The present invention relates to the use of a phospholipase enzyme for improving functional properties of an egg white composition. In particular, the present invention relates to a process for providing an egg white composition having said improved functional properties, wherein the process comprises the steps of providing an egg white com- position, adding a phospholipase enzyme to the egg white composition; and allowing the phospholipase enzyme to react with the egg white composition. The present invention further relates to an egg white product comprising at least the egg white composition having improved functional properties, and to a kit for improving said functional properties of an egg white composition comprising a phospholipase enzyme and instructions for carrying out the process according to the present invention.

Background of the invention

Eggs from birds such as hen and chicken are widely used in the preparation of numerous types of food products. In particular, eggs are used as a food ingredient for the formulation of processed food products (e.g. mayonnaise, dressings, desserts, confectionaries, cakes and meringues) and the addition may provide various functional properties such as emulsifying properties and/or foaming properties to these food products. The eggs from hens are most widely used. The eggs consist of a protective eggshell, egg white, and egg yolk, each separately contained within thin membranes. Prior to application in food products the eggs undergo an initial processing, where the eggs are washed and cracked and the protective eggshell is discarded. Optionally, the egg yolk and egg white may be separated. Depending on the required functional properties of the food product in question the egg ingredient may consist of the whole egg, the egg yolk or egg white. The egg yolk is a major source of vitamins and minerals. It contains protein, choline, carbohy- drates and cholesterol as well as all of the egg's fat, including approximately 9% phospholipids (phosphatidyl-choline, phosphatidyl- ethanolamine). The egg white also denoted albumen consists mostly of the protein albumin and water.

Typically, the egg ingredient being whole egg, egg yolk or egg white is prepared prior to addition to a food product. Preparation may for instance involve whipping and/or beating the egg ingredient so that it is brought to a form particularly suitable for the food product in question. To improve said form the egg ingredient may be subjected to a pre- treatment prior to said preparation. The pre-treatment may aim at providing improved functional properties of the egg ingredient, such as whipping ability and foam stability. Such pre-treatment processes may include enzymatic treatment. For example US 5,080,911 relates to a process for modifying the properties of egg yolk, which comprises treat- ing egg yolk with an effective amount of phospholipase D derived from a microorganism, thereby converting phospholipids contained in the egg yolk and resulting in higher heat-gelation and emulsification properties. In JP 2003-102439 a method for providing a processed egg product is described, where the method includes enzymatically treating egg yolk with phospholipase A2 to increase emulsifying ability.

In a variety of egg-containing food products, however, it is desirable to add only the egg white, hence denoted egg white food products. In particular, egg white is added due to the very good foaming properties. The egg white may be aerated by whipping and/or beating, and adding such aerated egg white to a food product may provide the food product with unique properties with respect to texture, taste and appearance, which is particularly appreciated in food products such as desserts, confectionaries, cakes and meringues. It is, however, difficult to keep the aerated state of the egg white, which may in term result in a deterioration of the final egg white food product. This is mainly attributed to insufficient functional properties of the egg white ingredient. Accordingly, functional properties such as foam stability of an egg white are paramount for the quality of the food products, and thus, a prolonged stabilisation of the aerated state of egg white is desired. EP 1 438 902 relates to providing such prolonged stabilisation of an aerated egg white composition. In particular, EP 1 438 902 relates to providing a sugar alcohol composition for the use of stabilizing egg foam of aerated liquid egg, comprising alpha-D-glucopyranosyl-l,6-sorbitol. However, in many cases it may be desirable to avoid the use of additives such as sugar alcohol compositions, thus, providing the need for an alternative method of increasing the whipping ability of egg whites and stabilizing the egg white foam.

A major problem affecting the functional properties of egg whites is contamination of the egg white with egg yolk. It is readily recognised within the art that any contamination of the egg white with egg yolk will significantly reduce the functional properties of the egg white, e.g. the foam stability, foam density and whipping ability. This is true even for minute remnants of the egg yolk in the egg white. Thus, during the initial processing it is desirable to ensure a complete separation of the egg white from the egg yolk. Nevertheless this might not always be possible. Especially, in large scale processing of eggs a completely un- contaminated egg white is virtually impossible to obtain. In addition, storage of eggs over a period of time also results in migration of lipids from the egg yolk to the egg white and even such small amounts have a negative effect on the functional properties of the egg white, thereby rendering attempts for complete separation by simple mechanical means impossible. Therefore, on industrial scale a need exists for modifying or neutralizing contaminating lipid residues of egg yolk from the egg white.

The exact mechanism as to how lipids impede the functional properties of egg white has not yet been established, and diverging opinions exist as to the impact of phospholipids and triglyceride fractions. It is, however, well-established that some functional properties of yolk-contaminated egg white can be wholly or partly recovered by re- moval of lipids using lipase enzymes. Macherey et al. (J Food Sci. 2011 May;76(4) :C651-5. doi : 10.1111/j.1750-3841.2011.02138.x. Epub 2011 Apr 13.) suggest that phospholipases may also be used for the recovery of functional properties of yolk-contaminated egg white. However, they also recognise that neither attempts nor successful use of phospholipase have yet been reported. For use of lipase a limit exists as to the amount of yolk contamination, which the presently available enzymatic methods can recover, and not all functional properties can be recovered at levels of contamination, which is typically found in egg white obtained by in- dustrial scale separation methods. US 2,892,720 relates to a process for reducing the lipid content of egg albumen which comprises subjecting the albumen containing a small amount of yolk to the action of an added lipase preparation free from zymases and proteases to substantially reduce the lipid content. Macherey et al. have also investigated the use of lipases for improving the functional properties of yolk-contaminated egg whites. The functional properties include foam capacity, foam drainage rate, foaming power, foam stability, foam volume, and foam height. It was found that lipases of different origin have different effect. Pancreatic lipases have shown effective whereas lipase isolated from wheat germ has not been successful. Macherey et al. further found that lipase from Mucor miehei (EC 3.1.1.3) did not function unless colipase, a protein co- factor, was present. No general explanation is presently offered as to why some lipases work and others do not, and there is no theoretical approach as to identify enzymes which can successfully improve the functional properties of yolk-contaminated egg white.

