Non-animal based protein sources with functional properties

Abstract

To provide non-animal based protein sources with functional properties.SOLUTION: There are herein provided compositions with enhanced protein content, compositions with functional proteins, protein combinations and methods for the preparation thereof. In some embodiments, there are herein provided ingredients for producing egg-free food items. The ingredient composition for producing an egg-free food item may comprise a recombinant ovalbumin (rOVA), wherein the rOVA provides to the egg-free food item at least one egg white characteristic selected from gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 888,674, filed on August 19, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, the content of which is hereby incorporated by reference in its entirety. The ASCII copy created on August 18, 2020, is named 49160 - 717.601_ST25.txt and is 287,890 bytes in size.

Background Art

[0003] Background of the Invention Proteins are important food nutrients and food ingredients. They can serve as a fuel source or as a source of amino acids, including essential amino acids that cannot be synthesized by the body. For healthy adults, the recommended daily protein intake is 10% - 35% of a person's total calorie requirement, and currently, for most humans, the majority of protein intake is from animal - based sources. In addition, proteins are used in a wide variety of foods and food ingredients. In many cases, these proteins are supplied from animals. Due to the increase in the world's population and the simultaneous increase in global food demand, there is a need to provide alternative sustainable non - animal - based protein sources as useful protein sources for daily diets, food ingredients, and food products.

Summary of the Invention

Means for Solving the Problems

[0004] Gist of the Invention Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As realized, the present disclosure is capable of other different embodiments, and some details thereof can all be modified in various obvious matters without departing from the present disclosure. Therefore, the drawings and description should be regarded essentially as exemplary and not as restrictive.

[0005] In some embodiments, raw materials for manufacturing egg-free foods are provided herein. The raw material composition for manufacturing egg-free foods may include recombinant ovalbumin (rOVA), the pH of rOVA may be from about 3.5 to about 7.0, and when present in egg-free foods, rOVA is in an amount of about 2% to about 15% (w / w). rOVA provides at least one egg white property selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturizing, water retention, clarification, and aggregability to egg-free foods.

[0006] In some cases, the composition may be dried or may be in powder form. In some cases, the composition may contain at least 75% rOVA (w / w of total protein or w / w of total composition). In some cases, the powder composition may be a concentrate. In some cases, the powder composition may be an isolate. In some cases, the powder composition may be at least about 75%, at least about 80%, at least about 85% or at least about 90% rOVA (w / w). In some cases, the powder composition is at least about 80%, at least about 85% or at least about 90% rOVA (w / w). In some cases, the powder is a concentrate. In some cases, the powder composition is an isolate.

[0007] In some cases, the composition may be a liquid. In some cases, the liquid composition may contain at least 50% rOVA (w / w of total protein or w / w of the composition). In some cases, the liquid composition contains at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85% or at least about 90% rOVA (w / w). The term w / w of total protein in the context of rOVA% means that rOVA comprises a defined percentage of the total protein in the composition. In one example, a composition containing at least 50% rOVA w / w of total protein would have at least half rOVA, which is the total protein, and about half the other protein, which is another protein. Thus, the total composition does not necessarily have to be at least 50% rOVA by weight, only the protein content of the composition has to be at least 50% rOVA.

[0008] In some cases, rOVA provides equivalent properties or improved properties compared to native egg white in similar foods. In some cases, rOVA provides a foam volume that is at least 20%, 30%, 40% or 50% higher than native egg white. In some cases, rOVA provides a time to foam that can be at least 20%, 30%, 40% or 50% faster than native egg white. In some cases, when solubilized, the pH of rOVA is about 3.5 to about 4.5. In some cases, rOVA provides a hardness that can be higher than native egg white to egg-free food compositions. In some cases, rOVA provides a chewiness that can be higher than native egg white to egg-free food compositions. In some cases, rOVA provides an elasticity comparable to native egg white.

