in an air/water surface dilatational elasticity that is at least 35% less than that of hydrophobin alone, where the dilatational measurement is made between 600 and 4000s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05 Hz, using the procedure as described in here; and

d. wherein the ratio of co-surfactant(s) to total hydrophobin(s) is in the range of 0.02 to 1.0.

2. The product of claim 1 wherein the hydrophobin(s) are class II hydrophobin(s) selected from the group consisting of HFBII, HFBI or Cerato Ulmin or blends thereof.

3. The product of claim 1 or 2 wherein the total hydrophobin(s) concentration is at most 1.5 wt.%

4. The product of any of claims 1 to 3 wherein the co-surfactant(s) is or are water soluble non-ionic surfactant(s).

5. The product of any of claims 1 to 4 wherein the co-surfactant(s) are selected from Polysorbates; polyglycerol esters of alkyl or alkenyl fatty acids, diacetyl tartartic acid esters of mono-/di- glycerides, sucrose esters with an HLB more than 8 or blends thereof.

6. The product of any of claims 1 to 5 wherein the co-surfactant(s) has a minimum effective HLB value of 8.

7. The product of any of claims 1 to 6 wherein after 6 weeks storage at 5°C no more than 10 bubbles of a size greater than 6 mm in diameter are observed in a sample size of 9437 mm squared area where the sample is in a 10 mm deep container and had an initial overrun between 30 and 200% overrun.

8. The process of making a non-frozen aerated product comprising the steps of:

a. Blending the food composition ingredients together and mixing;

b. Adding at least 0.01 wt.% of hydrophobin(s) and co-surfactant(s) in a total concentration range of 0.001 to less than 0.2 wt% to the chilled mix of step (a);

wherein the one or more co-surfactant(s) are ingredients which, when mixed in an aqueous solution containing 0.001 wt.% hydrophobin and between 0.0015 and 0.2 wt.% of at least one non-hydrophobin (secondary) protein at a concentration effective to confer an air/water surface dilatational elasticity that is at least 30% of that of 0.001 wt.% pure hydrophobin, absent the co- surfactant, measured between 600 and 7200^" s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05 Hz using the procedure as described in here; and

c. Aerating the mix of step (b) to produce the aerated product;

d. wherein the ratio of co-surfactant(s) to total hydrophobin(s) is in the range of 0.02 to 1.0;

e. wherein the aerated product contains one or more secondary protein(s) different from hydrophobin in a total concentration range of 0.25 to less than 6.0 wt%, and;

wherein the one or more secondary protein(s) are defined as non- hydrophobin proteins that when mixed with 0.001 wt.% hydrophobin in aqueous solution at a concentration of 0.04 wt.% results in an air/water surface dilatational elasticity that is at least 35% less than that of hydrophobin alone, where the dilatational measurement is made between 600 and 4000s at 5°C for an air droplet in water subject to a continuous area change of between 2.5 and 3.5% oscillated at a frequency of 0.05 Hz, using the procedure as described in here; and

f. wherein pasteurisation may be accomplished after step (a) and/or step (b).