A drawback with the presently available enzyme treatments is that the amount of contaminating egg yolk must be limited, typically below 1% (w/w), in order to sufficiently improve the functional properties of the egg white. Accordingly, an object of the present invention is to provide a treatment which may recover functional properties of yolk- contaminated egg white having a higher amount of contamination. Even still, the lipase enzymatic treatment of egg white having yolk contamination below 1% (w/w) does not offer full recovery of all functional properties. In particular, foam stability cannot be fully recovered even for egg whites having yolk contamination below 0.2% (w/w). Accordingly, it is a further object of the present invention to provide a treatment for recovering the functional properties of egg white having contaminating egg yolk lipid remnants in order to arrive at functional properties of the egg white corresponding to compositions where no contamination is present and/or to even further improve the functional properties as compared to non-contaminated egg white. It is a further object of the invention that the treatment should accommodate large-scale processing requirements for recovering low-quality products. A further object is to improve prod- ucts comprising egg white compositions.

Summary of the invention

These and further objects are solved by the present invention. Thus, a first aspect the present invention relates to a use of a phospholi- pase enzyme for improving functional properties of an egg white composition comprising more than 80% (w/w) of egg white, preferably more than 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white. In an embodiment of the present invention the egg white composition com- prises from about 0.001% to about 5% (w/w) of egg yolk, preferably from about 0.001% to about 4% (w/w) of egg yolk, more preferably from about 0.001% to about 3% (w/w) of egg yolk, even more preferably from about 0.001% to about 2.5% (w/w) of egg yolk, where the total sum of the egg white composition does not exceed 100% (w/w). In a preferred embodiment of the present invention the egg white composition comprises from about 0.1% to about 0.3% (w/w) of egg yolk.

The present inventors have surprisingly found that adding any phospholipase enzyme to an egg white composition contaminated with egg yolk lipids improves the functional properties of the egg white sig- nificantly as compared to compositions where no phospholipase enzyme is added. In particular, the functional properties are improved to such an extent that no further enzymatic and/or alternative treatment for improving the functional properties is needed. The present inventors have found this to be true even when the contamination exceeds concentra- tions of egg yolk, which can normally not be remedied, such as concentrations of egg yolk above about 1% (w/w). Depending on the contamination the use of the phospholipase enzyme improves the functional properties of the egg white composition to a level equal to the functional properties of an egg white composition, which is substantially free of egg yolk. That is, the use of the phospholipase enzyme may fully recover the functional properties of an industrial grade yolk-contaminated egg white composition. The present inventors have surprisingly found that the functional properties of an egg white having a yolk contamination as high as up to 1% (w/w) may be recovered to an extent where the egg white can be used in egg white products, which egg white product would normally require egg white substantially free of egg yolk. The present inventors further found that the functional properties of an egg white having a yolk contamination as high as up to 5% (w/w) may be im- proved, although to a lesser extent as compared to egg white, which is substantially free of egg yolk.

The present inventors have observed that using phospholipase enzyme on yolk-contaminated egg white compositions results in significant improvement of the whipping ability of the egg white composition. That is, an egg white composition with yolk-contamination, which clearly prevents the egg white composition from being aerated into foam by whipping and/or beating, can readily be treated with a phospholipase enzyme so that it may easily be aerated into foam. The present inventors found this to be true for egg white compositions with a yolk- contamination as high as about 3% (w/w), and that for an egg white composition with a yolk-contamination as high as about 1% (w/w), the quality of the foam may be recovered to an extent, which is equal to or better than an egg white composition substantially free of egg yolk contamination.

Moreover it was found that the use of the phospholipase enzyme even further improves the functional properties of a yolk- contaminated egg white to exceed those of an egg white composition substantially free of yolk contamination. Without being bound by any specific theory it is believed that the hydrolysis of phospholipids by the phospholipase enzyme provides for improved functional properties of the egg white composition, and that the product of this hydrolysis, which is not naturally present in egg white compositions, is responsible for the observed effect.

In an industrial scale separation process the resulting egg white composition will typically have a yolk contamination in the range from about 0.1% to about 0.3% (w/w), typically about 0.25% (w/w). The present inventors have found the use of phospholipase enzyme to be very effective in improving the functional properties of a yolk- contaminated egg white composition within this range of contamination. The present inventors have even observed that the functional properties of the egg white composition have improved beyond the functional properties of reference egg white compositions, which have been manually separated to ensure compositions substantially free of egg yolk. There- fore, in a preferred embodiment of the present invention the egg white composition comprises from about 0.1% to about 0.3% (w/w) of egg yolk.

It follows that in a preferred embodiment of the present invention the phospholipase enzyme is added to the egg white composition in order to improve the functional properties. By functional properties of the egg white composition is in particular meant all the properties within the aspect of providing a foamed and/or aerated egg white composition, wherein the quality of the functional properties in respect of foaming and/or aeration of the egg white composition is measured with parame- ters such as foam height, foam density, foam volume, foam capacity, foaming power, and foam stability. Accordingly, an embodiment of the present invention relates to the use of the phospholipase enzyme for improving functional properties of the egg white composition, wherein the functional properties are selected from foam height, foam density, foam volume, foam capacity, foaming power, and foam stability of the egg white composition.