[0009] In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. In some cases, the amino acid sequence of rOVA lacks the N-terminal methionine. In some cases, rOVA further contains the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus.

[0010] In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 - 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation.

[0011] In some cases, when solubilized, the pH may be about 6 - about 6.8. In some cases, when solubilized, the pH of rOVA may be less than about 6.1. In some cases, rOVA may be present in an amount of less than about 8% in an egg-free food. In some cases, rOVA may be present in an amount of about 7% or less in an egg-free food.

[0012] In some embodiments, baked goods are provided herein. The baked good food product may include (i) recombinant ovalbumin (rOVA), wherein when solubilized, the pH of rOVA can be about 3.5 - about 7.0, (ii) at least one fat or oil, (iii) at least one cereal starch, and (iv) at least one sweetener. rOVA provides at least one egg white property selected from binding, elasticity, air incorporation, browning, structuring, water retention and cohesiveness to the baked good food product, and the baked good food product does not contain any natural egg white protein or natural egg white.

[0013] In some cases, rOVA may be present in the product at about 2% to 15% (w / w of the total protein before baking or w / w of the total food product). In some cases, rOVA is present in the product at about 2% to about 5% (w / w). In some cases, the baked good may contain a milk component or a leavening agent, or a combination thereof. In some cases, the product may be a cake, bread, roll, pastry, cracker, muffin, scone, biscuit or cookie. In some cases, the baked good product may have a crumb structure equivalent to or better than that of a similar baked good product made using natural egg white or natural whole egg. In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, turkey OVA or chicken OVA. In some cases, the percentage of weight loss is lower in the baked good product made using rOVA when compared to an equivalent baked good product made using whole egg.

[0014] In some embodiments, an emulsified product is provided herein. The emulsified product may comprise (i) recombinant ovalbumin (rOVA), (ii) at least one fat or oil, and (iii) water, and rOVA may be present in the product at about 2% to 15% (w / w). In some cases, the emulsified product may contain an acidifying agent. In some cases, the product may be a salad dressing, sauce, mayonnaise, sandwich spread or gravy.

[0015] In some embodiments, a food product is provided that comprises (i) recombinant ovalbumin (rOVA) which, when solubilized, may have a pH of about 3.5 to about 7.0, (ii) at least one sweetener, and (iii) optionally, an ingestible liquid, wherein rOVA may be present in the food product at about 2% to about 15% (w / w), and wherein rOVA provides the food product with foaming properties, whipping properties, fluffiness or air miscibility.

[0016] In some cases, rOVA may further provide gelation to the food product. In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 - 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation. In some cases, the food product may be a meringue, a whipped dessert, a whipped topping or a soufflé. In some cases, rOVA may provide a foam volume to the food product that is at least 20%, 30%, 40% or 50% higher than native egg white. In some cases, rOVA may provide a food product with a time to foam that is at least 20%, 30%, 40% or 50% faster than native egg white. In some cases, when solubilized, the pH of rOVA is about 3.5 - about 4.5.

[0017] In some cases, rOVA is present in the food product at about 5% - about 10% (w / w). In some cases, rOVA is present in the food product at about 7% - about 8% (w / w). In some cases, rOVA is present in the food product at about 4%, about 7% or about 12% (w / w). In some cases, when solubilized, the pH of rOVA is about 6. In some cases, rOVA is present in the food product at about 9% - about 10% (w / w). In some cases, when solubilized, the pH of rOVA is about 7. In some cases, the product may be a beverage. In some cases, the beverage may be an ingestible alcohol. In some cases, rOVA provides foaming, whipability, fluffiness or air miscibility to an ingestible alcoholic beverage. In some cases, the beverage is a coffee drink. In some cases, rOVA provides foaming, whipability, fluffiness or air miscibility to a coffee drink. In some cases, the coffee drink lacks a milk component.

