WO2017125213A1

WO2017125213A1 - Aqueous composition in liquid form for quiescent freezing

Info

Publication number: WO2017125213A1
Application number: PCT/EP2016/081114
Authority: WO
Inventors: David John Judge; Penelope Eileen Knight; Loyd Wix
Original assignee: Unilever N.V.; Unilever Plc; Conopco, Inc., D.B.A. Unilever
Priority date: 2016-01-18
Filing date: 2016-12-15
Publication date: 2017-07-27

Abstract

The present invention relates to an aqueous composition in liquid form, which contains oil, milk protein, monosaccharides, disaccharides, and/or oligosaccharides, a hydrocolloid and a hydrophobin. The composition may be aerated, and may be used to be frozen quiescently to prepare a frozen confection. The invention also provides a method for preparation of the composition of the invention, and a method for freezing the aerated composition of the invention. The liquid composition can be distributed at temperatures above 0°C, and frozen at the point of use prior to consumption, such that much energy is saved as compared to distribution of frozen confections at temperatures below 0°C.

Description

AQUEOUS COMPOSITION IN LIQUID FORM FOR QUIESCENT FREEZING

The present invention relates to an aqueous composition in liquid form. The composition may be aerated, and may be used to be frozen quiescently to prepare an ice cream. The invention also provides a method for preparation of the composition of the invention, and a method for freezing the aerated composition of the invention.

BACKGROUND TO THE INVENTION

Usually ice cream is prepared in a factory, and stored and distributed from the factory to the consumer in frozen form. This has the disadvantage that much energy is required to keep the storage and distribution temperature below 0°C, or even below -10°C or -20°C, so that the consumer can consume a perfect ice cream. Much energy can be saved by supplying and distributing a composition at a higher temperature, and that the consumer can freeze at home, to consume a perfect ice cream. Such a distribution channel at temperatures higher than 0°C are much more sustainable and less energy consuming than the standard distribution channel at temperature below 0°C.

Therefore there is a need for the supply of compositions that can be distributed at temperatures higher than 0°C, and that the consumer can freeze at home to prepare a perfect ice cream.

WO 2007/039064 A1 discloses flowable aerated products containing hydrophobin and with reduced creaming. US 2014/0050836 A1 discloses an aerated frozen confectionery product containing hydrophobin, a co-surfactant, and a secondary protein (not hydrophobin). This leads to products wherein the microstructure of the freshly produced product is preferable since the air bubbles sizes are generally smaller than other products. Moreover, the microstructure of the product after storage and temperature abuse is preferable since the air bubbles sizes are more stable and do not grow (coarsen) to the same extent as comparative cases.

US 2014/0170292 A1 discloses an aerated chilled or ambient confectionery product product containing hydrophobin, a co-surfactant, and a secondary protein (not hydrophobin). This leads to products wherein the microstructure of the freshly produced product is preferable since the air bubbles sizes are generally smaller than other products. Moreover, the microstructure of the product after storage and temperature abuse is preferable since the air bubbles sizes are more stable and do not grow (coarsen) to the same extent as comparative cases.

WO 2012/1 10376 A1 and WO 2014/029574 A1 relate to packaged shelf- or chilled-stable mixes of ingredients for the preparation of frozen confections. The compositions contain as emulsifier a propylene glycol monoester of fatty acid, preferably propylene glycol

monostearate (PGMS).

WO 2008/009616 A2, WO 2008/009617 A1 , WO 2008/009618 A2, and WO 2008/009623 A1 relate to stable foams having a controlled fine air bubble size distribution and to edible products prepared therefrom having a low fat content. The compositions preferably contain a polyglycerol ester of fatty acids (PGE).

Also WO 2012/016852 A2 relates to unfrozen packaged confectionery products for the preparation of quiescently frozen confectionery products, preferably containing a polyglycerol ester of fatty acids (PGE).

WO 98/23169 discloses an ice cream formulation that can be conveniently stored, prior to use. Also these formulations preferably contain a polyglycerol ester of fatty acids (PGE).

ZA 9810254 relates to an ice cream mix which can be stored above 0°C and which can be frozen by the consumer at home. The mix preferably contains mono- and diglycerides of fatty acids.

WO 2008/019865 A1 relates to the incorporation of air or gas into viscous food matrices, which are meant to be consumed at temperatures above 0°C. The compositions may contain a wide range of surfactant particles or crystals.

US 5 925 392 relates to a method of producing and pre-distribution preparing unfrozen, expanded ice cream mix, containing an emulsifier mixture containing lactic acid ester of mono- or diglycerides of fatty acids (LACTEM), microcrystalline cellulose, modified starch, and gelatin.

EP 1 415 542 A1 relates to ice-cream dessert material which is in liquid form, is stable during storage and has an oil-in-water emulsion character. Non-pre-published documents WO 2016/075016 A1 and WO 2016/075017 A1 both relate to aqueous compositions in liquid form. The compositions may be aerated, and may be frozen quiescently to prepare frozen confections. SUMMARY OF THE INVENTION

Production, distribution and storage of ice cream costs much energy, because of the low temperatures that are required. Generally the entire supply chain, before the ice cream is consumed, is lower than -18°C. In order to substantially save on energy use, it would be very beneficial to prepare good quality compositions that can be distributed stored at

temperatures above 0°C, and that can be frozen by the consumer just before the point of consumption, to prepare a frozen confection. Such frozen confection should have the same properties as regular frozen confections, which are dynamically frozen in an ice cream factory. For example the structure, ice content, and size of ice crystals should be similar to the regular frozen confections. This means that the compositions should be stable with respect to its structure, until the point that the compositions are frozen.

