The present invention relates to a process for the preparation of an egg white composition.

Egg white is a desirable ingredient for many foods such as bakery products, meringues and meat products because of its excellent foaming and gelling properties (J. Food Engineering 83 (2007) 404-413). Egg white consists mainly of protein (10 % w/w) and 0.5-0.6 % w/w carbohydrates, mainly glucose. The protein part of egg white can be devided into several classes of proteins, such as ovalbumin, ovotransferrin, ovomucoid, ovomucin and lysozyme (Y. Mine, Trends in Food Sci Techn. 1995, vol 6, p 225-232).

On an industrial scale, egg white is usually de-sugared and spray-dried followed by a so called hot room treatment of about 4 weeks at about 70-85 degrees Celsius in order to reduce microbial contamination and restore functional property losses due to spray drying. A disadvantage of heating spray-dried egg white is that it is a very time and energy consuming process. The process from egg breaking to sales of spray dried egg white takes about 6 weeks.

The aim of the present invention is an improved process for the preparation of egg white with good gelling and/or foaming properties, which is less time and energy consuming than known in the art.

Summary of the invention

The present invention relates to a process for preparing an egg white composition, comprising incubating egg white with a protease such that an egg white composition is prepared that has an increased gelling temperature compared to egg white that has not been treated with a protease.

The invention further relates to an egg white composition obtainable by a process according to the present disclosure.

It was surprisingly found that when egg white was treated with a protease such that the resulting egg white composition had a higher gelling temperature than untreated egg white, the egg white composition still had desirable foaming and / or gelling properties.

An advantage of an egg white composition according to the present invention is that the egg white composition formed a gel at a higher temperature than untreated egg white and that it was able to pasteurize the egg white composition without loosing foaming and gelling properties. It was found that the egg white composition as disclosed herein had a gelling temperature that is 1 to 15 degrees Celsius higher, for example 2 to 14, or 3 to 12 degrees Celsius higher, than egg white that has not been incubated with a protease.

Detailed description

The present disclosure relates to a process for preparing an egg white composition, comprising incubating egg white with a protease such that an egg white composition is prepared that has an increased gelling temperature compared to egg white that has not been treated with a protease.

Gelling temperature of an egg white (composition) is defined herein as the temperature at which an egg white (composition) starts to form a gel, upon heating of the egg white (composition). In the context of the present invention the wording gelling and gelation have the same meaning and are used interchangeably. Gelling

temperature or gel point can be measured by rheometry, in an experiment where the temperature of egg white is slowly increased while the elastic modulus (G') and the loss or viscosity modulus (G") are measured. The gel point is the temperature at which G'=G". Above this temperature the material is considered a gel and the elastic modulus (G') is larger than the loss or viscosity modulus (G").

Egg white as used in the present disclosure is used to indicate the clear liquid contained within an egg. Egg white is usually obtained by separating fresh eggs in egg yolk and egg white. Egg white in the process according to the present invention may be any suitable egg white. Egg white may be homogenized before it is used in a process according to the present invention.

Egg white may be homogenized by incubating egg white at a temperature of 0- 10 degrees Celsius, for instance 1 to 8 degrees Celsius at a pH value of the egg white (pH 8.5-9.5). Said incubation step is usually followed by a step of centrifuging the egg white. The supernatant obtained by centrifuging egg white is egg white that is homogenized. Centrifuging egg white may be carried out by any suitable method known by the skilled person in the art.

Egg white may be incubated with any suitable protease in a process according to the present invention which, results in an increased gelling temperature of an egg white composition.

A protease is defined herein as an enzyme that hydrolyses the peptide bonds that link amino acids in a polypeptide chain that form a protein.

The internationally recognized schemes for the classification and nomenclature of all enzymes from IUMB include proteases. The updated IUMB text for protease EC numbers can be found at the internet site: http://www.chem.qmw/ac.uk/iubmb/enzyme/EC3/4/1 1/. In this system enzymes are defined by the fact that they catalyze a single reaction. This has the important implication that several different proteins are all described as the same enzyme, and a protein that catalyses more than one reaction is treated as more than one enzyme.

