WO2006034856A1

WO2006034856A1 - Activated globular protein and its use in edible compositions

Info

Publication number: WO2006034856A1
Application number: PCT/EP2005/010484
Authority: WO
Inventors: Lionel Bovetto; Christophe J. E. Schmitt; Martin Beaulieu; Nicolas Carlier; Eric Kolodziejczyk
Original assignee: Nestec S.A.
Priority date: 2004-09-29
Filing date: 2005-09-28
Publication date: 2006-04-06
Also published as: AR051046A1; AU2005289071A1; EP1841326A1; BRPI0515936A; TW200626073A; MX2007003633A

Abstract

The present invention pertains to activated globular protein preparations, a method for obtaining such preparations and to their use in edible compositions. In particular, the present invention relates to the use of activated globular protein as a gelling agent, a thickening agent, an emulsifying agent, a stabilizing agent, a whipping agent, a protein supplement and/or gelatin substitute.

Description

Activated globular protein and its use in edible compositions.

Background of the invention

Hydrocolloids, such as gelatin, starch or pectin, find an application in various fields of the food and pharmaceutical industry for stabilizing the product and for imparting additional advantageous product properties.

Since the occurrence of BSE cases in Europe and also in the US, consumers tend to disapprove proteins and hydrocolloids isolated from bovine meat and bone material, which applies to such kind of materials derived from animal in general as well. In order to meet the consumer's desire to avoid products of animal origin, gelatin has been replaced in most of the nutritional and pharmaceutical products by other ingredients. The said ingredients used for this purpose showed, however, not all of the valuable properties of gelatin so that their use often resulted in a loss of desired textural characteristics in the final product, as well as in the development of product defects over time, which are not considered to be acceptable and eventually resulted in a disapproval of the products by the consumers.

So far, no adequate replacement for gelatin is known. In GB 2 188 526 a protein material is disclosed, that may be used as a stabilizing agent or a supplement in dairy food or drink products. This protein material requires, however, a complicated multi- step isolation process, involving a time and cost intensive elution of fractions from an ion exchange medium, while nutritional products, to which such protein material has been added, have not been accepted by the consumers.

The patent application EP 0 696 426 Al pertains to a process for the preparation of co- precipitate. That enable the production of dairy products with a 100 % dairy composition and thus a product that can be called yogurt, that mean that gelatin has not to be included in the formula. A problem of this kind of substitute is that to obtain a final product with an acceptable texture, it is necessary to add about 2 to 6% of co- precipitate. Thus, there is still a need in the art to provide a replacement for gelatin showing essentially the same properties, e.g. allowing stabilization of the composition and adaptation of the textural characteristics thereof. Such replacement should not be derived from meat, tendons and/or bone material, and should be capable to be prepared on a large scale at relatively low costs. In addition, such replacement should be easily obtainable according to simple methods.

Summary of the invention

It is an object of the present invention to provide a gelatin substitute with improved nutritional properties.

It is an other object of the present invention to provide a gelatin substitute that shows at least the same physical properties.

It is an other object of the present invention to provide a substitute which is cost efficient by allowing the decrease of the protein concentration without any textural or mouthfeel losses.

The above problem has been solved by providing an activated whey protein preparation obtainable according to a method comprising the steps of subjecting a low salt solution containing whey proteins to a heat treatment at a temperature in the range of from 68 to 98°C for a time period of from 1 to 240 minutes, and having at least 20 % of accessible thiol groups on the basis of the total amount of thiol groups present in said activated whey protein, and a proportion of soluble aggregates of at least 20 %.

Such method can be applied for any other globular protein of interest, having a nutritional chemical index superior to gelatin, such as proteins form egg, cereals, oilseeds, or other vegetables.

Time, temperature and pH combination should be established in order to obtain a percentage of accessible thiol groups of at least 20 % in an upward or stable maximum plateau, (based on the total amount of thiol groups in the protein), having a proportion of high molecular weight soluble aggregates of at least 20 %, and a minimum increase of hydrophobicity of at least 100% when compared to the hydrophobicity of non-treated proteins. Then, the present heat activated globular composition enables the stabilisation and/or the formation of gels having at least the same properties that gels formed by using gelatin or gelatin substitute but simultaneously, said preparation can be used at a lower concentration and said preparation has a higher nutritional value.

The combination of both last features allows a cost reduction and a nutritional improvement in the final product.

Thus, the present invention pertains to a natural heat activated globular protein protein polymere characterised in that it has a protein efficiency ratio of at least 100, preferably 110. In particular, it pertains to a natural heat activated whey protein protein polymere characterised in that it has a protein efficiency ratio of at least 110.

Brief description of the Figures

FIG. 1(A.) shows the formation of soluble aggregates and disappearance of native β- lactoglobulin at 70⁰C at a protein concentration of 1% ; l(B) shows the formation and disappearance of soluble aggregates and disappearance of native β-lactoglobulin at 70°C at a protein concentration of 4%.