We have met this objective by a liquid composition containing hydrophobin, with a relatively low water content, relatively low milk protein content, and relatively high level of sugars and other saccharide sweeteners which have been combined in such a way to create a good level of sweetness which is not too sweet. The mixture of sugars and other saccharide sweeteners has a number average molecular weight <M>n ranging from 240 to 350 gram per mole; and the total solids content is at a concentration ranging from 42% to 60% by weight. The ice content of the compositions when they are frozen is relatively low. Such composition can be used to prepare an aerated composition, which is packaged, distributed and stored at temperatures above 0°C for a period of up to several months and quiescently frozen in such package, for instance by a consumer at home. Moreover, the relative amounts of the ingredients in the composition of the invention also leads to a desired structure and texture, when the composition has been quiescently frozen.

The hydrophobin in the composition of the invention HFB facilitates the formation of small and stable air bubbles. The gas bubbles are stable during storage of the liquid composition at ambient temperature and inhibit formation of large ice crystals when the composition is quiescently frozen. The relatively high solids content in the composition leads to a decrease of the phase volume of ice when the composition is frozen. Typically the critical ice range is below 40% ice when the composition is frozen at -18°C, in order to obtain a homogeneous frozen composition. Accordingly in a first aspect the invention provides a composition in liquid form comprising a continuous aqueous phase; and

triglycerides at a concentration ranging from 3% to 10% by weight, preferably from 5% to 10% by weight; and

milk protein at a concentration ranging from 0.8% to 5% by weight; and

one or more compounds selected from monosaccharides, disaccharides, and

oligosaccharides, at a concentration ranging from 32% to 50% by weight, and

wherein a mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, has a number average molecular weight <M>n ranging from 240 to 350 gram per mole; and

one or more hydrocolloids to provide an apparent yield stress of at least 1 Pa, comprising xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, alginate, pectin, and carboxy-methylcellulose, or any combination of these; and

a hydrophobin at a concentration of at least 0.03% by weight; and

total solids at a concentration ranging from 42% to 60% by weight.

Preferably, the first aspect of the invention provides a composition according to the first aspect of the invention containing gas bubbles at an overrun ranging from 30% to 200%.

In a second aspect the invention provides a method for preparation of a composition according to the first aspect of the invention containing gas bubbles, comprising the steps: a) Providing an unaerated composition according to the first aspect of the invention; b) Optionally homogenising the composition from step a);

c) Aerating the composition from step a) or step b); and

d) Optionally packing the composition from step c) in a container and sealing the

container.

The second aspect the invention also provides a method for preparation of a frozen aerated composition, wherein an aerated composition according to the first aspect of the invention is brought to a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C. Preferably the aerated composition is not agitated when brought to a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C. This means that preferably the aerated premix is quiescently frozen to prepare an ice cream. When the composition of the first aspect of the invention is quiescently frozen, the resultant microstructure resembles ice cream structure obtained by dynamic freezing. Such

microstructure deliver similar organoleptic properties and product qualities as regular dynamically frozen ice cream. The aerated composition of the invention has favourable properties. The first is that a fine/stable dispersion of gas can be created, that does not disproportionate, shows no significant coarsening and is prevented from separating from the composition, e.g. by creaming. Moreover, the aerated composition does not display other forms of phase separation such as syneresis. Another advantages of our invention is that part of the conventional sucrose is replaced by other saccharides. Products, and in particular ice cream with a high sucrose concentration can be conceived by consumers to be too sweet. By using other saccharides, the sweetness of the compositions is reduced, while the beneficial properties of the presence of saccharides is maintained. In particular the presence of saccharides leads to a favourable structure of the frozen aerated composition.

DETAILED DESCRIPTION OF THE INVENTION

Tests and Definitions

All percentages, unless otherwise stated, refer to the percentage by weight (wt%).

In the context of the present invention, an average droplet or gas bubble diameter is expressed as the D4,3 value, which is the volume weighted mean diameter. Gas bubbles or droplets in a product may not be perfect spheres. The volume based bubble or droplet diameter equals the diameter of a sphere that has the same volume as a given bubble or droplet.

Ambient temperature is considered to be a temperature between about 20°C and about 25°C, preferably between 20°C and 23°C. By the terms "flowable composition" or "flowable product", which are used interchangeably herein, we mean a composition where the composition will flow following a relatively small amount of agitation (e.g. shaking, stirring or sucking), as opposed to a solid or set composition. Flowable compositions include pourable compositions and semi-set

compositions. The temperature at which the flowability of the composition or product is considered is the temperature at which the product is normally served. For example, flowability of a chilled product is typically determined at 5°C whereas flowability of an ambient product is typically determined at room temperature (20°C). Flowability of ice-containing products is typically determined at -10°C. Measurements are generally carried out at 1 atm pressure. The term "aerated" means that gas has been intentionally incorporated into a composition, for example by mechanical means. The gas can be any gas, but is preferably, in the context of food products, a food-grade gas such as air, nitrogen, nitrous oxide, or carbon dioxide.