The system categorises the proteases into endo- and exoproteases. Endoproteases are those enzymes that hydrolyze internal peptide bonds of proteins and exoproteases are those enzymes that hydrolyze peptide bonds adjacent to a terminal oarmino group (so-called "aminopeptidases"), or a peptide bond between the terminal carboxyl group and the penultimate amino acid (so-called "carboxypeptidases"). The endoproteases are divided into sub-subclasses on the basis of catalytic mechanism. There are sub-subclasses of serine endoproteases (EC 3.4.21 ), cysteine endoproteases (EC 3.4.22), aspartic endoproteases (EC 3.4.23), metalloendoproteases (EC 3.4.24) and threonine endoproteases (EC 3.4.25).

A suitable protease in a process according to the present disclosure may for instance belong to a group selected from aspartic endoproteases, metalloendoproteases, and serine endoprotease. Suitable aspartic endoproteases may for instance be aspergillopepsin I (EC 3.4.23.18) or aspergillopepsin II (EC 3.4.23.19). Suitable metalloendoproteases may for instance be bacillolysin (EC. 3.4.24.28). A suitable serine endoprotease may for instance be subtilisin (EC 3.4.21 .62) Aspegillopepsin I and II are usually produced by a fungus belonging to the genus Aspergillus, for instance an A. niger. Bacillolysin and subtilisin may be produced by Bacillus sp. A protease in a process according to the present disclosure may comprise one or more proteases. For instance, a protease may comprise a mixture of aspergillopepsin I and aspergillopepsin II, or any other suitable mixtures of proteases.

In another embodiment a process as described herein comprises incubating egg white with a protease such that an egg white composition is prepared comprising a degree of hydrolysis of between 0.1 and 30%, for instance between 0.5 and 20%, for instance between 1 and 10%.

A method to determine the degree of hydrolysis is by using orthophtalic anhydride (OPA), which reacts with any terminal aminogroup, such as described in Nielsen et. al. (2001 ) J. Food Science Vol.66, No. 5, p. 642-646. The amount of reacted OPA can be quantified using UVA IS spectrometry. The degree of hydrolysis can be expressed as the value of hydrolysed protein versus intact protein.

Incubating egg white with a protease such that an egg white composition is prepared that has a higher gelling temperature than egg white may be performed in any suitable way.

In a preferred embodiment, incubating egg white with a protease as disclosed herein may take place during desugaring of egg white. Upon spray drying of egg white, sugars may cause browning by maillardation. Desugaring the egg white may prevent such browning to occur. Desugaring may be performed by incubating egg white with a glucose oxidase and a catalase or by incubating with yeast, which is known to a skilled person in the art (see for instance V. Lechevalier et. AL, J. Food Eng. 2007, 83, p. 404- 413).

Incubating egg white with a protease may be carried out at a pH which is not the optimum pH of a protease, for instance a pH of between 5 and 8. Preferably the pH is higher or lower than the isoelectric point of egg white proteins.

Suitable temperatures at which a egg white may be incubated with a protease in a process according to the present disclosure may be between 4 and 60 degrees Celsius, for instance between 10 and 55 degrees Celsius, for instance between 15 and 52 degrees Celsius.

In one embodiment a process as described herein comprises pasteurizing the egg white composition and obtaining a pasteurized egg white composition. An advantage of pasteurizing a liquid egg white composition is that it reduces microbial contamination and may make the time and energy consuming hot room treatment redundant, or can lower the temperature and/or time needed for a hot room treatment. Consequently, the time from breaking eggs to selling eggs can be shortened with weeks when using the process for preparing an egg white composition according to the present disclosure compared to a process for preparing egg white wherein a hot room treatment is applied.

Another advantage of pasteurizing an egg white composition in the process according to the present invention is that a microbial safe fluid egg white product can be prepared.

Pasteurization of an egg white composition may be carried out in any suitable way. Pasteurization may for instance comprise bringing the egg white composition to a temperature of between 60 to 75 degrees Celsius or 62 to 70 degrees Celsius, for instance for 10 sec to 10 min, for instance for 20 sec to 8 min, for instance for 1 min to 6 min.

In another embodiment, a process according to the present disclosure comprises a step of spray drying the egg white composition and obtaining a spray-dried egg white composition. Spray drying is a known technology to the skilled person in the art. Spray drying of egg white may be carried out after the egg white is incubated with a protease and heat pasteurized.