FIG. 2 is a flow chart of a process for the preparation of a fermented dairy product using the present invention.

FIG. 3. is a graph illustrating the milk and whey supernatants analysis after ultracentrifugation (lh/lOO.OOOg) by SEC on TSK G 2000 using Phosphate Buffer.

FIG. 4. is a graph illustrating the milk and whey supernatants analysis after acidification by GDL by SEC on TSK G 2000 using Phosphate Buffer.

FIG. 5. shows the evolution of the gel strength results on finished products during storage.

FIG. 6. shows the evolution of Back Extrusion results on finished products during storage. FIG. 7 A and C: show the structure of the gel as revealed after an immuno-labelling by anti βLG antibodies; 7 B and D are enlargements of the areas delineated respectively in images A and C.

Detailled description of the invention

During the extensive experiments leading to the present invention, the named inventors surprisingly noted that globular protein subjected for a particular period (the upward phase or plateau) of time to a temperature in the range of from 68 to 98 ⁰C, exhibits properties, clearly matching or even exceeding those of conventional gelatin or replacer.

This material is extremely suitable for use as e.g. a gelling agent, a thickening agent, an emulsifying agent, a stabilizing agent, a whipping agent, a protein supplement and/or gelatin substitute.

Moreover, it also has been found that the heat treatment of a globular protein isolate and subsequent mixing with milk yields a product different from subjecting a mixture of globular protein with milk to a heat treatment.

Hence, the preparation of the present invention may be used as an adequate replacement for gelatin in, for example, dairy products and in particular in low fat fermented dairy products can be expected to be highly accepted both by the consumers, since no material deemed to be potentially hazardous is utilized, as well as by the producers, since it represents an advantageous additive for the preparation of edible compositions.

The term "globular protein" as used in the present invention, comprises: from milk, egg, from cereals (Barley, Maize, Oats, Rice, Wheat, Sorghum, Millet), from legumes (Chickpea, Lentil, Lima bean, Pea, Field bean), from oilseeds (Cotton, Oilseed rape, Peanut, Sesame, soybean, Sunflower) and from pseudocereals (Buckwheat, Amaranth Quinoa) and also sweet potato isolate, white lupin (L.albus) and locus bean.

In the present invention, one kind of globular protein can be used but a mixture of globular proteins can also be used.

In the present invention any globular proteins, in particular those having a PER of at least 100 can be used, but in the following description whey proteins will be used as a preferred embodiment. Said whey proteins can be obtained by any process for the preparation of milk whey known in the art, as well as milk whey protein fractions prepared therefrom or the main proteins of milk whey, such as beta-lactoglobulin, alpha-lactalbumin and whey albumin. In particular, sweet whey obtained as a by-product in cheese manufacture, and acid whey as produced in acid casein manufacture or rennet whey as produced during rennet casein manufacture may be used as the whey protein. Preferably, in particular under cost aspects, whey protein preparation which has not been subjected to additional fractionation processes after its production is preferred as starting material. The present invention is not restricted to whey isolates from bovine origin, but pertains to whey isolates from all mammalian animal species, such as from sheep, goats, horses, and camels. E.g. a whey protein may comprise at least 10 wt.-%, preferably 50 wt.-% β- lactoglobulin and/or 10 wt.-, preferably 20 wt- oc-lactalbumin.

Whey proteins have a better protein efficiency ratio (PER) compared to casein 118/100

PER = body weight growth (g) / protein weight intake (g).

Examples: PER % Casem casein 3,2 100

Egg 3,8 118

Whey 3,8 118

Whole Soya 2,5 78

Wheat gluten 0,3 9

Whey protein is an excellent source of essential amino acids (AA) (45%). Rich in AA which requirements may be increased in case of stress and in elderly: compared to casein ( 0.3g cysteine/10Og protein) sweet whey proteins contain 7 times more cysteine and acid whey 10 times more cysteine. Cysteine is the rate limiting amino acid for glutathione synthesis, glutathione is a tripeptide made of glutamate cysteine and glycine. Glutathione has primary important functions in the defense of the body in case of stress.

The term "activated whey protein preparation" as used in the present invention, pertains to a whey protein preparation that has been subjected to a heat treatment after the isolation of whey protein from the respective precursor material, such as e.g. milk or compositions obtained during a processing of milk to dairy products and that also shows the properties as mentioned above. The heat treatment may be a heating step carried out at a temperature of from 68 ⁰C up to 98°C, preferably of from more than 80 to 95 ⁰C, even most preferably of from 85 to 95 ⁰C. Tliis preparation is very stable and could be stored in sterile conditions at +4°C during at least 6 months.

The whey protein, as well as the fractions and/or the major proteins thereof may be used in purified form or likewise in form of a crude product. The starting material for the preparation of the activated whey protein preparation is preferably low in divalent cations, the concentration in said cations is preferably less than 2.5% in its powder form, more preferably less than 2%.

