WO2011015504A2

WO2011015504A2 - Aerated products

Info

Publication number: WO2011015504A2
Application number: PCT/EP2010/060950
Authority: WO
Inventors: Theodorus Berend Jan Blijdenstein; Andrew Richard Cox; Hyun-Jung Kim; Simeon Dobrev Stoyanov
Original assignee: Unilever Plc; Unilever N.V.; Hindustan Unilever Limited
Priority date: 2009-08-07
Filing date: 2010-07-28
Publication date: 2011-02-10
Also published as: WO2011015504A3

Abstract

Aerated products containing crosslinked hydrophobin exhibit increased foam stability in presence of surface active agents.

Description

AERATED PRODUCTS

Field of the invention

[0001] The present invention relates to aerated products that include

hydrophobins and to a method for manufacturing the same.

Background to the invention

[0002] A wide variety of products contain introduced gas, such as air, nitrogen and/or carbon dioxide. Such products include frozen and chilled food products, for example ice cream and mousses, they also include shaving foams or hair foams. Two key considerations arise in the production and storage of aerated products, namely the ability to incorporate gas into the product during manufacture (foamability) and the subsequent stability of the gas bubbles during storage (foam stability). A number of additives are included in aerated products to assist in the creation and maintenance of foam. These include proteins such as sodium caseinate and whey, which are highly foamable, and biopolymers, such as carrageenans, guar gum, locust bean gum, pectins, alginates, xanthan, gellan, gelatin and mixtures thereof, which are good stabilisers. However, although stabilisers used in the art can often maintain the total foam volume, they are poor at inhibiting the coarsening of the foam microstructure, i.e. increase in gas bubble size by processes such as disproportionation and coalescence. Further, many of the ingredients used to stabilise the gas phase in aerated food products need to be added at fairly high levels which can have deleterious textural and/or calorific consequences.

[0003] EP1623631 describes the use of hydrophobin to create foams with good foam stability properties. Nonetheless, when put in presence of surface active agents, such foams tend to get collapsed by destabilization. There is therefore a need for an aerated product containing hydrophobin which is not completely destabilised by the presence and/or the introduction of surface active agents.

[0004] WO99/16983 describes the use of enzymatically cross-linked proteins but, whereas foams with higher overruns can be produced which are moreover more stable over a short period of time, they are not stable over a longer period of time as established in the comparative examples.

[0005] It has now been found that it is possible to increase the stability such a foam by crosslinking hydrophobin.

Tests and Definitions

Aerated products

[0006] The term "aerated" means that gas has been incorporated into the

product, such as by mechanical means. The gas can be any gas such as air, nitrogen or carbon dioxide. The extent of aeration is typically defined in terms of "overrun". In the context of the present invention, %overrun is defined in volume terms as:

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

X 100

[0007] The amount of overrun present in the product will vary depending on the desired product characteristics. For example, the level of overrun in ice cream is typically from about 70 to 100%, and in confectionery such as mousses the overrun can be as high as 200 to 250 wt%, whereas the overrun in water ices is from 25 to 30%. The level of overrun in some chilled products, ambient products and hot products can be lower, but generally over 10%, e.g. the level of overrun in milkshakes is typically from 10 to 40 wt%.

Hydrophobins:

[0008] 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-Xm (SEQ ID No. 1) where X represents any amino acid, and n and m independently represent an integer. Typically, a hydrophobin has a length of up to 125 amino acids. The cysteine residues (C) in the conserved sequence are part of disulphide bridges. In the context of the present invention, the term hydrophobin has a wider meaning to include functionally equivalent proteins still displaying the characteristic of self-assembly at a

hydrophobic-hydrophilic interface resulting in a protein film, such as proteins comprising the sequence:

X_n-C-Xi -50-C-X0-5-C-X1 -100-C-X1 -100-C-X1 -50-C-X0-5-C-X1 -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, α-helix) (De

Vocht et al., 1998, Biophys. J. 74: 2059-68).

[0009] 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.