In one embodiment a process according to the present disclosure further comprises using the egg white composition in the preparation of a food product. Use of an egg white composition according the present disclosure was found particularly suitable in the preparation of a food product that does not undergo a pasteurization step after the egg white has been added to the food product. A suitable food product may for instance be surimi, puddings, pastry, cookies, meringues, cake, glazing on pastry and biscuits, as a binding agent in the meat industry, meat analogues, nougats, batter.

In one aspect the present disclosure relates to an egg white composition obtainable by a process as disclosed herein. It was surprisingly found that an egg white composition according to the present invention results in an increased gelling temperature, such as 1 to 15 degrees Celsius higher, for example 2 to 14, or 3 to 12 degrees Celsius higher, compared to egg white that has not been incubated with a protease.

The present disclosure further relates to the use of a protease to increase the gelling temperature of an egg white composition. A protease used to increase the gelling temperature of an egg white composition may be any suitable protease as defined herein above. The increase in gelling temperature may be 1 to 15 degrees Celsius, for example 2 to 14, or 3 to 12 degrees Celsius.

In another aspect the present invention relates to a process for preparing an egg white composition, wherein a protease is used to increase the gelling temperature of egg white and wherein the egg white composition with an increased gelling temperature is produced.

In yet another aspect the present invention relates to a food product comprising egg white obtainable by a process according to the present invention. A suitable food product may be surimi, bavarois, pudding, pastry, cookies, meringues, cake, glazing on pastry and biscuits, as a binding agent in the meat industry, meat analogues, nougats, batter.

EXAMPLES

Materials

Egg white preparation

Fresh shell eggs were broken and separated. The egg white was collected in a small buffer tank, from where it was pumped to the fermentation vessel. After that an intermediate buffering step took place at 0-4 degrees Celsius at given pH of the egg white (pH 8.5-9.5). The time period of this buffering step depends on when the egg white is needed fro further processing, usually between 1 to 96h. Subsequently, the egg white was pumped through a Westfalia plate centrifuge with 4700 rpm, 8000 liters/h for approximately 1 1 seconds centrifuge residence time to a fermentation tank. The supernatant obtained after this centrifuge step was the egg white used in these examples.

Methods

Pasteurization

After incubating the egg white with a protein hydrolysate, samples were pasteurized on lab scale in a water bath at 70 degrees Celsius while stirring and measuring the temperature of the egg white. The egg white was kept for 5 min at 60-65 degrees Celsius and subsequently cooled to 20 degrees Celsius after which the functionality tests were started.

Foam height, capacity and stability

To determine foaming properties, 450 ml liquid egg white was whipped in a Hobart Mixer (model C100) by mixing for 90 sec in the 2 ^nd gear followed by 90 sec in the 3 ^rd gear. Subsequently, the foam was levelled and used to determine the various foaming properties. The whipping level was measured with e.g. a knitting needle and ru ler. Th is resu lted i n a measu re for the foam height. Foam ing capacity was determined by weighing 100 ml foam. Foam stability was assessed via a drainage experiment. 75 g foam was weighed into a funnel with a perforated plate. The dripping albumen was collected and the amount of liquid (ml) after a dripping time of 70 minutes was determined.

Gel strength test

To determine gel strength, 200 grams liquid egg white was transferred in flexible tubes (Vinylidene chloride tubes, diameter 30 mm). After closing, these tubes were heated in a water bath at 90 degrees Celsius for 30 min. After cooling down to 25 degrees Celsius in cold water, the gels ("sausages") were taken out of the tubes and were cut in 30 mm slices. The gel strength was determined by a compression test on a Fudoh rheometer (a plunger with a 5 mm disc was used with a speed of 30 cm/min).

Rheometry

A gel ling curve was measured in an Anton Paar MCR301 rheometer. An oscillatory measurement was carried out with a cup and bob geometry (strain = 1 %; Freq = 1 Hz). The storage and loss modulus (G' and G" respectively) were measured in time as a function of temperature (40-90 degrees Celsius; temperature increase is 1 .5 degrees Celsius per minute). The crossover of G' and G" is defined as the temperature where gelling starts: the gelling point.