Heat activated whey protein preparation according to the present invention and treated accordingly exhibits at least 20 % of accessible thiol groups, on the basis of the total amount of thiol groups present in said activated whey protein. An accessible thiol group according to the present invention is a thiol group that react spontaneously with DTNB reagent without denaturing agent like SDS or urea or guanidine which could be used to determine total thiol content see material and method. (Spectrophotometric Method for Determination of Heat- Activated Sulfhydryl Groups of Skimmilk.Koka, M., Mikolajcid, E.M., Gould, I. A., Dairy Science 51, 217-219 (1968)) The calculation was done in relation to the theoretical protein concentration of 1% or 0.2% and supposing that each molecule of B-LG is able to expose one accessible thiol.

According to a preferred embodiment, the activated v^hey protein preparation according to the present invention has at least 20% of accessible thiol groups, preferably between 35 % and 60 % accessible thiol groups, more preferably between 40 % and 55 % accessible thiol groups.

Moreover, the activated whey protein preparation according to the present invention has a proportion of soluble aggregates of at least 20 %. Trie proportion of soluble aggregates may be obtained by determining the amount of soluble aggregates by SEC (size exclusion chromatography) (for example measured in gram) and determining the total amount of protein (for example measured in gram) according to the following equation:

proportion of soluble aggregates = amount of soluble aggregates / total amount of protein We can give a definition of soluble aggregates: non sedimented after 30 min centrifugation under 10.00Og, filterable on 0.45 micron filter and eluted in /or close to the exclusion volume corresponding to 1/3 of total volume column (this limit was estimated at 160.000 dalton using gama-globulin for calibration) for a TSK G2000 column used in the following conditions : HPLC analysis was performed on a TSKgel G 2000 SWXL 7.8 mm ID x 30 cm column, using a SWXL 6 mm ID x 4 cm guard column (TosoHaas GmbH, Stuttgart, Germany). The column was packed with macroporous silica spherical particles of 5 μm and 250 A pores. The HPLC Agilent 1100 system was composed of a thermostated well-plate sampler (+4°C), column oven (25°C) and DAD detector, set up at 215 ran. Elution was performed in isocratic mode at a flow rate of 0.5 mL min-1 during 40 minutes. The elution buffer contained 50 mM NaH₂PO₄-H₂O in Lichrosolv Water (Merck, Darmstadt, Germany) at pH 6.8. The buffer was filtered through 0.45 μm filters (Millipore).

According to a preferred embodiment, the activated whey protein preparation of the present invention has a proportion of soluble aggregates of between 60 % and 100 %, preferably between 65 % and 90 %, more preferably between 70 % and 85 %.

In addition, the activated whey protein preparation shows an increase of surface hydrophobicity of at least 100 % as compared to the protein prior to subjecting it to the heat activation. The term "hydrophobicity" as used in the present invention designates the surface hydrophobicity evaluated by measuring the maxinrun of fluorescence intensity obtained by addition of fluorescent probe (ANS). The more ANS molecules adsorb to protein surface the more signal of fluorescence increase. During heat treatment protein surface interacting with ANS increases.

The hydrophobicity of the protein may be determined according to the following method: Hydrophobicity was evaluated by spectrofluorometric titration curve to reach the asymptotic fluorescence maximun intencity (Fmax). Adapted from: Bonomi, F., Iametti, S., Pagliarini, E., Peri, C: A spectrofluorimetric approach to the estimation of the surface hydrophobicity modification in milk proteins upon thermal treatment. Milchwissenschaft 43 (5) 281-285 (1998).

Preferably, the increase in hydrophobicity in the activated whey protein as compared to the starting whey protein is from 150 % to 300 %, preferably of from 200 % to 300 %, more preferably of from 220 % to 280 %. The activated whey protein preparation of the present invention may be present in form of a viscous liquid compatible with pumping from tank to tank and to be integrated- into /mixed with the respective edible composition. As desired, the activated whey protein preparation may be clear, but also opaque or turbid, depending on the product, into which it is to be integrated/mixed with. When desired a gelling of an activated whey protein preparation according to the present invention may be induced by any method known in the art, such as cryo-concentration, phase separation, acidification or calcium addition.

Without wishing to be bound to any theory, it is presently assumed that the selection of the specific temperature and time window results in a particular and specific denatured condition of the whey protein (in the upward phase or plateau), which is due to a temperature induced unfolding of the proteinaceous material including a potential interaction with other proteins present.

