METHOD FOR SEPARATION OF FAT AND PROTEIN IN EGG AND EGG PRODUCTS

RELATED APPLICATION

[0001] This application claims priority to US Provisional Application 63/322,436 filed on March 22, 2022, the content of which is incorporated herein by reference in its entirety for all purposes.

BACKGROUND

1. Field of the Invention

[0002] This disclosure relates to methods for separating protein and fat egg products. More particularly, the disclosure relates to a process for preparing a protein composition from egg products.

2. Description of Related Art

[0003] Eggs are a complex blend of several components. The most abundant components in eggs are proteins and fat.

[0004] The egg white, or albumen, is approximately 66% of the liquid weight of an egg. 90% of that weight is coming from water and the remaining weight of the egg white comes primarily from protein (10-15%), along with small quantities of fats (less than 0.4%), minerals, vitamins, and glucose. The most common proteins in egg white are Ovalbumen, Ovotransferrin, Ovomucoid, Globulins, Lysozyme, Ovomucin, and Avidin.

[0005] The egg yolk makes up approximately 33% of the liquid weight of an egg and is high in fat. As used herein, the term “fat” refers to lipids. The most prevalent lipids in egg yolk include: unsaturated fatty acids (Oleic acid, Linoleic acid, Palmitoleic acid, and Linolenic acid) and saturated fatty acids (Palmitic acid, Stearic acid, and Myristic acid). Egg yolks also contain some proteins, typically on the order of 10-20%.

[0006] Egg proteins and egg fats can be used for various applications, for example food and feed applications, either for their nutrition content or functional properties such as emulsifier (fats), gelling agent (protein), foaming agent (protein) etc. Due to their different uses, it is often desirable to separate egg fats and egg proteins from one another.

[0007] Various systems and methods have been developed for separation of eggs into yolks and whites. Separation of yolk and white from whole eggs can be done at high speeds under automated conditions, and can very effectively separate the yolks and eggs. [0008] However, a need exists for a means for further separation of egg components into more isolated components (such as proteins and lipids).

[0009] The proteins and fat in eggs together can act as an emulsifier. Whole broken eggs used in the industry are usually in a state of a stable mixture, which makes it very difficult to separate the egg proteins from the lipids.

[0010] A method is disclosed in US 11/971,802, in which lipids are separated from an egg mixture by using a cross-linking reagent. The cross-linking reagent, such as cyclobetadextran, silicon dioxide, colloidal silica material, fumed silica materials, and synthetic calcium silicate hydrates, is added to the egg mixture and the thus obtained crosslinked lipids can be separated from the proteins. However, for various reasons, the use of cross-linking agents is not desired.

[0011] Another method is disclosed in US 14/070, 120, in which egg fats and egg proteins are separated by using microfiltration.

[0012] A need exists for alternative and efficient methods and equipment for separating egg components into proteins and fats.

SUMMARY

[0013] The instrumentalities disclosed herein overcome the problems outlined above by providing a method for separating protein and fat in an egg product.

[0014] As used herein the term “protein” refers to organic compounds made of amino acids (polypeptides) and includes, but is not limited to, proteins such as Ovalbumen, Ovotransferrin, Ovomucoid, Globulins, Lysozyme, Ovomucin, and Avidin. As used herein, the term “fat” can be used interchangeably with “lipid” and refers to water-insoluble components such as fatty acids, steroids, such as cholesterol, glycolipids, lipoproteins and phospholipids, among others.

[0015] In certain embodiments, a process is disclosed for separating protein and fat in a starting material comprising eggs proteins and eggs fats, comprising

[0016] (a) adjusting the pH of starting material to a pH between 5 and 7,

[0017] (b) adding an effective amount of lipase to the starting material to form a mixture,

[0018] (c) incubating the mixture of step (b) at a temperature between 30°C and

60°C for a time period at least 10 min to obtain a digested mixture,

[0019] (d) subjecting the digested mixture to a separation step, forming at least two phases, said at least two phases comprising a protein phase and a lipid phase, and [0020] (e) collecting the protein phase and the lipid phase separately, thereby obtaining a protein composition and a lipid composition.

