EP3107406A1 - Stabilisiertes, durchlüftetes konfekt mit hydrophobin - Google Patents

Stabilisiertes, durchlüftetes konfekt mit hydrophobin

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Publication number
EP3107406A1
EP3107406A1 EP15713650.8A EP15713650A EP3107406A1 EP 3107406 A1 EP3107406 A1 EP 3107406A1 EP 15713650 A EP15713650 A EP 15713650A EP 3107406 A1 EP3107406 A1 EP 3107406A1
Authority
EP
European Patent Office
Prior art keywords
hydrophobin
surfactant
product
protein
total
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15713650.8A
Other languages
English (en)
French (fr)
Inventor
Andrew Richard Cox
Nicholas David Hedges
Penelope Eileen Knight
Damiano Rossetti
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unilever PLC
Unilever NV
Original Assignee
Unilever PLC
Unilever NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unilever PLC, Unilever NV filed Critical Unilever PLC
Publication of EP3107406A1 publication Critical patent/EP3107406A1/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/44—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P30/00—Shaping or working of foodstuffs characterised by the process or apparatus
    • A23P30/40—Foaming or whipping
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • 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).
  • CSF co-surfactant
  • 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.
  • 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.
  • 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.
  • a chill or ambient aerated food composition including but not limited to:
  • 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.
  • 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;
  • 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.%) .
  • an aerated food composition including but not limited to the steps of:
  • step (a) Adding hydrophobin and a Co-surfactant or Co-surfactants to the chilled mix of step (a);
  • step (b) Aerating the mix of step (b) to produce the aerated product
  • pasteurisation may be accomplished after step (a) and/or step (b).
  • Figure 1 is a graphical representation of Elastic modulus for Example 1 , samples A) HFBII ( ⁇ ), (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 ( ⁇ ), 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 ( ⁇ ), 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 ( ⁇ ), 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.
  • a chill or ambient aerated food composition including but not limited to:
  • 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.%) .
  • the total hydrophobin(s) concentration is at most 1.5 wt.%.
  • 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.
  • the Co- surfactant(s) is selected from Tweens 20, 60 or 80, PGE-O-80; Panodan-Visco Lo 2000 and blends thereof.
  • 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.
  • the average bubble size is at least 20, 30, 40 or 50 % smaller.
  • 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.
  • step (a) adding hydrophobin and a Co-surfactant or Co-surfactants to the chilled mix of step (a);
  • step (b) Aerating the mix of step (b) to produce the aerated and frozen product; and d. Optionally further ingredients could be added after aeration
  • pasteurisation may be accomplished after step (a) and/or step (b).
  • 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 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.
  • 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.
  • a chill product is one that is typically distributed and stored at temperatures between 0°C and 10°C
  • 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 are a well-defined class of proteins (Wessels, 1997, Adv. Microb.
  • hydrophobin has a length of up to 125 amino acids.
  • the cysteine residues (C) in the conserved sequence are part of disulphide bridges.
  • 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:
  • 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).
  • 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.
  • 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).
  • hydrophobins can be obtained by the use of recombinant technology.
  • 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
  • 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).
  • 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).
  • 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.
  • 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
  • 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.
  • the hydrophobin used in the present invention can be a Class I or a Class II hydrophobin.
  • the hydrophobin used is a Class II hydrophobin.
  • the hydrophobin used is HFBI, HFBII, or CU (cerato ulmin).
  • 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.%.
  • 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:
  • weights refer to a fixed volume of mix or product. Overrun is measured at atmospheric pressure.
  • 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.
  • 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.
  • the use of the interfacial tension ( ⁇ ) response to relative area variation ( ⁇ / ⁇ ) provides for the definition of the dilational viscoelasticity.
  • 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).
  • E e elastic
  • Ev viscous
  • One criterion to reduce bubble coarsening e.g. coalescence and/or disproportionation
  • a Co-surfactant is defined as:
  • 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.
  • 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.%.
  • Co-surfactant is chosen as one of more of the following:
  • Polysorbates including Polysorbate 20, 60 and/or 80 also known as Tween
  • Polyglycerol esters of fatty acids particularly PGE-O-80 as supplied by Danisco - Diacetyl tartartic acid esters of mono-/di- glycerides, particularly Panodan
  • 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.
  • inventive confectionary product(s) 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.
  • 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.
  • Example 1 Surface dilatational rheology measurements to demonstrate the effect of one inventive Co-surfactant
  • Evaluating the extent of the recovery (if any) of the elastic modulus (E e i) in presence of a Co-surfactant over the comparative hydrophobin + SMP case 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).
  • Example 3 Use of a Co-surfactant with Class II hydrophobin HFBI and CU in the presence of milk protein
  • SMP pure Class II hydrophobin HFBI
  • concentration of HFBI 0.2/200 wt.%
  • Example 4 Stability of foams at chill temperature in the presence and absence of a preferred level of co-surfactant
  • Table 3 Formulations of chilled aerated confections for macroscope images shown in Figure 5.
  • FIG. 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
  • 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.
  • 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.
  • 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.%.
  • Co-surfactant Tween 20 in the Co-surfactant to HFBI I ratio of 0.1 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.
  • 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.
  • Solutions were prepared using combinations of hydrophobin, Secondary protein e.g. a milk protein, and an added Co-surfactant.
  • the microstructure of each product was visualised using Low Temperature Scanning Electron Microscopy (LTSEM).
  • LTSEM Low Temperature Scanning Electron Microscopy
  • 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 : OCTTM 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.
  • 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.
  • 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:
  • 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.
  • 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:
  • 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
  • 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.
  • 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).
  • 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).
  • bubbles as small as 2 ⁇ and as large as 83 ⁇ 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.
  • 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
  • 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 ⁇ ,83 ⁇ ])
  • B(j) the j th bin covering the diameter range : [j W, (j + 1) W)
  • 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 ⁇ .
  • 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).
  • 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.
  • 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.
  • 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;
  • Image type Greyscale
  • 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:
  • each foam 100cm 3 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.
  • 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.
  • 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.
  • SMP Skim Milk Protein
  • polyglycerol ester is mainly di-, tri-, and tetraglycerol; Iodine value ⁇ 55; Saponification value 1 15-135.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Mycology (AREA)
  • Inorganic Chemistry (AREA)
  • Confectionery (AREA)
EP15713650.8A 2014-02-18 2015-02-06 Stabilisiertes, durchlüftetes konfekt mit hydrophobin Withdrawn EP3107406A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14155587 2014-02-18
PCT/EP2015/052556 WO2015124447A1 (en) 2014-02-18 2015-02-06 Stabilized aerated confection containing hydrophobin