The whey protein may be present in the aqueous solution in an amount of 0.1 wt.-% to 15 wt.-%, preferably in an amount of 0.2 wt.-% to 12 wt.-%, more preferably in an amount of 0.5 wt-% to 10 wt.-%, in particular in an amount of 2 wt.-% to 8 wt-%.. each on the basis of the total weight of the solution. In case another globular protein is used, it may be present in the aqueous solution in an amount of 0.1 wt.-% to 12 wt.-%, preferably in an amount of 0.2 wt.-% to 11 wt.-%, each on the basis of the total weight of the solution

The aqueous solution of the whey protein preparation as present before the activation step may also comprise additional compounds, such as by-products of the respective whey production processes, such as e.g. other proteins (proteose peptones, caseino- glyco-macropeptide (CGMP)), hydrocoloids, or carbohydrates. The amount of^" such additional compounds should not exceed 40 wt.-%, preferably 10 wt.-% of the weight of the whey protein contained in the whey protein isolate for ionic charged substances. The amount of such additional compounds should not exceed 300 wt.-%, preferably 100 wt- % of the weight of the whey protein contained in the whey protein isolate for non ionic substances like glucose, lactose, maltose, galactose, fructose or maltodextrine with high dextrose equivalent (30-45). The protective effect of lactose at a concentration between 5% and 25% can be used to prevent decrease of soluble aggregate and gelation of the whey protein during the heat treatment. In Phosphate buffer and depending on protein concentration, according to a preferred embodiment, the heating step is carried out during a time period of 5 to 60 min, preferably between 10 and 30 min. According to a more preferred embodiment the heating step is carried out at a time period of between 14 and 17 min and at a temperature of between 84 ⁰C and 86 ⁰C.

In absence of Phosphate buffer (just by adjusting pH by NaOH or HCl and for WPC, WPI with low mineral content especially Ca and Mg, the heating step is, preferably, carried out at a temperature of between more than 80 ⁰C and up to 98 °C, more preferably between 82°C and 95 ⁰C, more preferably at a temperature of between 85°C and 92 °C, in particular at a temperature of 92⁰C. It has been shown that temperatures between more than 80 ⁰C, i.e. about 81 ⁰C and 92 ⁰C lead to activated whey proteins having the above mentioned traits of accessible thiol groups and proportion of soluble aggregates, and which prove to even out compete the properties of commercial milk protein based functional ingredient (co precipitate) as a gelling agent, a thickening agent, an emulsifying agent, a stabilizing agent, or a whipping agent.

In said preferred embodiment, the heating step is carried out during a time period of 14 to 25 min, preferably between 14 and 20 min. According to a more preferred embodiment the heating step is carried out at a time period of between 14 and 17 min and at a temperature of between 90 C and 95 ⁰C.

The process is carried out in an essentially salt free condition, since salt is known to influence the folding and aggregative properties of proteins. Thus the present process may also comprise a desalting step of the starting material, which may be effected by methods known in the art to yield a starting material having a salt content of less than about 2.5% of salt, on the basis of the total composition. Low salt content allows working at acidic pH below pH 7.0

Alternatively, additional buffers may be added and/or the pH of the aqueous composition may be adjusted to a pH value in the range of from 6.2 to 8.4, preferably to 6.2 to 7.5, more preferred to a pH of 7.2 to 7.4, in absence or with at least one buffer. The buffers may be chosen from the group consisting of acetic acid and salts thereof, such as e.g. sodium acetate or potassium acetate, phosphoric acid and salts thereof, such as e.g. NaH₂PO₄, Na₂HPO₄, KH₂PO₄, K₂HPO₄, CH₃COOH.

Surprisingly, the present inventors also noted that addition of particular buffers to the starting material prior to the heating step allows to tailor properties of the whey protein preparation. For example, addition of CH₃COONaZCH₃COOH increases the amount of accessible thiols and an increase in hydrophobicity, both of which parameters for the unfolding status of the protein, while addition of Na₂HPO₄/NaH₂PO₄ has been found to favor/increase the percentual formation of soluble aggregates.

Depending on molecular weight, The buffer component(s) may be present in an amount of at least 0.1 wt.-%, preferably in an amount of at least 0.3 wt-%, more preferably in an amount of 0.8 to 2 wt.-%, on the basis of the weight of the total aqueous solution. As example a phosphate buffer prepared with K₂HPO₄ (MW: 174.18) 50 mM concentration correspond to 8.7g/l (0.87%), acetate buffer prepare with acetic acid (CH₃COOHiMW 60) 50 mM concentration corresponds to 0.3%.

Finally, inventors note that the present heat activated whey protein according to the present invention improves the nutritional value of the final product because the closest gelatin subsitute having the higher nutritional value is composed by 60% of caseins and 40% of whey proteins. It is well known that the caseins have a nutritional chemical index, PER of 100 and that the whey proteins have a PER of 118. Thus the gelatin substitute of the present invention has a higher nutritional chemical index when it is composed only with whey proteins. It can be used for the preparation of any kind of food product, food supplement, nutritional composition or pharmaceutical composition, in particular in low fat or essentially fat free dairy products, or also where it finds application as a gelling agent, thickening agent, emulsifying agent, stabilizing agent, whipping agent or an agent for mimicking fat, or as a protein supplement and/or gelatin substitute in food or drinkable products. Examples for products, where the present activated protein may find application are exemplarily yoghurt, fermented milks, milk-based fermented products, mousses, foams, emulsions, ice creams, fermented cereal based products, milk based powders, infant formulae, diet fortifications, pet food, tablets, liquid bacterial suspensions, dried oral supplement, wet oral supplement.