[0018] In some cases, rOVA may comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may comprise the amino acid sequence of duck OVA, goose OVA or chicken OVA. In some cases, rOVA does not contaminate food products with Salmonella. In some cases, the food product is a protein bar, energy bar, nutrition bar or granola bar. In some cases, the food product contains about 4% to about 8% (w / w) of rOVA. In some cases, the bar is baked or unbaked.

[0019] In some embodiments, meat analogue food products are described herein. The meat analogue food products can include (i) recombinant ovalbumin (rOVA), (ii) at least one fat or oil, and (iii) a plant-derived protein, wherein rOVA can be present in the food product at about 2% to about 15% (w / w), and rOVA acts as a binder or gelling agent, or a combination thereof.

[0020] In some cases, the plant protein may be an extruded plant protein. In some cases, the plant protein may be a non-extruded plant protein. In some cases, the meat analogue food products may be selected from hamburgers, patties, sausages, hot dogs, sliced deli meats, jerky, bacon, nuggets, ground meat-like compositions and formed meat-like compositions. In some cases, rOVA may provide the food product with a hardness that can be higher than that of native egg white. In some cases, rOVA may provide the food product with a chewiness that can be higher than that of native egg white. In some cases, rOVA may provide an elasticity comparable to that of native egg white.

[0021] In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 - 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation. In some cases, rOVA is present in a food product at about 4%, about 5% or about 6% (w / w). In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, ostrich OVA or chicken OVA.

[0022] In some embodiments, an egg white substitute is provided herein. The egg white substitute can include (i) recombinant ovalbumin (rOVA), (ii) at least one fat or oil, and (iii) a polysaccharide or a polysaccharide-containing raw material. rOVA can be present in the composition at about 2% - 15% (ww). The composition can have one or more properties selected from hardness, adhesiveness, fracturability, cohesiveness, tackiness and chewiness. When the egg white substitute can be cooked, the one or more properties are equivalent to or improved compared to natural egg white.

[0023] In some cases, the egg white substitute may further include a flavoring agent or a coloring agent, or a combination thereof. In some cases, the polysaccharide or the polysaccharide-containing raw material may be starch. In some cases, the polysaccharide or the polysaccharide-containing raw material may be selected from gellan gum, sodium alginate and psyllium, or any combination thereof. In some cases, rOVA may provide a food product with a hardness that can be higher than that of native egg white.

[0024] In some cases, rOVA may provide a more satisfying chewiness to food products than native egg white. In some cases, rOVA may provide the same degree of adhesiveness and / or elasticity as native egg white. In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 - 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation. In some cases, rOVA is present in the food product at about 10% - about 12% (w / w).

[0025] In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, ostrich OVA or chicken OVA.

[0026] In some embodiments, a powdered raw material composition is described herein. The powdered raw material composition may contain recombinant ovalbumin (rOVA). When solubilized, the pH of rOVA may be about 3.5 - about 7.0. rOVA may be at least 75% w / w of the composition. rOVA may contain one or more N-linked glycosylation sites having mannose linked to N-acetylglucosamine, and the N-linked glycosylation sites lack galactose. In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. In some cases, the amino acid sequence of rOVA lacks an N-terminal methionine. In some cases, rOVA further contains an EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. In some cases, the composition contains at least about 80%, at least about 85% or at least about 90% rOVA (w / w).

[0027] In some embodiments, the liquid composition may contain recombinant ovalbumin (rOVA), and the composition may contain at least 50% rOVA (w / w of total protein or w / w of total composition). In some cases, the composition may contain at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85% or at least about 90% rOVA (w / w).

[0028] In some cases, rOVA may contain the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may contain the amino acid sequence of duck OVA, turkey OVA or chicken OVA.

[0029] In some cases, the amino acid sequence of rOVA lacks the N-terminal methionine. In some cases, rOVA further contains the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. In some cases, the pH of solubilized rOVA may be from about 3.5 to about 7.0. In some cases, the pH of solubilized rOVA may be from about 6 to about 6.8. In some cases, the pH of solubilized rOVA may be less than about 6.1.