In a particular embodiment of the present invention the use of the phospholipase enzyme is to improve the functional property of foam stability, where the foam stability is defined as the volume of drainage per 75 g of foam, and where the volume of drainage is the volume of the liquid phase collected 70 minutes after whipping the egg white composition into foam. The present inventors have observed that the foam stability is significantly increased for a yolk-contaminated egg white composition by adding phospholipase enzyme as compared to when no enzyme is added. Furthermore, the foam stability is improved further as compared to similar results provided by alternative enzymatic treatments, such as enzymatic treatments using lipase. This is particularly true for yolk-contaminated egg white compositions, wherein the egg white com- position comprises from about 0.001% to about 3% (w/w) of egg yolk, preferably from about 0.01% to about 2% (w/w) of egg yolk, more preferably from about 0.01% to about 1% (w/w) of egg yolk, even more preferably from about 0.1% to about 1% (w/w) of egg yolk. For egg yolk contamination in concentrations as high as about 1% (w/w) the foam stability was found to be fully recovered to a level as if the egg white was initially substantially free of yolk contamination. Hence, depending on the contamination the foam stability may be fully recovered after use of phospholipase enzyme. Accordingly, in another embodiment of the present invention the foam stability of the egg white composition is im- proved by a factor of at least 1.5 as compared to compositions where the phospholipase enzyme is not used, and where the egg white composition has an egg yolk contamination of from about 0.01% to about 2.5% (w/w). In an embodiment the egg white composition has a foam stability of at least 2 times as high, preferably 2.5 times as high, more preferably 3 times as high as if the phospholipase enzyme is not used, where the egg white composition has an egg yolk contamination of from about 0.05% to about 2% (w/w). The present inventors found that the improved foam stability effectively protects against over-whipping and/or over-beating of the egg white composition. Continued whipping of the egg white composition after it has reached the optimum foam volume may otherwise result in a collapse of the foam, i.e. a decrease in foam volume and/or changed consistency. However, with the improved foam stability provided by the use of phospholipase enzyme the egg white composition substantially retains the optimum foam volume even when subject to over-whipping and/or over-beating. In another particular embodiment of the present invention the use of the phospholipase enzyme is to improve the functional property of foam density, and the use results in a foam density below about 0.1 g/ml, preferably below about 0.08 g/ml, where the egg white composition has an egg yolk con- tamination of from about 0.001% to about 1% (w/w).

The use of the phospholipase enzyme provides an egg white composition, which is suitable for being aerated into foam by whipping and/or beating. Adding the aerated egg white composition, which has been treated with phospholipase enzyme, to an egg white product has shown to provide the egg white product with unique properties with respect to texture, taste and appearance, which is particularly appreciated by consumers in egg white food products such as desserts, confection- aries, cakes and meringues. Thus, the unique properties of the egg white product are inherited from the improved functional properties of the egg white composition and it has been shown that the functional properties are not adversely affected by admixing further ingredients in the egg white composition. Therefore, an embodiment of the present invention relates to the use of the phospholipase for improving functional proper- ties of the egg white composition, wherein the egg white composition is comprised in an egg white product having additional ingredients. The present inventors have surprisingly found that egg white food products based on foamed egg white compositions treated with phospholipase enzyme retain the volume in the egg white food product to a greater ex- tent so that the egg white food product remains aerated.

The present inventors have also realised that the improved foam stability obtained by the use of the phospholipase enzyme according to the invention provides for an increased flexibility in the preparation processes of egg white food products. Hence, with the improved foam stability the foam will retain its volume for a longer period of time making other simultaneous and/or successive preparations of ingredients less dependent on when the foamed egg white composition is prepared. The aerated egg white foam does not substantially reduce its volume over an increased period of time such as up to about 1 hour. Thus, a high yield in food products can still be obtained even in situations where the aerated egg white foam cannot readily be used in the further food preparation process. With less stabile foam this would lead to a reduction in the yield of food product as the egg white foam volume would have reduced. Accordingly, a person skilled in the art of manufacturing egg white food products such as desserts, confectionaries, cakes and meringues will readily appreciate the flexibility in the preparation process conferred by the improved foam stability.

The present inventors have further found that the use of a phospholipase enzyme, rather than e.g. a lipase, facilitates the processing of egg white products and improves the overall durability of the food products. This is believed to be due to the high specificity of the phospholipase. Lipase in general has very low specificity for the substrate, whereas a phospholipase only catalyses the hydrolysis of phospholipids. Consequently, when producing an egg white product by adding additional ingredients to an egg white composition treated with lipase, the lipase may still hydrolyse any lipids present in the added ingredients, and such hydrolysis may render the egg white product less desirable. Therefore, it may be necessary to inactivate the lipase following treatment of the egg white composition. It was found that when using phospholipases the final egg white product was not compromised. Using phospholipase, however, the high specificity has turned out to ensure that the enzyme in the egg white composition is less likely to react with the additional ingredients thereby facilitating the process and avoiding degradation of the egg white product. Thereby a more simple industrial process is provided for as there is no need for quenching the action of the added enzyme.

In a preferred embodiment the phospholipase enzyme is a phospholipase A2 enzyme. The present inventors have surprisingly found that phospholipases of the type A2 exhibit particularly good effect on the functional properties of yolk-contaminated egg whites.