[0021] The starting material is prepared or derived from eggs. The term “egg” refers to any bird egg, preferably poultry eggs. In one aspect, the starting material is a liquid mixture. In another aspect, the starting material contains egg proteins and egg fats.

[0022] In one embodiment, the starting material is substantially free of cross-linking reagent, such as cyclobetadextran, silicon dioxide, colloidal silica material, fumed silica materials, and synthetic calcium silicate hydrates. Indeed, the present invention does not require any crosslinking reagent.

[0023] The starting material contains egg products. In certain embodiments, the starting material consists of egg products. Advantageously, the egg products can be a mixture of egg white (albumen) and egg yolk. In certain embodiments, the egg products can be egg white only or egg yolk only, even if these embodiments are not the preferred ones in the present invention.

[0024] In one embodiment, the starting material does not contain any (added) water. In another embodiment, the starting material contains water. For example, the starting material contains from 20% to 95%, or from 50% to 90% water by total weight.

[0025] In certain embodiments, the starting material is a stable mixture of egg proteins and egg fats, or an emulsion of egg proteins and egg fats. In a stable mixture, such as an emulsion, it is difficult to separate the proteins and fats by using mechanical separation only. In certain embodiment, the starting material is an aqueous mixture of egg proteins and egg fats.

[0026] Variations in the white and yolk (if both are present), and in the protein and fat contents occur depending upon the starting material and the egg source: sources that are high in egg whites, such as from spinning of egg shells, will be high in protein; while those that have more yolk based material, such as whole eggs from hatcheries or from breakers, will have relatively higher fat levels and relatively lower protein levels.

[0027] In one embodiment, the starting material is prepared from eggs graded as not suitable for direct human consumption. For purpose of this disclosure, the phrase “not suitable for direct human consumption” as applied to eggs, means the eggs are not to be consumed by human without some additional biochemical processing, for example, those eggs cannot by boiled and served directly to human or those eggs cannot be cracked open and added directly into a cooking or baking process.

[0028] In one embodiment, the starting material is prepared from unhatched eggs that have been subject to a hatching process or from liquid egg from an egg-breaking plant. [0029] As above explained, the starting material can contain egg yolk, alone or in mixture with egg white. The starting material may contain more than 0.2%, or more than 0.3%, or more than 0.5%, or more than 1%, or more than 2%, or more than 5%, or more than 10%, or more than 20%, or more than 30% or more than 40% egg yolk by weight of total solids.

[0030] The starting material may contain less than 70%, or less than 60%, or less than 55%, or less than 50%, or less than 45% egg yolk by weight of total solids.

[0031] As above explained, the starting material can contain egg white, alone or in mixture with egg yolk. The starting material may contain more than 45%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90% egg white by weight of total solids.

[0032] The starting material may contain less than 99.5%, or less than 99%, or less 98%, or less than 97%, or less than 96%, or less than 95%, or less than 90%, or less than 80%, or less than 70% egg white by weight of total solids.

[0033] The starting material may contain more than 40%, or more than 45% proteins (% based on total solids).

[0034] The starting material may contain less than 85%, or less than 80% proteins (% based on total solids).

[0035] The starting material may contain 40% to 85%, or 45% to 80% proteins (% based on total solids).

[0036] The starting material may contain more than 8%, or more than 10%, or more than 15% fats (% based on total solids).

[0037] The starting material may contain less than 40%, or less than 35% fats (% based on total solids).

[0038] In certain embodiments, the pH of the starting material is measured prior to step (a) above. The pH of the starting material may have a pH between 7 and 9, or between 7 and 8. In step (a), the starting material is adjusted to a pH between 5 and 7, or between 5 and 6.5, or between 6 and 6.5, or about 6 or about 6.5. Typically, egg whites have a pH that is slightly basic, between 7.5 and 9, while egg yolk usually is more acidic, having a pH between 6-7. It is generally very difficult to separate the fats and proteins, for example in mixtures of egg white and yolk which tend to form a very stable mixture. It was surprisingly discovered that when lipases are used at this pH, the resultant digested lipid components are easier to separate from the protein fraction (even if this is not the recommended optimal pH of the lipase). [0039] This pH adjustment can be performed by adding one or more pH adjusting agent. In certain embodiments, food grade pH adjusting agent is used. Examples of pH adjusting agent may include but are not limited to citric acid, acetic acid, phosphoric acid, potassium carbonate, potassium citrate.