[0010] 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 generally relatively insoluble whereas those of class Il hydrophobins readily dissolve in a variety of solvents. Preferably the hydrophobin is a class Il hydrophobin. Preferably the hydrophobin is soluble in water, by which is meant that it is at least 0.1 % soluble in water, preferably at least 0.5%. By at least 0.1 % soluble is meant that no hydrophobin precipitates when 0.1g of hydrophobin in 99.9 ml_ of water is subjected to 30,000 g centrifugation for 30 minutes at 2O⁰C.

[0011] Hydrophobin-like proteins (e.g."chaplins") have also been identified in

filamentous bacteria, such as Actinomycete and Streptomyces sp.

(WO01/74864; Talbot, 2003, Curr. Biol, 13: R696-R698). These bacterial proteins by contrast to fungal hydrophobins, may form only up to one disulphide bridge since they may 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.

[0012] 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).

[0013] Alternatively, hydrophobins can be obtained by the use of recombinant technology. For example host cells, typically micro-organisms, may be modified to express hydrophobins and the hydrophobins can then be isolated and used in accordance with the present invention. Techniques for introducing nucleic acid constructs encoding hydrophobins into host cells are well known in the art. More than 34 genes coding for

hydrophobins have been cloned, from over 16 fungal species (see for example WO96/41882 which gives the sequence of hydrophobins identified in Agaricus bisporus; and Wosten, 2001 , Annu Rev. Microbiol. 55: 625-646). Recombinant technology can also be used to modify hydrophobin sequences or synthesise novel hydrophobins having desired/improved properties.

[0014] 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.

[0015] 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).

[0016] 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.

[0017] 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".

[0018] 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.

Glutaraldehvde (GAH)

[0019] GAH (C5H8O2, Mw 100.12 g mol^"1) reacts with protein and will chemically cross-link these molecules. The sites of cross-linking between aldehydes and proteins are likely to be lysine, histidine, cysteine and tyrosine.

Figure 1. The structure of glutaraldehyde

Sodium dodecyl sulphate (SDS)

[0020] SDS (NaCi₂H₂SSO₄ Mw 288.38 g moP¹) is a highly effective surface

active agent used in any task requiring the removal of oily stains and residues. It is known to be highly surface active and can adsorb to the a/w surface and replace other adsorbed molecules that have a lower surface affinity.

Xanthan solution

[0021] Xanthan (CPKelco Keltrol RD) has been dissolved in double distilled water to 0.5 w/w %. This is to adjust overrun of HFB foam into 100%. Xanthan was used as a thickener.

Cross linked hvdrophobin

[0022] Cross linked hydrophobin can be detected most easily by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and then the amount can be read by densitometer.

[0023] Cross linking can be done chemically or enzymatically, as well known in the art and as described in "Protein crosslinking in foods: methods, consequences, applications" Julier A. Gerrard, Trends in Food Science & Technology - 13 (2002) 389-397.

[0024] Preferably, in the present invention, cross linking is done chemically.

Surface active agents

[0025] Surface active agents means a substance which lowers the surface

tension of the medium in which it is dissolved, and/or the interfacial tension with other phases, and, accordingly, is positively adsorbed at the liquid/vapour and/or at other interfaces. Examples of surface active agents include proteins, detergents, and soaps, syndets, emulsifiers, and foaming agents.

Summary of the invention

[0026] It is a first object of the present invention to provide an aerated product comprising cross-linked hydrophobin. In a related aspect, the present invention provides an aerated product in which the air phase is at least partially stabilised with cross-linked hydrophobin. In another related aspect, the present invention provides an aerated product comprising cross-linked hydrophobin in which the cross-linked hydrophobin is associated with the air phase.

[0027] Preferably, the aerated product is an edible food product.

[0028] Preferably the overrun of the aerated product is over 10%, more preferably over 30%.