Hence the term "aeration" is not limited to aeration using air, and encompasses the

"gasification" with other gases as well. The extent of aeration is measured in terms of "overrun" (with unit "%"), which is defined as:

volume of aerated product - volume of initial mix „„„_0/

overrun = - χ 100% (1 )

Volume of initial mix where the volumes refer to the volumes of aerated product and unaerated initial mix (from which the aerated product is made). Overrun is measured at atmospheric pressure.

The overrun of an aerated product and the volume fraction of gas in the aerated product generally relate in the following way:

volume fraction gas (in %) = 100% x [overrun / (100% + overrun)] (2) The terms "fat" or "oil" as used herein refers to lipids selected from triglycerides, diglycerides, monoglycerides and combinations thereof. The oil may be solid or liquid at ambient temperature. The terms "fat" and "oil" may be used interchangeably throughout this specification, and refer to the same type of materials. Preferably the oil in the context of this invention comprises at least 90 wt% of triglycerides, more preferably at least 95 wt%.

Frozen confection

As used herein, the term "frozen confection" refers to a sweet-tasting fabricated foodstuff intended for consumption in the frozen state (i.e. under conditions wherein the temperature of the foodstuff is less than 0°C, and preferably under conditions wherein the foodstuff comprises significant amounts of ice). Typical examples of frozen confections include ice creams, water ices and sorbets.

Saccharides and sugar alcohols

A "monosaccharide" is the basic unit of a carbohydrate, and they are the simplest form of sugars. Examples of monosaccharides are glucose, and fructose. A "disaccharide" is a chemical compound which is formed by the reaction between two monosaccharides.

As used herein, the term "oligosaccharide" refers to saccharides with a degree of

polymerisation (DP) of at least 3 to 9. "Polysaccharides" refers to saccharides having a degree of polymerization of at least 10. "Sugar alcohols" are alcohols prepared from saccharides, and they are a class of the polyols. Examples of these compounds are glycerol, erythritol, xylitol, sorbitol, and lactitol.

Saccharides and sugar alcohols molecular weight

A method to determine the average molecular weight for sugars comprising a mixture of mono-, di- and/or oligosaccharides and/or sugar alcohols has been described by

EP 1 676 486 A1 . The average molecular weight is defined by the number average molecular weight <M>„ (in g/mole):

Where is the mass of saccharide or sugar alcohol i, M-, is the molar mass of saccharide or sugar alcohol i and N, is the number of moles of saccharide or sugar alcohol i of molar mass

Glucose syrups (or "corn syrups" as they are sometimes called) are complex multi- component digestible saccharides derived from starch. The dextrose equivalent (DE) is a common industrial means of classification. Since they are complex mixtures their number average molecular weight <M>„ can be calculated from equation 4 (J. Chirife et al, 1997, Journal of Food Engineering, 33, p. 221 -226):

< M

Relative sweetness

Generally the relative sweetness of saccharides is determined relative to sucrose. Sucrose is taken as the reference with a sweetness of 1 . A 10% solution of sucrose in water has a relative sweetness of 0.1 . A relative sweetness of 0.33 is equivalent to 33% sucrose in water. The total relative sweetness (TRS) can be calculated using the following formula:

TRS = (MS1/100 x RSS1) + (MS2/100 x RSS2) + (MS3/100 x RSS3) +... Herein MS1 is the concentration of sugar 1 in water, and RSS1 is the relative sweetness of sugar 1. Similarly for sugars 2 and 3.

The sweetness of other sugars is expressed as relative sweetness to that of sucrose.

Relative sweetness of fructose = 1.7

Therefore sweetness of a 10% fructose solution = 10/100x1.7 = 0.17

Relative sweetness of dextrose monohydrate is = 0.73

Therefore sweetness of a 10% dextrose monohydrate solution = 10/100x0.73 = 0.073 Thus a formulation containing 10% sucrose, 10% fructose and 10% dextrose monohydrate has the following relative sweetness:

TRS = 0.10 + 0.17 + 0.073 =0.343

Composition in liquid form

In a first aspect the present invention provides a composition in liquid form comprising a continuous aqueous phase; and

milk protein at a concentration ranging from 0.8% to 5% by weight; and

one or more compounds selected from monosaccharides, disaccharides, and

oligosaccharides, at a concentration ranging from 32% to 50% by weight, and

a hydrophobin at a concentration of at least 0.03% by weight; and

total solids at a concentration ranging from 42% to 60% by weight.

Preferably the composition according to the invention is an edible composition. The composition is in liquid form, which means that the composition is a flowable composition. In the context of the present invention, the composition is in liquid form at a temperature above 0°C, and at these temperatures the composition is a flowable product. Preferably the composition according to the first aspect of the invention is ambient stable or chilled stable, meaning that it can be stored and kept at a temperature of at least 0°C, and preferably maximally 40°C, preferably maximally 30°C, preferably maximally 25°C. Preferably the composition is ambient stable or chilled stable during a period of at least 2 weeks, preferably at least 4 weeks, preferably at least 8 weeks. With stable is meant that the composition can be stored without noticeable or only small deterioration of quality during the storage period.