[0040] Step (a) can be performed before and/or after step (b). In one embodiment, step (a) above is performed before step (b), or in other words, the pH of the mixture is adjusted to a pH between 5 and 7, or between 5 and 6.5, or between 6 and 6.5, or around 6.5 by adding one or more pH adjusting agent, and then the lipase is added to the starting material to form a mixture. In another embodiment, step (b) above is performed before step (a), or in other words, the lipase is added to the starting material to form a mixture and then the pH of the mixture is adjusted to a pH between 5 and 7, or between 5 and 6.5, or between 6 and 6.5, or around 6.5 by adding one or more pH adjusting agent, followed by step (c) above. In another embodiment, step (a) above is performed before and after step (b), or in other words, and the pH of the mixture is adjusted to a pH between 5 and 7, the lipase is added to the starting material to form a mixture, and the pH is further adjusted to a pH between 5 and 7.

[0041] Any lipase may be used in the process and may be derived from a microbial source such as Mucor sp., Rhizopus oryzae, Candida cylindracea, Penicillium sp. Or from animal origin such as pancreatic lipase from porcine pancreas. Preferably, the lipase is a microbial lipase, such as a microbial lipase isolated from fungi. In one embodiment, the lipase is derived from a microbial selected from Mucor javanicus, Rhizopus oryzae, Candida cylindracea, Aspergillus niger, Aspergillus oryzae, and preferably from Mucor javanicus, Rhizopus oryzae, Candida cylindracea. In one embodiment, the term “lipase” encompasses phospholipase. In another preferred embodiment, the term “lipase” does not encompass phospholipase. In one embodiment, the lipase may be an alkaline lipase, or in other words a lipase with an optimal lipolyzing activity at a pH of 7 or higher. In an advantageous embodiment, the lipase is an alkaline lipase (having an optimal pH at 7 or higher as recommended by the manufacturer) but is used in the instant invention at a pH between 5 and 6.5, or between 6 and 6.5, or about 6 or about 6.5.

[0042] In certain embodiments, only one lipase is used. In certain embodiments, no co-lipase is used.

[0043] In certain embodiments, the amount of lipase used in step (b) is 0.01% to 0.3%, or 0.05% to 0.3%, or 0.06% to 0.3%, or 0.1% to 0.3% by weight of the total starting material. In one aspect, the amount of lipase used in step (b) is higher than 0.01%, or 0.05%, or 0.06% by weight of the total starting material. [0044] In some other embodiments, the starting material is desugared before or after step (b), such as by a fermentation process or by an enzymatic reaction with an enzyme as glucose oxidase. In certain embodiments, the starting material is not desugared before or after step (b).

[0045] The temperature of the incubation step (c) is not usually above 60°C because of the temperature denaturation of the enzymes, and is generally from 30°C to 60°C, preferably from 35°C to 50°C, preferably from 35°C to 45°C, preferably from 37°C to 45°C.

[0046] The duration of the incubation step may vary widely depending, for example, on the particular lipase used, the concentration of the lipase and the temperature. In certain embodiments, the incubation time in step (b) is at least 10 min. In certain embodiments, the incubation time in step (b) is 10-60 min, or 10-120 min, or longer.

[0047] In one aspect, the pH and/or temperature of the material are monitored during step (c). In one embodiment, the pH is further adjusted during step (c). In one embodiment, no pH adjustment is performed during step (c), or between step (c) and step (d).

[0048] In some embodiment, the separation in step (d) may be achieved by centrifugation. This can be performed with any appropriate equipment, such as a centrifuge and/or decanter. A centrifugal force between 5000 and 10,000G could be used. The temperature of step (d) of separation can be between 1°C and 60°C. However, while literature (as Stoke Law) and experts’ recommend increasing the temperature for a better separation, it was surprisingly discovered that lower temperatures could advantageously enhance fat and protein separation. Therefore, a temperature of step (d) between 1°C and 40°C, or between 5°C and 25°C, or between 5°C and 10°C is preferred.