Degree of hydrolysis

The degree of hydrolysis was determined using the method described by Nielsen et al. [Nielsen P.M. et al., Improved Method for Determining Food Protein Degree of Hydrolysis, Journal of Food Science, Vol. 66, No.5, 2001 .] and is expressed as DH = h htot ^* 100%, where h _to t is the total number of peptide bonds per protein equivalent and h is the number of hydrolized bonds.

Preparation of Aspergillopepsin I and Aspergillopepsin II

Aspergillopepsin I (protein sequence SEQ ID NO: 128 of WO02/068623) and Aspergillopepsin II (protein sequence SEQ ID NO: 131 of WO 02/068623) were obtained in industrially relevant quantities by overexpression of the encoding genes in an A. niger host cell using methods specified in the prior art. As the protease is efficiently secreted by the A. niger host cell, recovery of the crude enzyme is relatively simple: after fermentation the broth was filtered, the filtrate was sterile filtered and then concentrated on a Pellicon XL Casette Biomax 10 ("Millipore"). The subsequent chromatographic purification was carried out by an initial chromatography over a Q- sepharose XK 16/20 column at pH 5.6 followed by a CAIEX chromatography over a SP- Sepharose XK 16/20 column at a pH of 4.2. In both cases the enzyme was eluted using a gradient towards 1 mol/l NaCI. Fractions were collected based on the occurrence of bands of a specific molecular weight detected upon SDS-PAGE followed by Coomassie blue staining.

The specific activity aspergillopepsin I and aspergillopepsin II was checked in Tecan using E(Edans)-AAXAAK-(Dabcyl)-NH2 fluorescent kit, where X = His. Substrate stock solutions were prepared in DMSO in 5 mM concentration. The reaction mix contained: 195 mkl buffer (100 mM Na-acetate buffer, pH 4.0 or 100 mM Tris-HCI, pH 7.0), 2 mkl substrate stock solution and 5 mkl ZBJ sample (0.25 mg/ml in 50 mM Naacetate, pH 5.6). The reaction mix was incubated at 37 degrees Celsius in Tecan for 60 min (lambda ex=340nm, lambda em=485 nm). The enzyme activity was determined in relative fluorescent units (rfu) per minute per mg of protein.

Activity of Aspergillopepsin II determined by hydrolysis of a hemoglobin substrate

A solution with hemoglobin substrate (20g/L, Sigma Aldrich catalogue number H2625) was incubated with aspergillopepsin II enzyme solution at 40 degrees Celsius and a pH of 1 .75. The reaction was terminated by adding trichloric acetic acid (Sigma-Aldrich 27242). The optical density of the supernatant after centrifugation was measured at a wavelength of 275 nm (by UV-VIS Spectrophotometer Hitachi U2910) and was compared to an L-tyrosine solution (Merck, 1 .08371 ) of known strength. The obtained value is a measure for the activity.

Unit definition. The activity is expressed in units per gram. One unit is the amount of enzyme that hydrolyses an amount of substrate per minute, under the conditions of the test giving a solution with an optical density at 275 nm equal to the optical density of a solution containing 1 .10 μg tyrosine per mL in HCI solution of 0.006 mol/L.

SDS-PAGE

Egg white samples were diluted 20 times; reducing agent and buffer were added before running the SDS-PAGE for 30 minutes. The gel was stained with

SeeBlue for 1 hour and discolored in water for 1 day to decrease the blue background before an image was taken.

Desugaring of egg white

Egg white was manually separated from fresh eggs, mixed with a Silversion mixer for 10 minutes at 3500 rpm before centrifugation for 10 min at 5.000 rpm to remove debris and chelaza from the native egg white.

For desugaring the pH was adjusted from 9.1 to pH 7.2 and 5.6 for Validase and Aspergillopepsin II, respectively, with 0.5 M citric acid adding while stirring on a magnetic stirrer. Fresh 'bakers yeast' was used to desugar egg white with 0.06 % fresh yeast (which compares to 0.02 % Fermiol) according to the procedure which is common in factory processing, 6 hours incubation at 40 degrees Celcius.

After incubation the sugar was below the detection limit using the test for 'blood glucose' the BREEZE 2 monitor from Bayer. The highest incubation temperature of 50 degrees Celsius used here, was not limiting for yeast growth to remove the sugar in liquid egg white.