In a more preferred embodiment of the present invention the phospholipase enzyme originates from a microbial source. In an even more preferred embodiment the microbial source is selected from any of the strains Streptomyces violaceoruber or Aspergillus niger, preferably Aspergillus niger. In a particularly preferred embodiment the phospholipase A2 is phosphatide-2-acyl-hydrolase (E.C.3.1.1.4). The present inventors have found that the use of phosphatide-2-acyl-hydrolase results in a particularly good effect in the improvement of functional properties of yolk-contaminated egg white. Furthermore, phosphatide-2-acyl- hydrolase is produced by microbial fermentation of a selected strain of Aspergillus niger. This provides for a relatively simple synthesis and purification, and the phospholipase enzyme can easily be adapted for in- dustry. In addition, being produced by microbial fermentation the phos- phatide-2-acyl-hydrolase may readily be certified as both Kosher and Halal.

Another aspect of the present invention relates to a process for providing an egg white composition having improved functional proper- ties, wherein the process comprises the steps of:

i. providing an egg white composition comprising more than 80% (w/w) of egg white, preferably more tha n 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white;

ii. adding a phospholipase enzyme to the egg white composition; and

iii. allowing the phospholipase enzyme to react with the egg white composition.

In an embodiment of the process of the present invention from about 10 units to about 10 ⁶ units of phospholipase enzyme to 1 kg of egg white composition is added in step (ii), preferably from about 100 units to about 10 ⁵ units of phospholipase enzyme to 1 kg of egg white composition. In one embodiment according to the present invention the egg white composition comprises from about 0.001% to about 5% (w/w) of egg yolk, preferably from about 0.001% to about 4% (w/w) of egg yolk, more preferably from about 0.001% to about 3% (w/w) of egg yolk, even more preferably from about 0.001% to about 2.5% (w/w) of egg yolk.

A further aspect of the present invention relates to an egg white product comprising at least an egg white composition having improved functional properties, where the egg white product is obtainable by a process comprising the steps of:

i. providing an egg white composition comprising more than 80% (w/w) of egg white, preferably more than 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white;

ii. adding a phospholipase enzyme to the egg white composition; and

iii. allowing the phospholipase enzyme to react with the egg white composition;

iv. whipping the egg white composition of step iii); and

v. optionally adding additional ingredients to the egg white composition;

wherein the egg white composition has a foam stability of at least 1.5 times as high as the egg white composition provided in step i) without adding the phospholipase enzyme in step ii), where the egg white composition has an egg yolk contamination of from about 0.01% to about 2.5% (w/w), and wherein the foam stability is defined as the volume of drainage per 75 g of foam, where the volume of drainage is the volume of the liquid phase collected 70 minutes after whipping the egg white composition into foam. In another embodiment the egg white composition has a foam stability of at least 2 times as high, preferably 2.5 times as high, more preferably 3 times as high as the egg white composition provided in step i) without adding the phospholipase enzyme in step ii), where the egg white composition has an egg yolk contamination of from about 0.05% to about 1% (w/w). In a further embodiment the foam density of the egg white composition after whipping the egg white composition into foam is lower than the egg white composition provided in steps i) to iii) without adding the phospholipase enzyme in step ii). This is particularly true for yolk-contaminated egg white compositions, wherein a yolk contamination is present, i.e. wherein the egg white composition comprises from about 0.001% to about 3% (w/w) of egg yolk, preferably from about 0.01% to about 3% (w/w) of egg yolk, more preferably from about 0.01% to about 2% (w/w) of egg yolk, even more preferably from about 0.01% to about 1% (w/w) of egg yolk, and yet again even more preferably from about 0.1% to about 1% (w/w) of egg yolk.

Another aspect of the present invention relates to a kit for im- proving functional properties of an egg white composition comprising a phospholipase enzyme in a suitable container and instructions for the use and/or for carrying out the process according to the above- mentioned aspects of the present invention. In a preferred embodiment the phospholipase enzyme in the kit is phospholipase A2. In an even more preferred embodiment the phospholipase A2 enzyme originates from a microbial source. In a yet more preferred embodiment the microbial source is selected from any of the strains Streptomyces violaceoru- ber or Aspergillus niger, preferably Aspergillus niger. In a yet even more preferred embodiment the kit the phospholipase A2 enzyme is a phos- phatide-2-acyl-hydrolase, preferably phosphatide-2-acyl-hydrolase (E.C.3.1.1.4), which is also known under the trade name MAXAPAL® A2 provided by DSM Food Specialties B.V. Figures

The invention is explained in greater detail with the aid of examples of embodiments and with reference to the figures, in which

Figure 1 shows the results from a test of tolerance against over- whipping;

Figure 2 shows the foam stability of yolk-contaminated egg whites with and without phospholipase A2 enzyme.

Figure 3 depicts the effect of using phospholipase A2 enzyme on foam appearance after 180 s and 270 s, respectively, of whipping the egg white composition;

Figure 4 depicts the effect of using phospholipase A2 enzyme on foam appearance after 480 s of whipping the egg white composition.

Detailed description of the invention

The present invention is described in further detail. It should be understood that combinations of the features in the various embodiments shown and described are also contemplated, and that the various features, details and embodiments may be combined into other embodiments. In particular, it is contemplated that all features, details, and embodiments regarding the use, process, egg white composition, egg white product, and kit apply equally to one another.

Throughout the description and claims all percentages are (w/w) unless otherwise stated.

A first aspect of the present invention relates to a use of a phospholipase enzyme for im proving functional properties of an egg white composition comprising more than 80% (w/w) of egg white, preferably more than 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white.