Preferably the pH of the composition ranges from 4.5 to 7.5, preferably from 6.5 to 7.5.

Ingredients of the composition

The composition of the invention comprises oil at a concentration ranging from 3% to 10% by weight, preferably from 4% to 10% by weight, more preferred from 5% to 10% by weight, more preferred from 5.5% to 9% by weight. Preferred oils for use in the context of this invention are vegetable oils like coconut oil and palm oil, or fractions thereof. Another preferred fat is dairy fat, preferably milk fat, preferably cow's milk fat, or fractions thereof. Also combinations of these preferred oils and fats are within the scope of the present invention. The concentration of milk protein ranges from 0.8% to 5% by weight. Preferably the concentration of milk protein ranges from 1 % to 4% by weight. Preferably the concentration of milk solids excluding fat ranges from 2% to 15% by weight of the composition, more preferred from 2% to 12% by weight, more preferred from 3% to 12% by weight. The milk solids excluding fat generally may be added to the composition in the form of skimmed milk powder. Generally skimmed milk powder is dried defatted dairy milk, and generally comprises about 35% milk protein (casein and whey protein) and about 50% lactose. Also other milk protein sources may be used as source of milk protein.

The composition of the invention comprises one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, at a concentration ranging from 32% to 50% by weight, and wherein a mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, has a number average molecular weight <M>n ranging from 240 to 350 gram per mole This combination of sugars contributes to the stability of the liquid composition and to the microstructure of the frozen composition of the invention. In case lactose is added to the composition as part of skimmed milk powder, then this lactose is also included in the calculation of the total amount of saccharides.

Preferably the mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides has a number average molecular weight <M>n ranging from 250 to 350 gram per mole, preferably from 270 to 340 gram per mole. More preferred the number average molecular weight <M>n ranges from 270 to 320 gram per mole. This average molecular weight can be obtained by a mix of various sugar sources. Preferably a mixture of lactose from milk solids, sucrose, dextrose, and glucose syrup having a dextrose equivalent (DE) value ranging from 20 to 45, or maltodextrins is used. Preferably the composition comprises a glucose syrup having a DE value ranging from 25 to 35, more preferred a DE value ranging from 26 to 30. The composition may also comprise a mixture of glucose syrups with different DE values. The composition preferably also contains a glucose syrup or a maltodextrin having a DE value ranging from 35 to 45. In particular the higher molecular weight saccharides contribute to the stability of the composition of the invention and the microstructure of the frozen composition. Glucose syrups and corn syrups are considered to be synonyms.

Preferably the relative sweetness of the mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, is maximally 0.22, preferably maximally 0.2, preferably maximally 0.18. This way a mix of sweeteners can be used which does not make the compositions too sweet, while it has the advantage that it contributes to the stability of the liquid composition of the invention and the frozen products.

The composition may contain sugar alcohols, alone or in combination with one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides.

Preferably though the maximum concentration of sugar alcohols is maximally 10% by weight of the composition, more preferred maximally 8% by weight of the composition. More preferred the maximum concentration of sugar alcohols is 6% by weight. Alternatively and preferably sugar alcohols are absent from the composition. If present, then preferred sugar alcohols are erythritol, sorbitol, maltitol, lactilol, and xylitol, and more preferred maltitol and erythritol. The composition may also contain soluble fibres like inulin and/or polydextrose and/or oligofructosaccharides in addition to or to replace part of the oligosaccharides.

The total amount of sweeteners is relatively high, therefore also the total solids content is relatively high, it ranges from 42% to 60%. Preferably the total solids concentration ranges from 42% to 55% by weight, preferably from 43% to 50% by weight.

Hydrophobin

The composition of the invention comprises a hydrophobin. Hydrophobins are a well-defined class of proteins (Wessels, 1997, Advances in Microbial Physiology 38: 1 -45; Wosten, 2001 , Annual Reviews of Microbiology 55: 625-646) that are capable of self-assembly at a hydrophobic/hydrophilic interface, and having a conserved sequence: X_n-C-X5-9-C-C-Xl 1-39-C-X8-23-C-X5-9-C-C-X6-18-C-X_m (5) 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 (6) 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, o 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 are generally classified 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

Actinomycetes and Streptomyces sp. (WO 01/74864). 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 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 WO 01/57076 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.

Hydrophobins are excellent foaming agents that lead to good foamability and good foam stability of a product to which the hydrophobin is added, in products that contain water.

Hydrophobins are generally rather expensive, hence the product developer has the tendency to keep the concentration of hydrophobin as low as possible. This not only leads to cost reduction, but also to saving of resources, as the production of the hydrophobin naturally costs energy.

Hydrophobins may be added to products as aqueous solution or dispersion, or may be added in dry form. In that case the hydrophobins may be freeze-dried, or may be

agglomerated with a suitable material such as maltodextrin, in order to make the

hydrophobin better storage stable. Such agglomerated hydrophobin may have various particle sizes.