[0049] The digested mixture forms at least two phases after the separation step (d): one protein phase, containing at least protein, and one lipid phase (or fat phase), containing at least fat. In certain embodiments, the digested mixture forms three phases after the separation step (d): one protein phase and two lipid phases, one with low density fat and one with high density fat.

[0050] Step (e) consists of collecting the protein phase and the lipid phase(s) separately, thereby separating egg proteins and egg fats by obtaining a protein composition and a lipid composition.

[0051] In a preferred embodiment, the protein composition contains proteins that are derived from the egg yolk and the egg white. In another embodiment, the protein composition contains proteins that are derived from the egg yolk only, in particular when the egg products in the starting material consists of egg yolks. In another embodiment, the protein composition contains proteins that are derived from the egg white only, in particular when the egg products in the starting material consists of egg white.

[0052] In certain embodiments, the protein composition obtained in step (e) comprises less than 20%, or less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 1%, or less than 0.5% of fat by weight of total solids.

[0053] In certain embodiments, the protein composition obtained in step (e) comprises about 50% to 99%, or about 55% to 95%, or about 55% to 90%, or about 70% to about 90%, or more than 70%, more than 80%, or more than 90% of protein by weight of total solids.

[0054] The fat phase may be collected for other commercial applications different from the protein phase. In one embodiment, the lipid composition contains fat that are derived from the egg yolk and the egg white. In another embodiment, the lipid composition contains fats that are derived from the egg yolk only, in particular when the egg products in the starting material consists of egg yolks. In another embodiment, the lipid composition contains fats that are derived from the egg white only, in particular when the egg products in the starting material consists of egg white. In one embodiment, the lipid composition can be obtained from one lipid phase. In another embodiment, the lipid composition can be obtained from two lipid phases.

[0055] In certain embodiments, the fat (lipid) phase obtained in step (e) comprises about 40% to 99%, or about 45% to 90%, or more than 70%, more than 80%, or more than 90% of fat by weight of total solids.

[0056] In certain embodiment, the fat (lipid) phase obtained in step (e) comprises less than 45% of protein by weight of total solids.

[0057] In certain embodiments, the fat (lipid) phase obtained in step (e) comprises less than 5%, or less than 1% egg impurities by weight of total solids.

[0058] The present invention also concerns a lipid composition obtained by the process of separation.

[0059] The present invention also concerns a protein composition obtained by the process of separation. This protein composition has excellent binding properties. This protein composition is a liquid protein composition. The protein composition can be further concentrated, for example by evaporation, by reverse osmosis (RO) and/or nanofiltration. In one embodiment, the protein solution is concentrated so as to contain approximately 15%-35% by weight. The protein composition can be further dried. The protein composition can be further submitted to a heat treatment to improve gel functionality. [0060] Therefore, a further aspect of the present invention concerns a process for preparing a protein composition from a starting material comprising eggs proteins and eggs fats, comprising:

[0061] (a) adjusting the pH of starting material to a pH between 5 and 7,

[0062] (b) adding an effective amount of lipase to the starting material to form a mixture,

[0063] (c) incubating the mixture of step (b) at a temperature between 30°C and

60°C for a time period at least 10 min to obtain a digested mixture,

[0064] (d) subjecting the digested mixture to a separation step, forming at least two phases, said at least two phases comprising a protein phase and a lipid phase,

[0065] (e) collecting the protein phase, thereby obtaining a protein composition,

[0066] (f) optionally concentrating the protein phase, thereby providing an concentrated protein composition,

[0067] (g) optionally, drying said high protein composition, thereby providing a dry protein composition.

[0068] (h) optionally, submitting the dry protein composition to a heat treatment, thereby obtaining a heat-treated dry protein composition.

[0069] The above-mentioned technical features recited for the process for separating protein and fat also apply to the process for preparing a protein composition.

[0070] In certain embodiments, step (f) of concentrating is performed by filtration, more particularly by evaporation and/or reverse osmosis (RO) and/or nanofiltration. The protein composition can be deashed during this step (f).