The term "egg" is meant to embrace all eggs consisting of a protective eggshell, egg white, and egg yolk, contained within various thin membranes. Such eggs typically originate from birds and reptiles. The present invention is believed to be applicable to all eggs of aforementioned general type. However, in a preferred embodiment of the present invention the egg originates from birds, in a more preferred embodiment the egg originates from hens and/or chickens and/or ducks, most preferably from hens and/or chickens. Thus, in a preferred embodiment of the present invention the egg white composition originates from hens and/or chickens.

By the term "egg white composition" is meant the egg white from an egg, which has been separated from the rest of the egg. Hence, the "egg white composition" mainly comprises egg white. However, in large scale processing of eggs a complete separation of egg white from the egg yolk is very difficult to obtain. Furthermore, storage of eggs over a period of time also results in migration of lipids from the egg yolk to the egg white. Accordingly, the process of separation of egg white from egg yolk typically results in an "egg white composition" comprising egg white and egg yolk, wherein the "egg white composition" according to the present invention comprises more than 80% (w/w) of egg white, preferably more than 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white. In an embodiment of the present invention the egg white composition consists of 100% (w/w) of egg white, i.e. egg white substantially free of yolk contamination. It follows that the terms "egg white contaminated with yolk", "egg white contam inated with egg yolk" and "yolk-contaminated egg white composition" may be used interchangeably and refer to an egg white composition comprising egg yolk in an amount, which may impede the functional properties of the egg white. In an embodiment of the present invention the egg white composition comprises from about 0.001% to about 3% (w/w) of egg yolk, preferably from about 0.001% to about 2% (w/w) of egg yolk, more preferably from about 0.001% to about 1% (w/w) of egg yolk, even more preferably from about 0.01% to about 1% (w/w) of egg yolk, where the total sum of egg white and egg yolk does not exceed 100% (w/w). Therefore, an egg white composition according to the invention may consist of egg white and egg yolk, more specifically the egg white composition consists of egg white and egg yolk, wherein the egg white composition consists of from about 0.001% to about 3% (w/w) of egg yolk, preferably from about 0.001% to about 2% (w/w) of egg yolk, more preferably from about 0.001% to about 1% (w/w) of egg yolk, even more preferably from about 0.01% to about 1% (w/w) of egg yolk, and wherein the egg white constitutes the remaining part of the egg white composition, not exceeding 100% (w/w). In an industrial scale separation process the resulting egg white com position will typically have a yolk contamination in the range from about 0.01% to about 1% (w/w), typically from about 0.05% (w/w) to about 0.8% (w/w). By the term "i nd ustria l grade yolk-contaminated egg white composition" is meant an egg white composition having a yolk contamination within this range.

The terms "non-contaminated egg white" and "egg white composition substantially free of egg yolk" may be used interchangeably and refer to egg whites, which typically have been separated manually, and which do not have comprise egg yolk in amounts that would impede the functional properties. The egg white composition may, however, comprise some yolk due to migration over the membrane.

The terms "phospholipase" and "phospholipase enzyme" are well recognised within the art as a group of enzymes that hydrolyses phospholipids. In an embodiment of the present invention the phosphol- ipase enzyme originates from a microbial source. In a preferred embodiment the microbial source is selected from any of the strains Strep- tomyces violaceoruber or Aspergillus niger, preferably Aspergillus niger. In an embodiment of the present invention phospholipase A2 (IUB 3.1.1.4) manufactured by Nagase ChemteX Corporation under the trade name PLA2 NAGASE is used. The enzyme originates from a strain of Streptomyces violaceoruber. In another embodiment the phospholipase A2 enzyme is CakeZyme® Smart and/or CakeZyme® Smart L, which are both manufactured by DSM Food Specialties B.V. In a preferred em- bodiment the phosphol ipase A2 enzyme is a phosphatide-2-acyl- hydrolase, in particular the phosphatide-2-acyl-hydrolase (E.C.3.1.1.4), which is also known under the trade name MAXAPAL® A2 provided by DSM Food Specialties B.V. Phosphatide-2-acyl-hydrolase is produced by microbial fermentation of a selected strain of Aspergillus niger.

The phospholipase enzyme may be provided in a ny suita ble form, such as in a liquid suspension or as a powder, which is reconstituted prior to and/or during mixing. The present inventors believe that the phospholipase enzyme must have a high purity so that limited side- reactions occur. MAXAPAL® A2 complies with such high purity demands. However, the phospholipase enzyme may be provided in a suitable enzyme composition, which may comprise additional ingredients such as salts, sugars such as maltodextrin, and glycerol, and preservatives.

By "functional properties" of an egg white composition is meant the quality of the foam into which the egg white composition is trans- formed when aerated by whipping and/or beating. The egg white composition may be aerated into foam by whipping and/or beating the egg white composition using e.g. an eggbeater, a whisk, whirewhip, mixer, etc. Without being bound by theory the present inventors believe that the physical stress of beating an egg white composition creates the foam . There are two types of physical stress caused by beating them with e.g. a whisk, the first being that the whisk drags the liquid through itself, creating a force that unfolds the protein molecules. This process is called denaturation. The second stress comes from the mixing of air into the egg white composition, which causes the proteins to come out of their natural state. These denatured proteins gather together where the air and water meet and create multiple bonds with the other unravelled proteins, and thus become a foam, holding the incorporated air in place. This process is called coagulation. When beating the egg white composi- tion it may be classified in three stages according to the peaks they form when the beater is lifted : soft, firm, and stiff peaks. By the term "over- beaten" or "over-whipped" the egg white composition take on a dry appearance, and will eventually collapse. The term "whi pping a bility" should be construed as the susceptibility of an egg white composition to being whipped and/or beat into foam according to the above-mentioned three stages by subjecting the egg white composition to a whipping procedure for a period of time. The functional properties of the egg white composition are measured by the quality parameters such as foam height, foam density, foam volume, foam capacity, foaming power, and foam stability. Accordingly, an embodiment of the present invention relates to the use of the phospholipase enzyme for improving functional properties of the egg white composition, wherein the functional properties are selected from foam height, foam density, foam volume, foam capacity, foaming power, and foam stability of the egg white composi- tion. In an embodiment of the present invention the functional property is foam stability.