Preferably the concentration of hydrophobin ranges from 0.03% by weight to 1 % by weight based on the weight of the composition. Preferably the concentration of hydrophobin is at least 0.05% by weight, more preferably at least 0.08% by weight, based on the weight of the composition. Preferably the concentration of hydrophobin is maximally 0.8% by weight, preferably at most 0.6% by weight, preferably at most 0.4% by weight, preferably at most 0.3% by weight based on the weight of the composition. The hydrophobin used in the present invention can be a class I or a class II hydrophobin, or can be a combination of a class I and a class II hydrophobin. Examples of class II

hydrophobins are HFBI, HFBII, and cerato-ulmin. Preferably, the hydrophobin comprises class II hydrophobin. Preferably the hydrophobin is in isolated form, which means that it is added as a separate ingredient, and not as an element of other ingredients or micro- organisms. Preferably the hydrophobin is soluble in water. Preferably the hydrophobin is a class II hydrophobin HFBII.

Hydrocolloids

A hydrocolloid as defined herein is a hydrophilic polymer which is dispersable in water. The one or more hydrocolloids to provide an apparent yield stress of at least 1 Pa preferably comprise a water-soluble compound. The one or more hydrocolloids to provide a yield stress of at least 1 Pa comprises xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, alginate, pectin, and carboxy-methylcellulose, or any combination of these. Preferably the one or more hydrocolloids to provide an apparent yield stress of at least 1 Pa is selected from xanthan gum, carrageenan, guar gum, and locust bean gum, and from any combination of these. Preferably the one or more hydrocolloids provide an apparent yield stress of at least 2 Pa, preferably at least 3 Pa, more preferably at least 4 Pa, more preferably at least 5 Pa. Preferably the apparent yield stress is maximally 20 Pa, more preferred maximally 15 Pa, more preferred maximally 10 Pa. With apparent yield stress is meant the continuous phase apparent yield stress, meaning that the liquid phase has an apparent yield stress of at least 1 Pa. The yield stress prevents or delays creaming of gas bubbles, in case the composition of the invention contains dispersed gas bubbles. The yield stress is the force required to keep a bubble stationary in the liquid, counteracting the buoyancy. The hydrocolloid can increase the viscosity at zero shear or during flow. On mild agitation (e.g. manually gently shaking), the composition may flow due to shear thinning effect. The apparent yield stress can be determined as described in WO 2007/039064 A1. The dynamic viscosity of the liquid composition preferably ranges from 90 to 200 mPa-s at a shear rate of 0.1 s^"1 and a temperature of 20°C. The concentration of the hydrocolloid materials preferably ranges from 0.1 to 2% by weight. The composition of the invention preferably comprises one or more hydrocolloid selected from the group of xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, alginate, pectin, and carboxy-methylcellulose, and from any combination of these at a concentration ranging from 0.2 to 2% by weight, more preferred from 0.3 to 1.5% by weight, more preferred from 0.4 to 1 % by weight. In case xanthan gum is used as the single hydrocolloid, the concentration of xanthan gum ranges from 0.4% to 0.8% by weight, preferably from 0.5% to 0.8% by weight, preferably from 0.5% to 0.7% by weight. In case the hydrocolloid comprises carrageenan, then preferably the carrageenan comprises iota- carrageenan. In case iota-carrageenan is used as the single hydrocolloid, the concentration of iota-carrageenan ranges from 0.3% to 0.6% by weight, preferably from 0.3% to 0.5% by weight.

Preferably the present invention provides a composition in liquid form comprising a continuous aqueous phase; and

milk protein at a concentration ranging from 0.8% to 5% by weight; and

one or more compounds selected from monosaccharides, disaccharides, and

oligosaccharides, at a concentration ranging from 32% to 50% by weight, and

one or more hydrocolloids selected from xanthan gum, carrageenan, guar gum, locust bean gum, tara gum, alginate, pectin, and carboxy-methylcellulose, and from any combination of these at a concentration ranging from 0.1 to 2% by weight; and

a hydrophobin at a concentration of at least 0.001 % by weight; and

total solids at a concentration ranging from 42% to 60% by weight.

Emulsifier

Preferably the composition of the invention additionally contains one or more emulsifiers. In combination with the hydrophobin, the one or more emulsifiers facilitate the formation and stabilisation of gas bubbles when the composition of the invention is aerated and comprises a dispersed gas phase. In such case, preferably the aerated product will lose no more than 20% of the total overrun when stored at 5°C for 2 weeks. More preferably, the aerated product will lose no more than 10% of the total overrun when stored at 5°C for 2 weeks. The one or more emulsifiers that are preferably present in the composition are the only isolated emulsifiers. The phrase "isolated emulsifiers" means that the composition contains only the one or more emulsifiers as emulsifiers which have been added to the composition to act as an emulsifier. This does not exclude the possibility that the composition may contain emulsifiers which unintentionally are added to the composition as part of another ingredient. For example, the oil may contain minute amounts of monoglycerides or diglycerides which may act as emulsifiers. Such compounds are not included in the definition of the "isolated emulsifier". In the context of the present invention, the hydrophobin is not encompassed by the term "emulsifier".