[0029] Preferably also, the overrun is below 1000%, more preferably below

600%, even more preferably below 300%

[0030] Preferably also the pH of the aerated product is between 2 and 10

[0031] Preferably the hydrophobin is a class Il hydrophobin, more preferably the hydrophobin is HFB II.

[0032] In a preferred embodiment, the cross-linked hydrophobin is present in an amount of at least 0.001 wt%, more preferably at least 0.01 wt%.

[0033] Preferably the cross-linked hydrophobin is an enzymatically cross linked hydrophobin

[0034] In another preferred embodiment of the invention, the cross-linked

hydrophobin is a chemically cross linked hydrophobin.

[0035] In a more preferred embodiment of the invention the aerated product

comprises one or more surface active agents on top of hydrophobin.

[0036] It is a second object of the present invention to provide a process for

producing an aerated product wherein hydrophobin a composition containing at least 0.001 wt%, more preferably at least 0.01 wt%, hydrophobin is subjected to an aeration step characterised in that at least part of the hydrophobin is subjected to a cross linking step after the aeration step.

[0037] Preferably the aeration step brings the aerated product to an overrun of at least 10%, more preferably greater than 20% and most preferably greater than 50%.

[0038] In a preferred embodiment of the invention, the cross linking steo is an enzymatic cross linking step. [0039] In another preferred embodiment of the invention, the cross-linking step is a chemical cross-linking step. More, preferably the cross linking process is done by adding glutaraldehyde. Even more preferably the ratio (w/w) of glutaraldehyde to hydrophobin is between 10:1 and 1 :100, even more preferably above 1 :10, most preferably above 1 :3

[0040] Crross linking with glutaraldehyde for better stabilizing foam is a long

chemical reaction and in a preferred alternative of the invention,

glutaraldehyde is added during the aeration step. In another preferred alternative, glutaraldehyde is added after the aeration step.

[0041] Typically, the hydrophobin is added to the product in a form such it is

capable of self-assembly at an air-liquid surface. In one embodiment the product is a food product, In another embodiment this is an aerated personal or household care product, e.g. shampoo, conditioner, detergent, face wash.

[0042] Typically, the hydrophobin is added to the product of the invention in an isolated form, typically at least partially purified, such as at least 10% pure, based on weight of solids. By "added in isolated form", we mean that the hydrophobin is not added as part of a naturally-occurring organism, such as a mushroom, which naturally expresses hydrophobins. Instead, the hydrophobin will typically either have been extracted from a

naturally-occurring source or obtained by recombinant expression in a host organism.

[0043] The amount of hydrophobin added will generally vary depending on the product formulation and volume of the air phase. Typically, it will be added at least 0.001 wt%, hydrophobin, more preferably at least 0.005 or

0.01 wt%. Typically it will be added less than 1 wt% hydrophobin. The hydrophobin may be from a single source or a plurality of sources e.g. the hydrophobin can a mixture of two or more different hydrophobin

polypeptides.

[0044] Preferably the hydrophobin is a class Il hydrophobin, more preferably HFB II.

[0045] In one embodiment, the hydrophobin is added to the aerated product or compositions of the invention in an isolated form, typically at least partially purified.

Detailed description of the invention

[0046] The present invention will now be described further with reference to the following examples which are illustrative only and non-limiting.

Comparative examples: Preparation of foam with enzvmaticallv cross linked proteins

[0047] As according to WO99/16983 foams were produced using enzymatically cross linked proteins.

[0048] The proteins were ovalbumine, BSA and skimmed milk powder. The

results are summarised in the following tables:

[0049]

Table 1

[0050] The overrun decrease over time was as follows:

SMP

[0051]

Table 2

BSA

[0052]

Table 3

Ovalbumine

[0053] Table 4

[0054] Therefore, enzymatically crosslinking proteins as in WO99/16983 leads to foams which can have a higher overrun initially and which over a short period of time have a greater stability, but after less than 19 hours (1140 minutes) the foam has already collapsed,

Preparation of foam with enzvmaticallv cross linked hvdrophobin

[0055] A foam were produced using enzymatically cross linked hydrophobin.