The term "foam stability" according to the present invention is defined as the volume of drainage per 75 g of foam, where the volume of drainage is the volume of the liquid phase collected 70 minutes after whipping the egg white composition into foam. A person skilled in the art will readily recognise such a method for determining the foam stability. The present inventors have provided a method for measuring the foam stability, which method comprises the steps of

i. providing 450 ml of egg white composition;

ii. adjusting the temperature to 25°C;

iii. whipping the egg white composition for 90 s in 2nd gear and then 90 s in 3rd gear using a Hobart mixer CIOO;

iv. putting 75 g of foam in a funnel; and

v. measuring the volume of drained liquid from the foam after 70 minutes.

The precise method of measuring foam stability is not paramount. Any method suitable for determining the foam stability may be used. The scope of the present invention may readily be determined by comparison of foam stability as long as the same method of determining foam stability is used for all samples compared. For example the foam stability may be defined as

FS = (volume of initial liquid phase - volume of drainage) / (volume of initial liquid phase) x 100, where the volume of the initial liquid phase is the volume of the egg white composition before whipping the egg white composition into foam, and the volume of drainage is the volume of the liquid phase of the egg white composition collected 1 hour after whipping the egg white composition into foam.

By the term "foam height" of an egg white composition is meant measuring the foam height following the steps of whipping an egg white composition for a specific amount of time under specific conditions with respect to whipping time and whipping power; adding a predefined amount of the whipped egg white composition to a container with means for measuring the height of the foam; levelling the foam; and noting the height of the foam. Specific conditions with respect to whipping time and whipping power may be according to the examples of the present appli- cation.

The term "foam volume" of the egg white composition may be defi ned accordi ng to the pri nci ple of determ in ing the foa m heig ht, wherein the cross section of the container is noted and the volume calculated according the standard geometry.

In an embodiment of the present invention the use of the phos- pholipase enzyme is to improve the functional property of foam density, and the use results in a foam density below about 0.1 g/ml, preferably below about 0.08 g/ml, where the egg white composition has an egg yolk contamination of from about 0.001% to about 1% (w/w). By the term "foam density" of the egg white composition is meant determining the foam volume according to the principle of determining the foam volume, and calculating the density by the following equation : Foam density = (weight of foam) / (volume of foam), where both the weight of foam and the volume of foam are measured at the same time. A person skilled in the art will readily recognise such a method for determining the foam density. The present inventors have provided a method for measuring the foam density, which method comprises the steps of

i. providing 450 ml of egg white composition;

ii. adjusting the temperature to 25°C;

iii. whipping the egg white composition for 90 s in 2nd gear and then 90 s in 3rd gear using a Hobart mixer CIOO;

iv. putting 200 ml of foam in a measuring cup;

v. measuring the weight of the foam in the measuring cup; and vi. dividing the weight by 200.

By the term "foam capacity" of the egg white composition is meant the amount of interfacial area that can be created by the foam . Foam capacity may be calculated by the following equation :

Foam capacity = (volume of foam) / (volume of initial liquid phase) x 100, where the volume of foam is the final foam volume determined after whipping, and the volume of the initial liquid phase is the volume of egg whites in ml prior to whipping.

By the term "foaming power" of the egg white composition is meant

Foaming power = (volume of gas incorporated) / (volume of liquid) x 100, where the volume of gas incorporated during whipping is the volume of the egg whites after whipping minus the volume of egg whites before whipping. The volume of liquid is the volume of egg whites in ml before whipping.

It is true for all of the above-defined functional properties that the precise methods for measuring are not of high importance. Any method suitable for determining each of the functional properties may be applied. The scope of the present invention may readily be determined by comparison of the functional properties as long as the same method is used for a reference sample. A person skilled in the art will readily recognise the appropriate methods for any such comparative experiments.

According to the present invention by the term "improving" the functional properties is meant obtaining an egg white composition hav- ing functional properties most suitable for use in food products. This implies increased foam height, reduced foam density, increased foam volume, increased foam capacity, increased foaming power, and increased foam stability.

By the term "use" is meant facilitating the reaction, which oc- curs between the phospholipase enzyme and substrate, i .e. lipids, in particular phospholipids, which results in the hydrolysis of the phospholipids. Therefore, the use implies bringing the phospholipase enzyme into proximity of an egg white composition whereby the enzyme is able to catalyse the reaction in accordance with the common general principles of enzymology. Accordingly, by the term "use of a phospholipase enzyme" is to be understood as the steps of adding the phospholipase enzyme to an egg white composition, sufficiently mixing the phospholipase enzyme with the egg white composition in order to bring it into contact with the phospholipids of the egg white composition, providing suitable reaction conditions, and allowing a period of time for the phospholipase enzyme to hydrolyse the phospholipids in the egg white composition to a sufficient extent. A person skilled in the art will know how to ascertain suitable reaction conditions as well as to establish the period of time needed for the reaction in order to arrive at an egg white composition, wherein the phospholipids have been hydroiysed to a sufficient extent for the purpose in question.