Preferably, the one or more emulsifiers comprise polysorbate, polyglycerol esters of fatty acids, diacetyl tartartic acid esters of mono- and/or diglycerides, or sucrose fatty acid esters having a HLB-value of at least 8. More preferred the one or more emulsifier comprises diacetyl tartartic acid esters of mono- and/or diglycerides.

Polysorbates are a class of emulsifiers which comprise: Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate); Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate); Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and Polysorbate 80

(polyoxyethylene (20) sorbitan monooleate). The number 20 following the "polyoxyethylene" part refers to the total number of oxyethylene (CH2CH2O) groups found in the molecule. A suitable source for the polysorbates are the Tweens (Tween 20, 40, 60, 80) ex Croda International.

Diacetyl tartartic acid esters of mono- and/or diglycerides are also known as DATEM. A suitable source is Panodan DATEM ex DuPont Danisco.

A suitable source of polyglycerol fatty acid ester is Grindsted PGE ex DuPont Danisco.

Sucrose fatty acid esters having a HLB-value of at least 8 are esters of sucrose and one or more fatty acids. Sucrose fatty acid esters can be prepared with any type of fatty acid.

Suitable fatty acids may vary both in alkyl chain length and in degree of unsaturation.

Suitable fatty acids are saturated fatty acids including but not limited to capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, behenic acid. Likewise, monounsaturated fatty acids including but not limited to lauroleic acid, myristoleic acid, palmitoleic acid, oleic acid, are also suitable. Similarly, polyunsaturated fatty acids including but not limited to linoleic acid, linolenic acid, or arachidonic acid are suitable too. Sucrose fatty acid esters can also be mixtures of different compounds, for example mixtures in terms of compounds with a different degree of substitution, or of compounds with different types of fatty acids, or a combination of these. Commercially, food-grade sucrose fatty acid esters may be obtained from suppliers like Mitsubishi-Kagaku (Tokyo, Japan) or Sisterna

(Roosendaal, The Netherlands). Apart of their structure, sucrose fatty acid esters or mixtures of sucrose fatty acid esters may also be characterised by their properties. The most noteworthy property is their hydrophilic- lipophilic balance or HLB value. HLB values are a well-known classification of surfactants or mixtures of surfactants, based on the ratio of the hydrophilic and hydrophic portions of the surfactant molecules. HLB values of surfactants range from 0 to 20, where HLB 0 is the hydrophobic and HLB 20 the hydrophilic end of the range.

Preferably the concentration of the emulsifier is lower than that of the hydrophobin.

Preferably the weight ratio of emulsifier to hydrophobin ranges from 1 :10 to 1 :1 . More preferred the ratio of emulsifier to hydrophobin ranges from 2:10 to 8:10.

Aerated composition in liquid form

Preferably the composition of the invention has been aerated such that the product has a foamy structure and appearance. Aeration can be done with any gas normally used in food products, like air, oxygen, nitrogen, and carbon dioxide. Preferably aeration is done using air or nitrogen. In case the composition has been aerated, then preferably the composition contains gas bubbles at an overrun ranging from 30% to 200%. Preferably the overrun ranges from 30% to 150%. Preferably the overrun ranges from 30% to 150%. In such case gas bubbles will be dispersed in the continuous liquid phase. Preferably the gas bubbles have an average diameter D4,3 ranging from 5 to 100 micrometer. Preferably the gas bubbles have an average diameter D4,3 ranging from 5 to 90 micrometer, preferably ranging from 10 to 80 micrometer. Preferably at least 50% of the number of gas bubbles has a diameter smaller than 100 micrometer, more preferred at least 75% of the gas bubbles. Preferably at least 50% of the number of gas bubbles has a diameter smaller than

50 micrometer, more preferred at least 75% of the gas bubbles. Incidentally also larger bubbles may be present.

Also the aerated composition according to the invention is in liquid form, which means that it is a flowable composition. In the context of the present invention, the aerated composition is in liquid form at a temperature above 0°C, and at these temperatures the composition is a flowable product. Preferably such aerated composition is packaged in a closed package. Preferably, the composition is packaged aseptically. Preferably the package is sealed. This way the aerated composition of the invention can be distributed at a temperature above 0°C. Therefore preferably the composition is at a temperature of 0°C or higher, preferably at a temperature of maximally 40°C, preferably maximally 35°C.

Preferably the composition according to the invention is free from ethanol and starch.

Frozen composition

The packaged aerated composition of the invention can be frozen, by putting the packaged composition in a freezer at a temperature below 0°C. This way the composition will be quiescently frozen, meaning without agitation or aeration during the freezing process, to yield a frozen confection. Preferably the aerated composition of the invention is at a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C. Preferably the temperature at which the aerated composition is frozen is maximally -18°C. Preferably the ice content ranges from 30% to 40% by weight at -18°C, preferably from 30% to 39% by weight. The ice content of the frozen composition is relatively low. This way a quiescently frozen structure is prepared, that mimics that of a dynamically frozen ice cream. The advantage of the invention is that the structure of the frozen composition resembles that of a dynamically frozen composition.