[0071] In one embodiment, the protein composition obtained in step (e) or (f) is dried, preferably by spray drying.

[0072] In one embodiment, the protein composition obtained in step (e) or (f) is dried, preferably by spray drying, and is further submitted to a heat treatment, thereby obtaining a heat-treated dry protein composition. For example, the dry protein composition can be placed in a hot humid room with a temperature from 60°C to 80°C and humidity of 30% to 40% for at least 3 days. Advantageously, proteins are denatured, and gel strength is increased. In one embodiment, it has a gel strength of a least 100, or between 100 and 1500.

[0073] The invention is further directed to the protein composition obtained by this process, and/or the concentrated protein composition obtained in step (f), and/or the dry protein composition obtained in step (g), and/or the heat-treated dry protein composition obtained in step (h).

[0074] In one embodiment, the dry protein composition or the heat-treated dry composition comprises at least about 70%, or at least 80% of protein by dry weight. In one embodiment, the dry protein composition comprises less than about 20%, or less than 10% of fat by dry weight.

[0075] The protein composition obtained, in any form, may be used in a food, such as a human food or animal food. The protein composition may be used to enhance feeds for nutritional purpose, or functional purpose (for example, for binding), or both.

[0076] A further aspect of the present invention thus concerns a food comprising the protein composition obtained by a process herein disclosed. The food can be a human food or an animal food.

[0077] In one embodiment, the food is an animal food. The term “animal” may include but is not limited to cats, dogs, shrimp, pigs, fish. The animal feed can be of any form, including but not limited to liquid, wet form, canned, or dry form. In one aspect, dry forms can include but are not limited to pellet, kibble, treats and chunks. In some embodiments, the animal food comprises at least 0.1%, or at least 0.5%, or at least 1%, or at least 2% by weight of a protein composition disclosed herein, such as the dry protein composition.

DETAILED DESCRIPTION

[0078] This disclosure provides a process for preparing high protein composition from egg products. More particularly, the disclosure relates to methods for separating protein and fat in egg and egg products.

[0079] By way of example, several embodiments of the disclosed processes are described below:

[0080] Item 1. A process for separating protein and fat in a starting material comprising eggs proteins and eggs fats, comprising

[0081] (a) adjusting the pH of starting material to a pH between 5 and 7,

[0082] (b) adding an effective amount of lipase to starting material to form a mixture,

[0083] (c) incubating the mixture of step (b) at a temperature between 30°C and

60°C for a time period at least 10 min to obtain a digested mixture, [0084] (d) subjecting the digested mixture to a separation process, forming at least two phases, said at least two phases comprising a protein phase and a lipid phase, and

[0085] (e) collecting the protein phase and the lipid phase separately, thereby obtaining a protein composition and a lipid composition.

[0086] Item 2. The process of Item 1, wherein said starting material comprises egg white and egg yolk, and wherein the egg yolk in the starting material constitutes more than 0.2% by weight of total solids in the starting material.

[0087] Item 3. The process of Item 1 or 2, wherein the starting material is prepared from eggs graded as not suitable for direct human consumption.

[0088] Item 4. The process according to any one of Items 1 to 3, wherein the starting material is prepared from unhatched eggs that have been subject to a hatching process or from liquid egg from an egg-breaking plant.

[0089] Item 5. The process according to any one of Items 1 to 4, wherein said starting material prior to step (a) comprises more than 1% egg yolk by weight of total solids.

[0090] Item 6. The process according to any one of Items 1 to 5, wherein food grade agent is used for adjusting the pH in step (a).

[0091] Item 7. The process according to any one of Items 1 to 6, wherein the amount of lipase used in step (b) is 0.01% to 0.3% by weight of total starting material.

[0092] Item 8. The process according to any one of Items 1 to 7, wherein the starting material is not subject to a fermentation process before or after the lipase is added.

[0093] Item 9. The process according to any one of Items 1 to 8, wherein the protein phase obtained in step (e) comprises less than 20% of fat by weight of total solids.

[0094] Item 10. The process according to any one of Items 1 to 9, wherein the pH is adjusted to between 5 and 6.5 in step (a).