[0030] In some cases, rOVA may provide at least one egg white property selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturization, water retention, clarification and aggregability to egg-free foods. In some cases, rOVA may provide equivalent properties or improved properties compared to native egg white in similar egg-free foods. In some cases, rOVA may provide a foam volume that is at least 20%, 30%, 40% or 50% higher than native egg white to egg-free foods.

[0031] In some cases, rOVA may provide a time to foaming that is at least 20%, 30%, 40% or 50% faster than native egg white in egg-free foods. In some cases, rOVA may provide a hardness that is higher than native egg white in egg-free foods. In some cases, when solubilized, the pH of rOVA is from about 3.5 to about 4.5. In some cases, rOVA is present in egg-free foods at about 5% to about 10% (w / w). In some cases, rOVA is present in egg-free foods at about 7% to about 8% (w / w). In some cases, rOVA is present in egg-free foods at about 4%, about 7% or about 12% (w / w). In some cases, when solubilized, the pH of rOVA is about 6. In some cases, rOVA may provide a chewiness that is higher than native egg white in egg-free foods. In some cases, rOVA may provide an elasticity comparable to native egg white in egg-free foods.

[0032] In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation. In some cases, rOVA does not contaminate egg-free foods with Salmonella.

[0033] In some embodiments, a dry or powdered composition comprising recombinant ovalbumin (rOVA), wherein the composition may comprise at least 50% rOVA (w / w of total protein or w / w of total composition), is described herein. In some cases, the composition may comprise at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% rOVA (w / w). In some cases, rOVA may comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity to SEQ ID NO: 2 or SEQ ID NO: 1.

[0034] In some cases, rOVA may comprise the amino acid sequence of duck OVA, turkey OVA or chicken OVA. In some cases, the amino acid sequence of rOVA lacks an N-terminal methionine. In some cases, rOVA further comprises the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. In some cases, rOVA provides at least one egg white property selected from gelling, foaming, whipping, fluffiness, binding, elasticity, air incorporation, coating, film formation, emulsification, browning, thickening, texturizing, water retention, clarification and aggregation to an egg-free food. In some cases, rOVA may provide equivalent properties or improved properties compared to native egg white in a similar egg-free food.

[0035] In some cases, rOVA may provide a foam volume that is at least 20%, 30%, 40% or 50% higher than that of native egg white to an egg-free food. In some cases, rOVA may provide a time to foam that can be at least 20%, 30%, 40% or 50% faster than that of native egg white to an egg-free food. In some cases, when solubilized, the pH of rOVA is about 3.5 to about 4.5. In some cases, rOVA is present in an egg-free food at about 4%, about 7% or about 12% (w / w). In some cases, when solubilized, the pH of rOVA is about 6.

[0036] In some cases, rOVA may provide a hardness that can be higher than that of native egg white to egg-free foods. In some cases, rOVA may provide a chewiness that can be higher than that of native egg white to egg-free foods. In some cases, rOVA may provide an elasticity comparable to that of native egg white to egg-free foods. In some cases, when rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 - 7.0 when solubilized, rOVA provides improved gelation. In some cases, when rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, rOVA provides improved gelation.

[0037] In some embodiments, a method of making a food product is provided herein. The method of making a food product can include the steps of: (i) providing recombinant ovalbumin (rOVA) that has a pH of about 3.5 to about 7.0 when solubilized; and (ii) combining the rOVA in an amount of 2% - 15% (w / w) with one or more edible ingredients to form a food product, wherein the rOVA can provide at least one egg white property selected from gelling, foaming, whipping, fluffiness, binding, elasticity, air incorporation, coating, film formation, emulsification, browning, thickening, texturizing, water retention, clarification, and agglomeration to the food product.