The liquid composition of the invention can be distributed at temperatures above 0°C, and can be quiescently frozen, while providing a good texture, and without becoming too sweet. This way an excellent ice cream can be prepared by and served to a consumer, without the need of a distribution chain at -18°C or lower. Production and distribution of the aerated liquid composition of the invention can be done at temperatures above 0°C. Consequently this leads to a strong reduction of energy consumption, compared to the traditional supply chain of ice cream. Method for preparation of composition

In a second aspect the invention provides a method for preparation of the composition of the invention. An unaerated composition according to the first aspect of the invention can be prepared using any common equipment usually used for preparing liquid mixtures. The preparation method encompasses preferably two distinct unit operations:

1. Mixing stage - the ingredients of the composition of the invention are brought together, solubilised in water and mixed. 2. Optionally a homogenisation stage - to disperse the fat in small emulsion droplets for stability, at 0 to 150 bar in a high pressure homogenizer.

In case an aerated product according to the invention is prepared, the second aspect of the invention provides a method for preparation of a composition according to the first aspect of the invention containing gas bubbles, comprising the steps:

a) Providing an unaerated composition according to the first aspect of the invention; b) Optionally homogenising the composition from step a);

c) Aerating the composition from step a) or step b); and

container.

In step a) a premix of the composition is provided, which usually has been prepared in a mixed vessel. Preferably this is done by first mixing the part of saccharides, stabilisers and emulsifiers, and mixing this with heated water, preferably at a temperature of at least 70°C, preferably at least 75°C. Preferably milk protein is added to the mix when the temperature of the mix is lower than 75°C, after which optional glucose syrup is added. Subsequently, oil which is melted may be added and emulsified. The product has not yet been intentionally aerated. Incidentally the composition may contain gas bubbles which may have been included in the composition during the preparation process. In this step oil droplets are dispersed in a continuous aqueous matrix and the hydrocolloid providing yield stress is dispersed. Preferably in this step the oil droplets are have a volume weighted geometric mean diameter D4,3 of less than 20 micrometer, preferably less than 15 micrometer. This mean diameter may suitably be determined using the method described by Goudappel et al. (Journal of Colloid and Interface Science 239, p. 535-542, 2001 ). Typically, 80 to 100% of the total volume of the oil droplets contained in the present emulsion have a diameter of less than 20 micrometer.

In step b) the composition optionally is homogenised to make a more homogeneous and smooth premix. Preferred equipment for homogenisation include a high-shear mixer, with an impeller for mixing. Another preferred equipment is a high pressure homogeniser that is used to homogenise, preferably operating at a pressure ranging from 10 to 200 bar, preferably from 20 to 150 bar. The composition from step a) or b) may be pasteurised or sterilised. In case the composition is pasteurised or sterilised, the hydrophobins will be added to the mixture after such pasteurisation or sterilisation step, after the composition is at a temperature of less than 70°C, preferably less than 60°C. This is required in order to prevent that hydrophobin denatures due to the heating step. In such case the hydrophobins may be added to the composition aseptically, to prevent contamination of the composition. This may be done by filtering a solution of hydrophobin through a 0.2 μηη filter in a sterile environment.

In step c) the composition containing hydrophobin is aerated to an overrun preferably ranging from 30% to 200%. Preferably the overrun ranges from 30% to 150%. Aeration of the composition may be done as a batch operation, with a mixer which disperses air bubbles in the mixture, or may be done in line, using a continuous aerator. Such aerators operate by continuously passing mix through a reactor head which has a whipping camber composed of a stator and a rotor. Aeration is achieved by injecting the desired level of gas into the incoming premix - gas bubble breakdown and mixing is accomplished through the high speed rotation of the rotor equipped with teeth which pass through narrow gaps formed by additional static teeth mounted on the stator. The temperature of the mixture during aeration preferably ranges from 5°C to 25°C. Preferably the aeration is done aseptically, such that the composition is not spoiled.

Finally in optional step d) the aerated composition is packaged in a container and then closed or sealed. The temperature of the aerated composition in this step d) preferably ranges from 5°C to 25°C. Preferably the packaging is done aseptically, such that the composition is not spoiled. This way the aerated composition of the invention can be distributed. The package that is used may be any package that is commonly used for liquid food compositions. Preferably the package can withstand temperatures between 0°C and -30°C.

The second aspect the invention also provides a method for preparation of a frozen aerated composition, wherein an aerated composition according to the first aspect of the invention is brought to a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C. Preferably the aerated composition of the invention is packaged in a closed container.

Preferably the aerated composition is not agitated when brought to a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C. This means that preferably the aerated premix is quiescently frozen to prepare an ice cream. This can be done by a consumer in house or at another point of consumption. The consumer purchases an aerated composition according to the first aspect of the invention, which is packaged in a closed container. The closed container is put in a freezer, to yield a frozen confection.

DESCRIPTION OF FIGURES

All figures: Scanning electron microscopy images of quiescently frozen compositions from the example below. The images show air bubbles in a continuous matrix of water, ice, sugars, and other ingredients. In all figures: Top image bar size δθθμηη; middle image bar size Ι ΟΟμηη; bottom image bar size δθμηη.

Figure 1 : Composition C12.

Figure 2: Composition C13.

Figure 3: Composition C14.

Figure 4: Composition A14.