It follows that another aspect of the present invention relates to a process for providing an egg white composition having improved func- tional properties, wherein the process comprises the steps of: (i) providing an egg white composition comprising more than 80% (w/w) of egg white, preferably more than 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white; (ii) adding a phospholipase enzyme to the egg white composition; and (iii) allowing the phospholipase enzyme to react with the egg white composition. In an embodiment of the process of the present invention from about 10 units to about 10 ⁶ units of phospholipase enzyme to 1 kg of egg white composition is added in step (ii), preferably from about 100 units to about 10 ⁵ units of phospholipase enzyme to 1 kg of egg white composition. In another embodiment of the process according to the present invention, the step iii) of allowing the phospholipase enzyme to react with the egg white composition further comprises providing suitable conditions, such as allowing sufficient reaction time, adjusting and maintaining the temperature, and adjusting and maintain- ing pH for the phospholipase enzyme to react with the egg white composition. In an embodiment, providing suitable conditions includes allowing for a reaction time from about 10 s to about 72 hours, adjusting and maintaining the temperature from about 0°C to about 60°C, and adjusting and maintaining pH from about 1 to about 12. In one embodiment the pH is from about 7 to about 9, the reaction time is from about 3 to 6 hours at a temperature of about 40°C to about 60°C. In another embodiment the pH is from about 7 to about 9, the reaction time is from about 20 to 30 hours at a temperature of about 3°C to about 6°C. A person skilled in the art will know that conditions for enzymatic reactions can be adjusted to the required performance. For example the amount of phospholipase enzyme added to the egg white composition may be increased in order to reduce the reaction time and/or the reaction temperature. A person skilled in the art will equally recognise that optional additives to the egg white composition may reduce or increase the per- formance of the enzymes.

The egg white composition may be further processed such as into powder. Several methods may exist for making the egg white composition into powder. In one embodiment the process of turning albumen into powder involves several steps. After breaking and separating the albumen from the yolk the albumen is cleaned efficiently by a clarifier to remove eggshell fragments from the liquid.

The raw product is accumulated in a tank and adjusted to approx. pH 7. In order to increase the dry matter contents of the albu- men two different principles may be applied - ultra filtration (UF) or reverse osmosis (RO). The concentration of the albumen is performed continuously in an Ultra-filtration plant (UF) or reverse osmosis plant (RF). Prior to concentration the albumen is heated up to fermentation temperature to increase the efficiency of the membranes. The energy con- sumption for drying will be reduced by 50% per batch and the powder output will be 50 % higher per hour. The liquid albumen is fermented in order to remove glucose. The glucose must be removed as it may react with the amino acids during the spray drying process causing a so-called "Mailliard" reaction, resulting in an undesirable brownish colour of the egg white powder. Fermentation can be done by applying yeast, enzymes or bacteria. After fermentation the albumen is cooled and stored and ready for spray drying. To avoid protein denaturation, the spray drying is performed at a low temperature and under as gentle conditions as physically possible and then packed into cardboard boxes. The last step is dry pasteurisation, which is traditionally performed in a hot room where the powder is kept for 2-4 weeks at 65-70°C in order to decimate the number of bacteria for use in food as well as non-food products and to improve the gel strength.

Another aspect of the present invention relates to an egg white product comprising at least an egg white composition having improved functional properties, where the egg white product is obtainable by a process comprising the steps of:

i. providing an egg white composition comprising more than 80% (w/w) of egg white, preferably more tha n 90% (w/w) of egg white, more preferably more than 95% (w/w) of egg white, even more preferably more than 97% (w/w) of egg white;

ii. adding a phospholipase enzyme to the egg white composition; and

iii. allowing the phospholipase enzyme to react with the egg white composition;

iv. whipping the egg white composition of step iii); and

v. optionally adding additional ingredients to the egg white composition;

wherein the egg white composition has a foam stability of at least 1.5 times as high as the egg white composition provided in step i) without adding the phospholipase enzyme in step ii), where the egg white composition has an egg yolk contamination of from about 0.01% to about 2.5% (w/w), preferably from about 0.01% to about 1% (w/w), and wherein the foam stability is defined as the volume of drainage per 75 g of foam, where the volume of drainage is the volume of the liquid phase collected 70 minutes after whipping the egg white composition into foam.

The present inventors find that initially adding phospholipase enzyme to the egg white composition prior to further mixing with additional ingredients of an egg white food product may be most convenient. However, the present inventors also envisage that some or all additional ingredients of an egg white food product may be added prior to and/or simultaneous to adding the phospholipase enzyme. A person skilled in the art will readily be able to ascertain the most suitable procedure according to the egg white food product in question. Therefore, the egg white composition comprised in the above-mentioned egg white product may be provided by carrying out the steps i) to v) in any suitable order.

By the terms "egg white product" and "egg white food product" are meant a food product which comprises an amount of an egg white composition, wherein the egg white composition is aerated into foam by e.g. whipping and/or beating, and wherein said food product may further comprise additional ingredients which may or may not have been added prior to whipping and/or beating. Examples of such food products are desserts, confectionaries, cakes, marshmallows, nougat, ice cream topping, and meringues.

Another aspect of the present invention relates to a kit for improving functional properties of an egg white composition comprising a phospholipase and instructions for carrying out the process according to the above-mentioned aspect of the present invention. In a preferred embodiment the phospholipase enzyme in the kit is phospholipase A2 enzyme. In an even more preferred embodiment the phospholipase A2 enzyme originates from a microbial source. In a yet more preferred em- bodiment the microbial source is selected from any of the strains Strep- tomyces violaceoruber or Aspergillus niger, preferably Aspergillus niger. In an embodiment of the present invention phospholipase A2 enzyme (IUB 3.1.1.4) manufactured by Nagase ChemteX Corporation under the trade name PLA2 NAGASE is provided. In another embodiment the phospholipase A2 enzyme is CakeZyme® Smart manufactured by DSM Food Specialties B.V. In an even more preferred embodiment the kit the phospholipase A2 enzyme is a phosphatide-2-acyl-hydrolase, preferably phosphatide-2-acyl-hydrolase (E.C.3.1.1.4), which is also known under the trade name MAXAPAL® A2 provided by DSM Food Specialties B.V. The phospholipase enzyme may be provided with the kit in any suitable form such as in liquid or powder form. Furthermore, the phospholipase may be provided in any suitable container.