The invention is further defined by reference to the following examples describing in detail the preparation of the heat activated proteins of the present invention. The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Preparation of heat activated BLG 0.2% in Phosphate buffer:

β-Lactoglobulin (lot JE002-8-922, 13-12-2000) was obtained from Davisco (Le Sueur, MN, USA). The protein was purified from sweet whey by ultra-filtration and ion exchange chromatography. The composition of the powder is 89.7 % protein, 8.85 % moisture, 1.36% ash (0.079 % Ca²⁺, 0.013 % Mg²⁺, 0.097 % K⁺, 0.576 % Na⁺, 0.050 % CF). All other reagents used were of analytical or HPLC grade (Merck Darmstadt, Germany) unless otherwise stated.

The protein solution was prepared at 0.2% concentration by solvation of β-lactoglobulin in phosphate buffer 5OmM pH 6.8, and stirring at 20 °C for 2 h. The solution was filled in 20 ml glass vials (Agilent Technologies) and sealed with aluminium capsules containing a silicon/PTFE sealing. The solution was heated at 68 ⁰C for 60 min (time to reach the temperature 2.30 - 2.50 min). After the heat treatment, the samples were cooled in ice water to 20 ⁰C.

The determination of the accessible thiol groups was done in the samples after heat treatment. The 5,5'-Dithiobis (2-nitrobenzoic acid) (3,3 '-6) DTNB solution (5mM) (Fluka, Buchs Switzerland) was prepared in 0.05 M NaH2PO4 buffer at pH 6.8. After mixing 2 ml of the protein solution with 100 μl of the DTNB solution (incubation time: 3 min) at 20 °C, the absorbance was measured at λ=412 nm. The concentration of accessible SH was determined using a standard curve (5 to 200 μM L-Cys). The calculation was done in relation to the theoretical protein concentration of 0.108 mM and supposing that each molecule of B-LG is able to expose one accessible thiol. In those conditions 25.46% of theoretical free thiol content were accessibles.

Example 2 : Preparation of heat activated protein with a commercial solution as starting material

Whey protein isolate WPI Bipro (Batch JE032- 1-420) was obtained from Davisco (Le Sueur, MN, USA). The composition of the powder is 90.1 % protein, (0.082 % Ca2+, 0.006 % Mg2+, 0.044 % K+, 0.650 % Na+, 0.049 Phosphorus All other reagents used were of analytical or HPLC grade (Merck Darmstadt, Germany) unless otherwise stated.

The protein solution was prepared at 3.4% protein by solvation of whey protein powder in Millipore water, and stirring at 20 °C for 2 h. The final pH was controlled 7.2. The solution was filled in 20 ml glass vials (Agilent Technologies) and sealed with aluminum capsules containing a silicon/PTFE sealing. The solutions were heated at 85 ⁰C for 15 min (time to reach the temperature 2.30 - 2.50 min). After the heat treatment, the samples were cooled in ice water to 20 °C.

Determination of soluble protein and soluble aggregates

After heat treatment, the samples have been centrifuged at 26900 g (Sorvall Instruments RC5C rotor Sorvall SS34) for 15 min and the supernatant was diluted to a protein concentration of ~0.2-2mg/ml. This solution was filtered through 0.45 μm microfilters (Orange Scientific, Waterloo, Belgium), before injection of 25 μL. HPLC analysis was performed on a TSKgel G 2000 SWXL 7.8 mm ID x 30 cm column, using a SWXL 6 mm ID x 4 cm guard column (TosoHaas GmbH, Stuttgart, Germany). The column was packed with macroporous silica spherical particles of 5 μm and 250 A pores. The HPLC Agilent 1100 system was composed of a thermostated well-plate sampler (+4°C), column oven (25°C) and DAD detector, set up at 215 nm. Elution was performed in isocratic mode at a flow rate of 0.5 mL min-1 during 40 minutes. The elution buffer contained 50 mM NaH2PO4 ^• H2O in Lichrosolv Water (Merck, Darmstadt, Germany) at pH 6.8. The buffer was filtered through 0.45 μm filters (Millipore).

The soluble fraction contained 75% of soluble aggregates

Example 3 : Preparation of heat activated protein with a commercial solution as starting material

Whey protein isolate Prolacta 90 (Batch 989/2) was obtained from from Lactalis( Retier France) The composition of the powder is 87.2 % protein, (0.921 % Ca2+, 0.040 % Mg2+, 0.109 % K+, 0.058 % Na+,0.104% Cl-, 0.232% phosphorus) All other reagents used were of analytical or HPLC grade (Merck Darmstadt, Germany) unless otherwise stated.