Example 1. Treatment of egg white with Aspergillopepsin II (EC 3.4.23.19)

The pH of the egg white was adjusted to pH 5.6, using 0.5 M citric acid. The egg white was put in a water bath at 50 degrees Celsius for 20 min. One part of this sample was used as a reference sample. The pH of this sample was adjusted to 7.0 using 0.2M NaOH. The other part is incubated with 0.002% (w/v) (2.1 ^* 10E5 rfu/mg.min) Aspergillopepsin II, prepared as described above at 50 degrees Celsius for 5 hrs. After incubation the pH was adjusted to 7.0 with the use of 0.2 M NaOH. After incubation, the degree of hydrolysis was 1 .70%.

Functionality tests were carried out after incubation with Aspergillopepsin II at pH 5.6 (but final pH = 6.2) (Table 1 ). It was found that foaming properties were comparable to untreated egg white. Gel strength of Aspergillopepsin I I treated egg white was slightly lower compared to the reference samples.

Table 1 : Results functionality tests for untreated egg white and Aspergillopepsin II treated egg white

Example 2. Treatment of egg white with Aspergillopepsin I (EC. 3.4.23.18)

The pH of the egg white was adjusted to pH 5.6, using 0.5 M citric acid. The egg white was put in a water bath at 50 degrees Celsius for 20 min. One part of this sample was used as a reference sample. The pH of this sample was adjusted to 7.0 using 0.2M NaOH. The other part is incubated with 0.002% (w/v) (7.6 ^* 10E5 rfu/mg. min) Aspergillopepsin I prepared as described above at 50 degrees Celsius for 5 hrs. After incubation the pH was adjusted to 7.0 with the use of 0.2 M NaOH and the degree of hydrolysis was 2.00%.

Functionality tests were carried out after incubation with Aspergillopepsin I at pH 5.6 (but final pH = 6.2) (Table 2). It was found that both foaming properties and gel strength were slightly improved for Aspergillopepsin I treated egg white compared to the reference sample.

Table 2: Results functionality tests for untreated egg white and Aspergillopepsin I treated egg white

Example 3. Treatment of egg white with Validase BNP-L (EC 3.4.24.28)

The pH of the egg white was adjusted to pH 7, using 0.5 M citric acid. The egg white was put in a water bath at 40 degrees Celsius for 10 min. One part of this sample was used as a reference sample. The other part is incubated with 0.1 % (w/w) Validase BNP-L (DSM, Lotnr. OH0091 H; activity: 2000 NPU/g) at 40 degrees Celsius for 2 hrs. After incubation the pH was adjusted to 7.0 with the use of 0.2 M NaOH. After incubation the degree of hydrolysis was 7.29%.

Functionality tests were carried out after incubation with Validase BNP-L at pH 7.6 and pasteurization (Table 3), before and after pasteurization. Pasteurization was carried out as described above. It was found that the foaming capacity of Validase BNP-L treated egg white was much better, i.e. a lower amount of protein is necessary to make 100 ml foam, compared to untreated egg white (for both samples determined after pasteurization). Also foam height improved significantly. According to this test method, a foam height above 152 mm is regarded as "prime" foam, which is the highest qualification for foam quality. Gel strength of Aspergillopepsin I treated egg white was lower compared to the reference sample, may be due to a lower gel density caused by air bubbles in the Aspergillopepsin I treated egg white gel.

Table 3: Results functionality tests for untreated egg white and Validase BNP-L treated egg white

Example 4. Treatment of egg white with Alcalase EC 3.4.21.62

The pH of the egg white was adjusted to pH 7, using 0.5 M citric acid. The egg white was put in a water bath at 40 degrees Celsius for 10 min. One part of this sample was used as a reference sample. The other part is incubated with 0.1 % (w/w) Alcalase AF 2.4L Novozymes (Lotnr. RM09481 1 U-09124) at 40 degrees Celsius for 2 hrs. After incubation the pH was adjusted to 7.0 with the use of 0.2 M NaOH.