[0095] Item 11. A protein composition obtained according to the process of any one of Items 1 to 10.

[0096] Item 12. A lipid composition obtained according to the process of any one of Items 1 to 10.

[0097] Item 13. An animal food comprising the protein composition of Item 11.

[0098] Item 14. A process for preparing a protein composition from a starting material comprising eggs proteins and eggs fats, comprising:

[0099] (a) adjusting the pH of starting material to a pH between 5 and 7,

[0100] (b) adding an effective amount of lipase to the starting material to form a mixture, [0101] (c) incubating the mixture of step (b) at a temperature between 30°C and

60°C for a time period at least 10 min to obtain a digested mixture,

[0102] (d) subjecting the digested mixture to a separation step, forming at least two phases, said at least two phases comprising a protein phase and a lipid phase,

[0103] (e) collecting the protein phase, thereby obtaining a protein composition,

[0104] (f) optionally concentrating the protein phase, thereby providing a concentrated protein composition,

[0105] (g) optionally, drying said high protein composition, thereby providing a dry protein composition.

[0106] (h) optionally, submitting the dry protein composition to a heat treatment, thereby obtaining a heat-treated dry protein composition.

[0107] Item 15. The process of Item 14, wherein said starting material comprises egg white and egg yolk, and wherein the starting material comprises more than 0.2% egg yolk by weight of total solids before it is subject to the process of step (a).

[0108] Item 16. A protein composition obtained according to the process of Item 14 or 15.

[0109] Item 17. A concentrated protein composition obtained according to the process of Item 14 or 15.

[0110] Item 18. A dry protein composition obtained according to the process of

Item 14 or 15.

[0111] Item 19. A heat-treated dry protein composition obtained according to the process of Item 14 or 15.

[0112] Item 20. An animal food comprising one or more of the following: the protein composition of Item 11 or 16, the concentrated protein composition of Item 17, the dry protein composition of Item 18, or the heat-treated dry protein composition of Item 19.

[0113] It is to be noted that, as used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a device" may include reference to one device, as well as two or more devices, unless the context clearly limits the reference to one device.

[0114] The terms “between” and “at least” as used herein are inclusive. For example, a range of “between 5 and 10” means any amount equal to or greater than 5 but equal to or smaller than 10. [0115] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, more preferably ±5%, even more preferably ±2% from the specified value, as such variations are appropriate to reproduce the disclosed methods and products. In a particular embodiment, the term “about” can be ignored so as the indicated values are exact values.

[0116] Unless otherwise specified, the percentage of certain component in a composition is by weight. Various commercially available products may have been described or used in this disclosure. It is to be recognized that these products are cited for purpose of illustration only. Certain physical and/or chemical properties and composition of the products may be modified without departing from the spirit of the present disclosure. One of ordinary skill in the art may appreciate that under certain circumstances, it may be more desirable or more convenient to alter the physical and/or chemical characteristics or composition of one or more of these products in order to achieve the same or similar objectives as taught by this disclosure.

Examples

[0117] The following examples are provided to illustrate the present invention but are not intended to be limiting. The reagents, materials and instruments are presented as typical components, and various substitutions or modifications may be made in view of the foregoing disclosure by one of skills in the art without departing from the principle and spirit of the present invention.

Example 1 pH for Digestion

[0118] The goal for these experiments is to separate fat and protein from whole liquid egg (a mix of egg yolk and whites), which is a very stable liquid mixture. The raw material was a mixture of whole egg which was approximately 20% egg whites and 10% yolk and 70% water by volume. This mixture contained about 44% of fats (% dry basis) and about 55% of proteins (% dry basis). All raw material was from whole eggs that are inedible for human. Several trials were performed with different enzymes, pH and temperatures. Four different enzymes: ENZECO® Esterase/Lipase (EDC, optimal recommended pH 7-8, enzymatic activity 425 FlP/g), ENZECO® LIPASE RO 2 (EDC, optimal recommended pH 5- 6, enzymatic activity 225-300 BGE/g), Lipomod 34P (BioCatalyst, optimal recommended pH 5-8, enzymatic activity 225 BGE/g), ENZECO® LIPASE RO Concentrate (EDC, optimal recommended pH 7-7.5, enzymatic activity 180 FlPU/mg) were tested. Briefly, the pH of the mix of egg yolk and whites was measured and when the pH was outside the range of 5-6.5, acetic acid (pH adjusting agent) was added to adjust the pH to 5-6.5. The enzyme was then added to the mix of egg yolk and whites and incubated at different temperatures and pH. After the digestion was done, the digested mixture was subject to centrifugation in a bench top centrifuge to separate the phases containing fat and protein. During these trials, 3 layers were obtained after centrifugation. The top phase contained mostly low-density fat (first fat phase), middle one containing water and protein (protein phase) and bottom one contained high-density lipid and egg impurities (second fat phase, named “pellet”).