The protein solution was prepared at 3.4% protein by solvation of whey protein powder in Millipore water, and stirring at 20 ⁰C for 2 h. The final pH was controlled 7.0 then adjusted to pH 8.4 by NaOH IN to avoid insoluble aggregate formation during heat treatment. The solution was filled in 20 ml glass vials (Agilent Technologies) and sealed with aluminum capsules containing a silicon/PTFE sealing. The solutions were heated at 85 ⁰C for 15 min (time to reach the temperature 2.30 - 2.50 min). After the heat treatment, the samples were cooled in ice water to 20 ⁰C.

Determination of soluble proteins. The heated whey proteins have been centrifuged at 20 ⁰C and 26900 g for 15 min. The supernatant was used to determine the protein concentration in quartz cuvettes at 280nm. Values are expressed as a percentage of the initial value before heat treatment. For those specific conditions, 100% soluble proteins were obtained.

Example 4: preparation of acid gel using conventional approach (milk + whey proteins pasteurized together) or invention (pasteurised milk and activated whey proteins).

Samples were prepared as follow for the conventional approach (milk + whey proteins pasteurized together):

Skim milk powder (SMP) and whey protein isolate (WPI) were dispersed in 50 ml water. In order to maintain total solid content stable at 11%, lactose has been added to the samples with a fraction of whey protein (see Table 1). After complete dispersion, volumes were adjusted to 100 ml. Solutions were stirred gently for two hours at room temperature and then stored overnight at 4°C to allow complete hydration. After reconstitution pH were close to 6.6-6.7. AU samples were pasteurized at 85°C for 15 minutes and stored in ice until use.

Samples were prepared as follow for the Invention (pasteurized milk + activated whey proteins):

Preparation of activated whey proteins solution: 6% whey protein solution in water (Bipro®) was stirred gently for two hours at room temperature and then stored overnight at 4°C to allow complete hydration. After reconstitution, measured pH value was 7.2. The soluble aggregates were obtained by heat treatment at 85°C for 15 minutes and stored in ice until use.

Preparation of the samples: Skim milk powder was dispersed in 50 ml water. In order to maintain total solid content stable at 11%, lactose has been added to the samples with a fraction of whey protein (detailed recipes are presented in table 1). After complete dispersion, volumes were adjusted to 83 ml. Solutions were stirred gently for two hours at room temperature and then stored overnight at 4°C to allow complete hydration. Samples were pasteurized at 85°C for 15 minutes, and then cooled down at 4⁰C. Activated whey proteins were added to the desired concentration and the final volume was adjusted to 100 ml with water and stored in ice until utilization.

¹Skimmed milk powder was obtained from Ingredia (France) Detailed composition of the powder is: 34% protein, 50.5% lactose, 7.94% ashes, 2.02 % citrate, 4.47 water. ²Whey protein isolate (Bipro^®, batch JE032- 1-420) was obtained from Davisco (Le Sueur, MN, USA). The composition of the powder is 90.1 % protein, (0.082 % Ca²⁺, 0.006 % Mg²⁺, 0.044 % K⁺, 0.650 % Na⁺, 0.049 Phosphorus. ³From a 6% stock solution of activated whey proteins.

Acid-induced gelation: Glucono-δ-lactone (GDL) was added (1.5% w/v) to ice-cold samples. Temperature was increased to 42°C (time zero) and acidification was monitored over time.

Characterization of activated whey proteins by size exclusion chromatography (SEC): After centrifugation (Ih, 100.00Og) supernatants were analyzed by SEC. Soluble aggregates are represented by the bottom part of the bar chart (figure 3). Samples prepared by new approach (milk 85°C/15' + soluble aggregates (whey 85°C/15')) contained up to 10 times more soluble aggregates compare to conventional approach (milk and whey 85°C/15').

After acid-induced gelation, samples were centrifuged (Ih, 100.00Og) and supernatants analyzed by SEC (Figure 4). The strong decrease in soluble aggregates observed for samples prepared by the new approach lead to conclude that soluble aggregates are now involved in the gel structure.

Rheological characterization of acid-induced gels: Small-deformation oscillatory measurements were performed, at a frequency of 1 Hz and strain amplitude of 0.5%, on a controlled-strain rheometer (ARES 100 FRT, Rheonietric Scientific, Piscataway, NJ, USA) equipped with a circulating water bath temperature controller. Couette device with a cup (35 mm diameter) and bob system (33 mm diameter, 35 mm length) was used. Samples were poured directly into the heated measuring system (42°C) of the rheometer. Samples were covered with a thin film of paraffin, oil in order to avoid evaporation during heat treatment. Complex modulus (G*) has been used to represent the solid-like behavior. G* is a representation of both storage modulus (G') and loss modulus (G") according to these equations: G'=G*^»cos δ, and G"=G*'sin δ. For a gelled system (G'»>G"), G* could be assimilated to G¹ considering that G" and δ are tending towards zero.

As expected from the SEC results for the solution fraction prior acidification (figure 3), obtained gels are firmer with the new process (higher G*). Measured G* values were 882 and 2187 Pa for addition levels of 0.5 and 1.0% of AWP, respectively. For the same addition level, conventional approach is yielding G* values of 538 and 777 Pa (see Figure 4).