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Publication number Priority date Publication date Assignee Title
WO1996041882A1 (en) 1995-06-12 1996-12-27 Proefstation Voor De Champignoncultuur Hydrophobins from edible fungi, genes, nucleotide sequences and dna-fragments encoding for said hydrophobins, and expression thereof
GB0002661D0 (en) 2000-02-04 2000-03-29 Biomade B V Method of stabilizing a hydrophobin-containing solution and a method of coating a surface with a hydrophobin
GB0007770D0 (en) 2000-03-30 2000-05-17 Biomade B V Protein capable of self-assembly at a hydrophobic hydrophillic interface, method of coating a surface, method of stabilizing a dispersion, method of stabilizi
CA2575319C (en) * 2004-07-27 2014-10-14 Unilever Plc Aerated food products containing hydrophobin
ATE417511T1 (de) 2005-09-23 2009-01-15 Unilever Nv Durchlüftete produkte mit niedrigem ph-wert
DE602005006829D1 (de) 2005-12-21 2008-06-26 Unilever Nv Gefrorene belüftete Süssspeisen
BRPI0705417B1 (pt) 2006-12-20 2016-08-16 Unilever Nv produto alimentício aerado e processos para a produção de um produto alimentício aerado
EP1938697B1 (de) * 2006-12-20 2016-07-06 Unilever PLC Mit luft durchsetzte lebensmittelprodukte und verfahren zu ihrer herstellung
AU2008229927B2 (en) 2007-10-25 2009-08-06 Unilever Plc Aerated fat-continuous products

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See also references of WO2015124447A1 *

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