[0119] In order to illustrate the determination of the optimal pH for the digestion, Table 1 shows the results when the pH of several batches of the starting materials is varied, and when ENZECO® Esterase/Lipase is added.

[0120] Fat extraction is calculated by (fat separated after centrifugation)/(fat in the initial raw material-egg) x 100%. A CEM Oracle NMR equipment was used to measure fat content. The protein (dry basis) in Table 1 is based on protein content in the middle phase. The protein was analyzed using a LECO combustion furnace nitrogen analyzer. The solids content was analyzed using a CEM Smart 6 microwave moisture analyzer.

[0121] Success in the trials was measured by how much fat and protein were separated.

Table 1 Comparison of protein/fat separation at different lipase digestion pH

[0122] As shown in Table 1, at lower pH, e.g., 6.5 or 6, the fat amount in fat phase and protein amount in protein phase were both substantially higher than those at pH 7 or 8.

[0123] Based on all trials, it was determined that the optimal pH for all enzymes was between 5-6.5, which is different from the optimal pH recommended by some of the suppliers. It was also determined that optimal temperature was 37-50°C, more particularly 37-45°C. Regarding dosage of the enzymes, the optimal usage rate was from 0.04 to 0.3% as calculated by weight of aqueous solution of whole egg. Example 2 Temperature during the phases separation

[0124] The goal for this experiment was to evaluate the impact of the temperature on the separation process (centrifugation). The raw material was a mixture of whole egg which was approximately 20% egg whites and 10% yolk and 70% water by volume. This mixture contained about 44% of fats (% dry basis) and about 55% of proteins (% dry basis). All the enzymatic digestion reactions were performed at the same temperature, pH and for the same amount of time. The only difference was the temperature at the separation step (in this case a bench top centrifuge was used).

[0125] The success was measured by the fat phase amount: the higher the fat phase content by weight, the better the separation.

Table 2

[0126] As shown in Table 2, the lower the temperature, the better the phase separation result. In this example at 10°C , there is 17% more separation than at 25°C and 25% more separation than at 40°C.

[0127] As mentioned above, the decrease in temperature goes against most of literature and expert’s recommendation (and from what is expected from Stoke Law). It is indeed common knowledge to increase temperature to improve separation of phases using mechanical separation.

Example 3 Statistical analysis of the disclosed methods using enzymes as compared to a control method without enzymes

[0128] The goal of this study was to compare the effectiveness of the method disclosed herein against a baseline method (processing the egg at the same conditions but without adding the enzyme). The goal was to determine whether the new method is significantly better than the baseline. To accomplish this, we conducted a Z-test.

[0129] For the disclosed method, 8 samples were tested at the optimized parameters (pH, reaction temperature, enzyme usage rate, separation temperature). The bottom phase (pellet) was measured after the separation and recorded as % of weight in. See the summary statistics below:

[0130] The baseline used was egg submitted to the same parameters (pH, reaction temperature, separation temperature), except for enzyme usage rate, which was 0. For this baseline test, the separation was 13.16%. Therefore, an optimistic value for the separation without the enzyme is 15%.

[0131] A Z-test was then performed at significance level of 0.001. With Z = 15.63, we reject the null hypothesis and conclude that the population mean is significantly greater than the specified value of 15% at a 0.1% significance level. This means that if no enzyme is used, the phase separation is below 15%. By contrast, when using the disclosed method, phase separation is above 15% with confidence level higher than 99.9%.