Example 5 : Preparation of a fermented dairy product with activated whey protein preparation and differences compared to the prior art:

Preparation of a fermented dairy product with activated whey protein preparation

Fermented dairy products with 0.5 w.t.-% activated whey protein were produced and compared to reference samples. Skimmed milk powder was added to adjust the total protein content to 4.3 w.t-% and the total solid content to 11.7%. For the preparation of samples according to the present invention, pasteurization of the initial blend (liquid rnilk, milk powder and lactose) was done without added activated whey protein isolates. In parallel, a heat activated whey protein solution (HAWP) is prepared as indicated in Example 4 (with a protein content of 10 % and a lactose content of 10%), cooled to 43°C and incorporated to the pasteurized blend just before ferments addition as indicated in Figure 2.

For control reference samples pasteurization of the blend of all ingredients (liquid milk, milk powder, whey protein isolates and lactose) was done before fermentation. This blend contains 0.5 w.t-% of unheated whey protein and a total content of protein of 4.3 w.t-%.

One recipe with activated proteins defined in the present invention has been compared to a conventional yogurt made with co-precipitate. Total protein content was to 5.1 % in the recipe with the co-precipitate for a total solid content of 13.9 % and only 4.3% in the recipe with activated whey proteins (HAWP) for a solid content of 11.7%.

As shown in the Figure 5, addition of activated whey protein increases the gel strength up to 30% compared to the product with unheated whey protein. Compared to the recipe with the coprecipitate, the gel strength is 1.4 more important with the recipe containing activated whey protein whereas the total protein content is 16% less important.

The back extrusion represented in the figure 6 illustrates the similarity of the recipes with coprecipitate and heated whey protein.

Sensory evaluation

Samples produced according to the fermented dairy product preparation method of the present invention provide superior properties when compared to the reference samples.

Sensory test results show that an addition of whey protein preparation after the pasteurization step results in an increased firmness and thickness compared to the references whatever the concentration added of activated protein (the more the quantity of whey protein, the more increase of texture) .

The following results come from sensory profiles done with a recipe containing 4.4 % of total protein whose 0.5% come from activated whey protein.

Fermented dairy products (yogurts) prepared according the present invention have the following properties compared to the reference (i.e. yogurts with unheated whey protein) - less serum

- less ropy

- more granular,

- less smooth - more firm

- more sticky

- more thick.

Sensory test show that yogurts prepared according to the fermented dairy product preparation method of the present invention obtain a more advantageous texture (thickness of product, strength of product), but these products can be also more granular. Advantageously, the flavor characteristics of the yogurt are essentially not influenced.

Yogurts produced according the present invention compared to the usual white base with co-precipitate are very close although the total protein is 14% less than the reference. However, products with activated whey protein remained more grainy but were perceived less powdery than, the product with co-precipitate.

Example 6 : Preparation of a whipped acidic dairy product

A dispersion of 3 w/w% heat-activated whey proteins (Bipro^®) prepared as described in example 3 has been mixed to a 1.5 w/w% acacia gum dispersion in water and 14% sucrose. The pH of the final mixture was adjusted to 7.0 by means on NaOH or HCl. A quantity of 0.5 w/w% of GDL was added to this mixture to produce in situ acidification. Concomitant whipping was achieved using a Creamtester or Hobbart mixer for 5 minutes in order to produced foam having an overrun ranging from 50 to 300%. The final pH of the aerated product was ranging between 5.0 to 3.6. This whipped product can be used as is or mixed with other ingredients.

Example 7 : Preparation of an acidic dairy "emulgel"

A dispersion of 6 w/w% heat-activated whey proteins (Bipro) prepared as described in example 2 has been mixed to 10 w/w% sunflower oil. The pH of the final mixture was adjusted to 7.0 by means on NaOH or HCl. A quantity of 1 w/w% of GDL was added to this mixture to produce in situ acidification. Concomitant emulsifϊcation was achieved using an Ultra Turrax T25 mixing device at maximum speed for 1. The final pH of the emulsified product was ranging between 5.5 to 4.0 after 2 hours. In these conditions, the final product was forming a self-supporting white gel, which could be use as is or submitted to subsequent shearing to produce a more creamy spreadable product.

Example 8 : Microscopic images of a fermented product after immunofluorescent labeling by an anti BLG antibody

Specimen preparation The specimens were sliced with a razor blade into 1-2 mm thick section and submitted to an overnight chemical fixation in a mixture of acetone (3 parts) and glacial acetic acid (1 part). Then the acetic acid was removed by incubating the sliced in pure acetone (8 hours) and the specimens were processed for embedding in historesin (50 % resin/50 % acetone, overnight) and pure historesin (overniglit). After polymerisation in teflon molds, the specimens were sliced into 4 μm thin section ( Ultracut, Leica).