Figure 5: Composition A15.

Figure 6: Composition B10.

EXAMPLES

The following non-limiting examples illustrate the present invention. Raw materials

· Cream: 48% fat, ex Meadow Foods (Chester, Cheshire, UK).

• Skimmed milk powder, ex Dairy crest (Esher, Surrey, UK); containing typically about 50% lactose and 35% protein.

• Dextrose monohydrate: C-Pharm Dex 02010 ex Cargill (Minneapolis, MN, USA.

• Sucrose, ex Tate and Lyle (London, UK).

· Xanthan gum: Keltrol F, ex CP Kelco (Nijmegen, The Netherlands).

• Carrageenan: Deltagel, ex Kerry Group (Kerry, Ireland)

• DATEM: Panodan DATEM, ex DuPont Danisco (Copenhagen, Denmark)

• Aqueous HFBII: Class II Hydrophobin HFBII ex DuPont Danisco (Copenhagen,

Denmark), produced by the fungus Trichoderma reesei, and isolated from fermentation broth. Concentration is 99.7 g/kg of hydrophobin in aqueous dispersion, containing potassium sorbate as preservative, pH about 3.5.

Preparation of aerated compositions and quiescently freezing the compositions

Liquid compositions were prepared with the following compositions. Recipe of liquid compositions, with amounts of the ingredients in weight

These mixtures were prepared by the following process. A premix of each composition was prepared in a mixed vessel. This was done by first mixing dry sugars, emulsifier, and stabiliser, and adding this dry mix to water at 82°C. After 2 minutes stirring and cooling, skimmed milk powder was added at 72°C. Then again 2 minutes stirring. After cooling down the aqueous HFBII and cream were added to the composition. The mixtures were aerated with air using a WCB inline aerator (WCB Ice Cream, Aarhus, Denmark) to an overrun of about 100%.

Table 2 Compositions of liquid compositions from Table 1.

After aeration the liquid compositions were visually assessed on stability during storage at room temperature or at 5°C. The factors assessed were the following: • Stability of overrun during storage: good when no or very small overrun loss, and bad when severe overrun loss during storage.

• Bubble growth during storage: good when no or very small coarsening of bubbles, and bad when severe coarsening of bubbles during storage.

· Phase separation (bubbles) during storage: good when no or very small creaming of bubbles, and bad when severe creaming of bubbles.

• Phase separation (serum) during storage: good when no or very small formation of serum layer on bottom of container, and bad when thick serum layer on bottom of container. The evaluation of the samples gave the following:

Table 3 Compositions of liquid compositions from Table 1.

^* RT = room temperature

Claims

1. A composition in liquid form comprising a continuous aqueous phase; and triglycerides at a concentration ranging from 3% to 10% by weight, preferably from 5% to 10% by weight; and

milk protein at a concentration ranging from 0.8% to 5% by weight; and

one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, at a concentration ranging from 32% to 50% by weight, and wherein a mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, has a number average molecular weight <M>n ranging from 240 to 350 gram per mole; and

a hydrophobin at a concentration of at least 0.03% by weight; and

total solids at a concentration ranging from 42% to 60% by weight.

2. A composition according to claim 1 , wherein the mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides has a number average molecular weight <M>n ranging from 250 to 350 gram per mole, preferably from 270 to 340 gram per mole.

3. A composition according to claim 1 or 2, wherein the relative sweetness of the mixture of the one or more compounds selected from monosaccharides, disaccharides, and oligosaccharides, is maximally 0.22, preferably maximally 0.2, preferably maximally 0.18.

4. A composition according to any of claims 1 to 3, wherein the hydrophobin comprises class II hydrophobin.

5. A composition according to any of claims 1 to 4, additionally containing one or more emulsifiers.

6. A composition according to claim 5, wherein the one or more emulsifiers comprise polysorbate, polyglycerol esters of fatty acids, diacetyl tartartic acid esters of mono- and/or diglycerides, or sucrose fatty acid esters having a HLB-value of at least 8.

7. A composition according to claim 5 or 6, wherein the weight ratio of emulsifier to hydrophobin ranges from 1 :10 to 1 :1.

8. A composition according to any of claims 1 to 7, containing gas bubbles at an overrun ranging from 30% to 200%.

9. A composition according to claim 8, wherein the gas bubbles have an average diameter D4,3 ranging from 5 to 100 micrometer.

10. A composition according to claim 8 or 9, wherein the composition is packaged in a closed package.

1 1. A composition according to any of claims 8 to 10, wherein the composition is at a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C.

12. A composition according to claim 1 1 , wherein the ice content ranges from 30% to 40% by weight at -18°C, preferably from 30% to 39% by weight.

13. A method for preparation of a composition according to claim 8 or 9, comprising the steps:

a) Providing an unaerated composition according to any of claims 1 to 7;

b) Optionally homogenising the composition from step a);

c) Aerating the composition from step a) or step b); and

d) Optionally packing the composition from step c) in a container and sealing the container.

14. A method for preparation of a frozen aerated composition according to claim 1 1 or 12, wherein a composition according to any of claims 8 to 10, is brought to a temperature below 0°C, preferably below -5°C, preferably between -10°C and -25°C.