(HFB II).

[0056]

Table 5

[0057] Here it is shown that even after 60 hours a foam with enzymatically cross linked hydrophobin is still stable.

Preparation of Hvdrophobin (HFBII) foam and 0.5% xanthan solution with

Glutaraldehvde (GAH in the rest of the description)

Experiment 1

[0058] 100 ml_ of a 0.1 w/w % HFB solution (pH -3.5) was aerated using a

Kenwood mixer (model KM220, Kenwood electronics) for 5 minutes at its maximum speed.

[0059] The initial overrun was 450% (Volume final-volume initial/volume initial X 100). And the overrun was adjusted to 100% by the addition of either pure xanthan solution or GAH containing xanthan solution. This is in order to compare 100% overruned foam stability further with and without

GAH/SDS. A solution of GAH in xanthan solution was made such that when mixed with the hydrophobin foam, the GAH to HFB ratio would be 1 :1. With this amount of GAH, the pH of xanthan solution was used 1 ) as it is 2) optimized to 7.8 (i.e. the optimum pH for cross-linking by GAH) or 3) adjusted to 10. The reaction took place at room temperature, 25⁰C.

Experiment 2

[0060] The pH of a 60 ml_ of 0.1 w/w % HFB solution was adjusted to pH 7.8 and then whipped by same Kenwood mixer for 8 min at its maximum speed. The initial overrun was 360%. The overrun was adjusted to 100% by addition of either pure xanthan solution or GAH containing xanthan solution. This is in order to compare 100% overruned foam stability further with and without GAH/SDS. A GAH amount in xanthan solution was adjusted to GAH to HFB ratio as 1 :10 or 1 :100. The reaction has been at 5 oC.

Cross link of HFB at air bubble surface

[0061] The overrun of the HFBII foam was 100% with the xanthan solution which contained 1) no GAH (comparative example) and 2) GAH (examples). 10 g of this foam without/with crosslinking agent was placed in two vials and stored at designated temeperature (at room temperature in experiment 1 and at 5⁰C in experiment 2) for 24 hours to ensure the reaction continued. After 24 hours, 0.5ml of either double distilled water or 10% SDS solution (which finally made SDS concentration as 0.5% in total) was added into each vial. The sample with distilled water is for control and the one with SDS is to monitor the replacement of HFBII from bubble surface. The foam stability and bubble size evolution were monitored over 15 days. By visual observation and using a Turbiscan.

Confirmation of the polymerization of HFBII

[0062] Foams with or without GAH/SDS were placed in Eppendorf tube for

centrifugation. Then centrifugation for 10 min separated foam and serum layer. Each part was carefully collected and applied Sodium Dodecyl Sulfate PolyacrylAmide Gel Electrophoresis (SDS Page) to trace protein with silver staining of protein.

Results

[0063] The results of the foam appearance after 15 days of crosslinker (GAH) and consequently after 14 days replacing reagent (SDS) treatment was as follows: [0064] The comparative example without GAH and without SDS) showed drainage but stable foam on top with no significant change in relative bubble size

[0065] The comparative example with SDS showed complete collapse of the

foam. The two examples with GAH and with GAH+SDS showed drastic difference from the comparative example. The addition of SDS on the crosslinked HFBII did not trigger any destabilization of foam.

Example 3: Aerated Low Fat mayonnaise comprising foam stabilised bv cross- linked HFBII-foam

[0066] In this example, the bubble size evolutions of a mayonnaise aerated with foam stabilised with cross-linked Hydrophobin will be compared to that of a mayonnaise which is aerated by hydrophobin which is not cross-linked. An approximation of the bubble size evolution is probed by turbidimetry using a Turbiscan Lab Expert apparatus (Formulaction, France). Sample volumes of 20ml of the foams were studied by turbidimetry in time using a Turbiscan Lab Expert (Formulaction, Toulouse, France). We interpret the average backscattering along the height of a sample with exclusion of the top and bottom parts where the backscattering is affected by edge effects. The backscattering (BS) is related to the transport mean free path of the light in the sample through

[0067] For an aerated mayonnaise we plot the relative increase of the transport mean free path λ (t)/ λ (0) as an approximation of the bubble size evolution, since mayonnaise contains a high concentration of oil droplets in addition to the air bubbles. Since the emulsion droplet size is known to be constant over time, we can interpret an increase in λ to be an

underestimating signal of bubble coarsening.