Then, the immuno-fluorescence technique was applied to label β-Lactoglobulin (BLG) using rabbit anti-BLG as primary antibody (1/200 in PBS, pH 7.4, overnight) as primary anti-body and FITC labelled goat anti-rabbit gamrna globulins (Sigma, 1/100, 1 hours) as secondary antibody.

The microscopy observations were achieved using the Confocal microscopy LSM510 from Zeiss using the objective x63.

Quantitative analysis

The quantitative analysis was carried out with, the in-house software Colibri. The following parameters were evaluated:

• The area fraction of the network (Vv).

• The surface density of the network (Sv). This parameter quantifies the amount of interfaces between two phases. Higher values indicate higher amount of interface (higher tortuosity) or smaller structures, which is better expressed by normalizing this paramater with the area fraction (Sv/Vv). ® The mean thickness of the protein strands

• The fractal dimension

Results: Qualitative analysis: The figures 7 highlight two important structural impact of the new process:

In the reference product 7(A), the protein network looks far coarser and thicker than in the sample submitted to the new process 7(C).

In figure 7(C), the plain arrows points to BLG aggregates found in the traditional product, but not in the new one. Moreover, in figure 7(B), the broken arrow highlights the presence of about 200 nm aggregates which form the body of the protein strand, and determine their striated appearance. In the new process such striation is hardly recognizable.

Quantitative analysis:

The results of the uantitative anal sis are iven in the table below:

As can be seen Sv as well as Sv/Vv, and the fractal dimension which are higher in the new product confirm the conclusions of the qualitative analysis. The protein strands are also thinner in the new product.

This study demonstrates clearly that the new process modifies profoundly the structure of the protein network, leading to less aggregated whey proteins.

Claims

1. An activated whey protein preparation, obtainable according to a method comprising the steps of subjecting a low salt solution containing whey proteins to a heat treatment at a temperature in the range of from 68° to 98 ⁰C for a time period of from 1 to 240 minutes, and having at least 20 % of accessible thiol groups on the basis of the total amount of thiol groups present in said activated whey protein, and a proportion of soluble aggregates of at least 20 %.

2. The activated whey protein preparation according to claim 1, wherein said activated whey protein preparation exhibits an increase of hydrophobicity of at least 100 %, when compared to the hydrophobicity of the non-heat treated protein.

3. The activated whey protein preparation according to claim 1 or 2, wherein said activated whey protein has between 30 % and 70 % accessible thiol groups, and/or showing a hydrophobicity having a minimal increase of between 200 % and 300 %, when compared to the hydrophobicity of the non-activated protein, and/or having a proportion of soluble aggregates of between 60 % and 100 °/6.

4 . An activated protein preparation, according to one of claims 1 to 3, wherein the whey protein is replaced by a globular protein, in particular from egg, cereals, oilseeds, or other vegetables.

5. A method for preparing an activated whey protein, said method comprising heating whey protein in a low salt aqueous medium during 10 to 30 min at a temperature of from 75°C to 90 ⁰C.

6. The method according to claim 5, wherein the heating is performed in the presence of at least one component being selected from the group consisting of acetic acid and salts thereof, phosphoric acid and salts.

7. The method according to any of the claims 4 to 6, wherein the mixture of said whey protein in said aqueous medium has prior to the heating step a pH of from 6.2 to 8.4, preferably a pH of 6.6 to 7.4, in absence of at least one buffer component, and/or a pH of 6 to 8, preferably of 6.2 to 7.5 in the presence of at least one buffer component.

8. The method according to any of the claims 4 to 7, wherein said whey protein is present in an amount of 0.1 wt.-% to 15 wt.-%, preferably in an amount of 0.2 wt.-% to 12 wt.-%, more preferably in an amount of 2 wt.-% to 8 wt % each on the basis of the weight of the total weight of the solution.

9. Use of an activated protein according to any of the claims 1 to 4, for the preparation of a food product, a food supplement, a nutritional or pharmaceutical composition.

10. The use according to claim 9, wherein said nutritional or pharmaceutical composition is selected from the group consisting of nutritional foam composition and dairy composition.

11. The use according to claim 9 or 10, wherein said activated protein preparation is used as a gelling agent, thickening agent, emulsifying agent, stabilizing agent, whipping agent or an agent for mimicking fat, or as a protein supplement and/or gelatin substitute in food or drinkable products.

12. A dairy product comprising a heat activated protein preparation according to any of the claims 1 to 4.

13. A foam composition comprising a heat activated protein preparation according to any of the claims 1 to 4.

14. The foam composition according to claim 13, wherein said foam comprises a milk-based matrix and/or comprises at least one gum material and/or at least one polysaccharide, preferably acacia gum.

15. A method for preparing a fermented dairy product, said method comprising the steps of: providing an activated whey protein preparation according to any of the claims

1 to 3, mixing said whey protein preparation with a dairy composition and allowing hydration, inoculating the mixture with a suitable strain for fermentation, and allowing fermentation.

16. The method according to claim 15, further comprising the steps of homogenizing and/or cooling prior to the inoculation step.