Examples

The following examples of embodiments of the invention should not be construed as limiting the scope of the invention. A series of tests on egg white compositions were conducted. In particular, foam density was tested for egg white compositions having different level of yolk contamination. The tolerance of the egg white composition against over- whipping was tested, and the foam stability over time was tested. In all the examples eggs originating from hens and/or chickens were used.

Example 1

Enzyme modification of egg white compositions For these tests was used an enzyme dosage of 2500-5000 units of enzyme/litre of egg white. The enzyme treatment was performed at a temperature of 4-5°C for 24-48 hours or at a temperature of 45-50°C for 4-8 Hours.

Method for testing foam density

Whipping test was performed using a Hobart CIOO mixing machine equipped with a wire whip. The test was performed according to a standard whipping test consisting of the following steps:

i. providing 450 ml of egg white;

ii. adjusting to 25°C;

iii. whipping for 90 s in 2 gear and then 90 s in 3rd gear on the Hobart CIOO mixing machine;

iv. putting 200 ml of foam in a measuring cup;

v. measuring the weight of the foam in the measuring cup; and vi. dividing the measured weight by 200.

Method for testing tolerance against over-whipping

For testing the tolerance against over-whipping the method for testing foam density was continued with the following step:

vii. whipping the foam for an additional term of 90 s;

viii. putting 200 ml of foam in a measuring cup;

ix. measuring the weight of the foam in the measuring cup; and x. dividing the measured weight by 200.

Method for testing foam stability

Foam stability test was performed using a Hobart CIOO mixing machine equipped with a wire whip. The test was performed according to a standard whipping test consisting of the following steps:

i. providing 450 ml of egg white;

ii. adjusting to 25°C;

iii. whipping for 90 s in 2 gear and then 90 s in 3rd gear on the Hobart CIOO mixing machine;

iv. putting 75 g of foam in a funnel; and v. measuring the volume of d rained liq uid from the foa m after 70 minutes.

Example 2

Reference Tests

For a reference test pure and yolk free ha nd sepa rated egg white was measured for foa m density, foa m stability and tolerance against over- whipping, using the above test methods. The results are presented in Table 1.

Table 1 Results of reference tests

Example 3

Tests of volk-contaminated egg white compositions and comparison with reference values from hand separated egg yolk

The results of the reference tests as presented in Example 1 were compared to tests of foam density, tolerance against over-whipping and foam stability for different yolk-contaminated egg white compositions. Results of the yolk-contaminated egg white compositions are presented in Table 2 both with and without enzymatic treatment with phos- pholipase enzyme. Table 2 Results of tests of yolk-contaminated egg white compositions

Test 180 s DenTolerance Tolerance Foam stasity g/ml over- over- bility whipping whipping ml/70 min 270 s. 360 s g/ml

g/ml

Sample

Egg white + 0,22% 0.116 0.135 0.224 14 yolk, no enzyme

Egg white + 0,25% 0.185 0.257 0.351 20.0 yolk, no enzyme

Egg white + 0,27% 0.185 0.259 0.355 24.0 yolk, no enzyme

Egg white + 0,9% 0.220 0.302 0.438 40.0 yolk, No enzyme

Egg white + 1,0% 0.285 0.307 0.490 47.0 yolk, no enzyme

Egg white + 2,0% 0.294 0.448 0.582 51.5 yolk, no enzyme

Egg white + 3,0 % 0.393 0.525 0.685 60.5 yolk, no enzyme

Egg white + 0,22% 0.079 0.080 0.067 5.5 yolk + Maxapal A2

Egg White + 0,25% 0.074 0.070 0.067 5.0 Yolk + Maxapal A2

Egg White + 0,25% 0.072 0.070 0.068 7.0 Yolk+ PLA2

Egg White + 0,25% 0.072 0.069 0.068 11.0 Yolk + Lipase

Egg White + 0,25% 0.076 0.074 0.069 12.0 Yolk + Cakezyme

Egg white + 0,27% 0.076 0.071 0.069 5.5 yolk + Maxapal A2 Test 180 s DenTolerance Tolerance Foam stasity g/ml over- over- bility whipping whipping ml/70 min 270 s. 360 s g/ml

g/ml

Sample

Egg white + 0,9% 0.089 0.075 0.070 6.0 yolk + Maxapal A2

Egg white + 1,0% 0.096 0.082 0.076 8.0 yolk + Maxapal A2

Egg white + 2,0% 0.177 0.117 0.093 11.0 yolk + Maxapal A2

Egg white + 3,0 % 0.266 0.212 0.138 20.0 yolk + Maxapal A2

By comparing the results from the tests made on hand separated egg whites with the results from the hydrolysed yolk contaminated egg whites, it can be concluded that hydrolysis with phospholipase A2 (PLA2) can restore the foaming properties regarding foam density, tolerance against over whipping and foam stability. This with yolk contents up to 1% (w/w). It also improves the foaming properties of egg yolk contaminated with even higher contents of egg yolk. The results are presented in Figure 1 and Figure 2. Furthermore, the effect of enzymatic treatment with PLA2 is shown in Figure 3 and Figure 4.