Functionality tests were carried out after incubation with Alcalase at pH 7.6 (Table 4). Foam height of Alcalase treated egg white was found to be higher compared to the reference sample. Gel strength of Alcalase treated egg white was lower compared to untreated egg white.

Table 4: Results functionality tests for untreated egg white and Alcalase treated egg white

Example 5. Gelling temperature of egg white compositions

Egg white compositions comprising Aspergillopepsin II, Aspergillopepsin I, Validase, or Alcalase were prepared as described in Examples 1 to 4. Subsequently, the gelling points, i.e. the temperature at which gelling starts were determined by rheometry as described above .The difference in gelling temperature is given in Table 5 below.

Table 5: Difference in gelling temperature between reference egg white and egg white with added protease

Protease added Difference in gelling temperature (degrees

Celsius) between reference egg white and egg white with added hydrolysate

Aspergillopepsin II 7

Aspergillopepsin I 4

Validase-BNP-L 10

Alcalase 7 The results in Examples 1 to 5 show that incubation of egg white with different proteases increased the gelling temperature of the egg white composition. The foaming properties of the protease treated egg white were as good as untreated egg white or were improved. The gel strength of egg white treated with aspergillopepsin was similar to untreated egg white; the gel strength of egg white treated with metalloendoprotease and serine endoprotease was lower than untreated egg white.

Example 6. Treatment of egg white with different doses of Aspergillopepsin II (EC 3.4.23.19) during desugaring of egg white

Egg white was brought to pH 5.6 with 0.5 M citric acid and yeast suspension with 0.5 and 1 % of Aspergillopepsin II (batch VMJ1 146-70) were incubated with the egg white for 5 hours at 50°C. The protease activity of Aspergillopepsin II batch

VMJ1 146-70 was determined using hemoglobine as substrate described above and was 13300 U / g formulated liquid product. 1 % enzyme compares to 133000 U / kg egg white.

After incubation, the egg white was centrifuged for 10 minutes at 5.000 rpm to remove the yeast. The supernatant was used for analysis and pasteurization followed by gelling. The gel points were measured twice; the values for the increase in gel point compared to reference egg white are presented in Table 6.

Table 6: Difference in gelling temperature of desugared egg white treated with Aspergillopepsin II and untreated desugared egg white at pH 5.6

Conclusion: Aspergillopepsin II is effective to increase the egg white gel point with about 5°C. The incubation can take place during desugaring of egg white.

SDS PAGE: SDS page showed that during treatment of egg white with aspergillopepsin II the proteins of between about 45 and 60 kDa (ovalbumin, globulins) remained mostly intact. Moreover, also the protein band of 65-80 kDa (ovotransferin) remained mostly intact. As the larger proteins remained mostly intact the gel strength remained substantial.

Example 7. Treatment of egg white with different doses of Validase BNP-L (EC3.4.24.28) during desugaring of egg white with Validase BNP

Egg white was brought to pH 7.2 with 0.5 M citric acid. Yeast suspension with 0.05% Validase BNP-L as described in Example 3 were added to the egg white and incubated for 5 hours at 50°C. In a second experiment 0.1 % Validase BNP-L was added the egg white two hours after addition of the yeast.

After incubation, the egg white was centrifuged for 10 minutes at 5.000 rpm to remove the yeast. The supernatant was used for several analysis and pasteurization followed by gelling. The gel points were measured and results are shown in Table 7.

Table 7: Gel point of desugared egg white pH 7.2 treated with Validase BNP-L

Conclusion: Validase-BNP-L (bacillolysin) is effective to increase the egg white gel point with about 10 degrees Celsius. The incubation can take place during desugaring of egg white.

SDS PAGE:

SDS PAGE showed that during treatment of egg white with Validase BNP-L at 0.1 % the protein bands of between 65 and 80 kDa (ovotransferrin) disappeared almost completely. In constrast these protein band of between 65 and 80 kDa (ovotransferrin) after treatment with 0.05% Validase remained intact. The protein bands of between 45 and 60 kDa (ovalbumin, globulins) were shifted to a lower molecular weight (about 5kDa) with treatment of 0.1 % Validase. In contrast during treatment with 0.05% Validase protein bands of between 45 and 60 kDa remained intact. As the larger proteins remained mostly intact the gel strength of the Validase BNP-L remained substantial.