[0068] 100 ml or 0.1 wt% HFBII solution was aerated using a Kenwood type of

Kitchen mixer and left to drain overnight. For the invention example, part of the dry foam was collected and mixed with 0.5wt% xanthan solution which contained glutaraldehyde to make a 1 :1 ratio of glutaraldehyde to hydrophobin at a pH of 7.8. The Overrun of this foam was 100%. This foam was incubated for the cross-linking reaction at 20°C for 24 hours. For a comparative sample, another part of the dry foam was collected and mixed with 0.5wt% xanthan solution without glutaraldehyde. The Overrun of this foam was also 100%. After the overnight reaction, the foams were three times diluted, gently stirred and creamed on top to collect it as pure as possible with a minimum residual amount of xanthan and

glutaraldehyde.

[0069] 20 ml of the washed and cross-linked foam was mixed to 20 ml Low Fat Mayonnaise (Hellmann's Light), which was degassed before the mixing. No volume decrease was observed during mixing and this resulted into an aerated mayonnaise comprising 25 vol% air.

[0070] Similarly, 20 ml of the comparative foam sample was mixed to 20 ml

degassed Low Fat Mayonnaise (Hellmann's Light), and also here no volume decrease was observed and thus this also yielded a mayonnaise with 25 vol% air.

[0071] Portions of App. 20 ml of the aerated mayonnaises were transferred into a glass tubes for turbidimetry. The bubble size evolution was monitored over a time lapse of 80 days.

[0072] Figure 1 shows the relative increase in transport mean free path λ (t)/ λ (0) of the invention product and the reference, measured over 81 days. Taking into account that much of the bubble size evolution is masked by the concentrated emulsion in the mayonnaise, still a significant difference in evolution can be observed between the two samples. The reference mayonnaise shows a 10% increase in optical path length after about 4 days, whereas for the crosslinked sample it takes about 80 days to reach this increase. This means that the average bubble size increases fastest in the reference mayonnaise.

[0073] This result clearly shows the improved stability to disproportionation of the aerated low fat mayonnaise comprising cross-linked hydrophobin foam compared to the product comprining non cross-linked hydrophobin foam.

Claims

1. An aerated product comprising cross-linked hydrophobin.

2. An aerated product according to claim 1 having an overrun of above 10%.

3. An aerated product according to claim 1 or claim 2 having a pH of between 2 and 10.

4. An aerated product according to any preceding claim wherein the cross-linked hydrophobin is present in an amount of at least 0.001 wt%, more preferably at least 0.01 wt%.

5. An aerated product according to any preceding claim containing a surface active agent.

6. An aerated product according to any preceding claim wherein the cross linked hydrophobin is chemically cross linked.

7. A process for producing an aerated product wherein a composition containing at least 0.001 wt%, more preferably at least 0.01 wt%, hydrophobin is subjected to an aeration step characterised in that at least part of the hydrophobin is subjected to a cross linking step after the aeration step.

8. A process according to claim 6 wherein the aeration step brings the aerated product to an overrun of at least 10%.

9. A process according to claim 7 or 8 characterised in that the crosslinking step is a chemical cross linking step

10. A process according to claim 8 wherein the cross linking step is done by

adding glutaraldehyde.

11. A process according to claim 8 wherein the ratio (w/w) of glutaraldehyde to hydrophobin is between 10:1 and 1 :100, preferably above 1 :10.

12. A process according to claim 11 wherein glutaraldehyde is added during the aeration step.

13. A process according to claim 11 wherein glutaraldehyde is added after the aeration step.