[0038] In some embodiments, a method of making an ingredient is provided herein. The method of manufacturing an ingredient composition can include the steps of: (i) expressing recombinant ovalbumin (rOVA) in microbial cells, wherein the rOVA can be secreted by the microbial cells into a liquid medium; (ii) recovering the liquid medium containing the secreted rOVA; (iii) performing a separation step at a pH of about 3.5; (iv) solubilizing the rOVA at a pH of about 12; and (v) adjusting the final pH of the rOVA to about 3.5 to about 7.0 to create an ingredient composition.

[0039] In some cases, the separation step may include ion exchange chromatography or ammonium sulfate precipitation. In some cases, the ion exchange chromatography may be cation exchange chromatography, anion exchange chromatography, or a combination thereof. In some cases, the method may further include a filtration step after the solubilization step. In some cases, the microbial cells may be fungal cells. In some cases, the fungal cells may be Pichia sp. In some cases, the microbial cells express a recombinant helper factor, and the helper factor enhances the level of expression or accumulation of rOVA.

[0040] In some cases, rOVA may include the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. In some cases, rOVA may include the amino acid sequence of duck OVA, turkey OVA, or chicken OVA. In some cases, the amino acid sequence of secreted rOVA lacks an N-terminal methionine. In some cases, secreted rOVA further includes the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus.

[0041] In some embodiments, the egg-free food product may contain recombinant ovalbumin (rOVA) in an amount of about 15% to about 25% (w / w of total protein or w / w of food product). In some cases, the egg-free food product may contain rOVA in an amount of up to about 23% (w / w).

[0042] In some embodiments, the use of recombinant ovalbumin (rOVA) is provided herein. Recombinant ovalbumin (rOVA) may be used as a raw material in the production of baked goods. rOVA may be used as a raw material in the production of egg-free food products. rOVA may be used as a raw material in the production of meat analog food products. rOVA may be used as a raw material in the production of egg white substitutes. rOVA may be used as an alternative egg liquid for baked goods products, and the alternative egg liquid may provide membrane formation equivalent to or better than an egg liquid that may contain natural egg white or natural whole egg.

[0043] rOVA may comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. rOVA may comprise the amino acid sequence of duck OVA, goose OVA or chicken OVA. In some cases, rOVA is present in an amount of 8% - 9% (w / w) in the egg liquid.

[0044] In some embodiments, the large-scale production of recombinant ovalbumin (rOVA) is described herein. The large-scale production of rOVA may include at least 1 liter of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production may include at least 10 liters of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production may include at least 100 liters of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production may include at least 1000 liters of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production includes at least 10,000 liters of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production includes at least 100,000 liters of liquid culture of microbial cells expressing rOVA. In some cases, the large-scale production includes approximately 200,000 liters of liquid culture of microbial cells expressing rOVA.

[0045] In some embodiments, a raw material composition for producing a food product that does not contain eggs containing recombinant ovalbumin may be provided herein. The recombinant ovalbumin may provide at least one egg white property selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturization, water retention, clarification, and aggregability.

[0046] The egg white properties provided by the recombinant ovalbumin may be substantially the same as or better than the same properties provided by native egg white. The composition may not contain any native egg white protein. The composition may not contain any animal products.

[0047] The composition may not contain any protein extracted from eggs. The color of the composition may have improved whiteness or be colorless as compared to native egg white. The recombinant ovalbumin may include a polypeptide sequence derived from the group consisting of chicken, goose, quail, ostrich, and duck.

[0048] The recombinant ovalbumin may be sensorially neutral with respect to taste, odor, mouthfeel, or any combination thereof. The recombinant ovalbumin may provide foaming and coagulation characteristics to the composition.

[0049] In some embodiments, a baked product containing the raw material composition provided herein is provided herein. The recombinant ovalbumin may provide structure, texture, or both structure and texture to the baked product. The recombinant ovalbumin may provide protein fortification to the baked product. The recombinant ovalbumin may be present in the baked product at a concentration of about 1% to about 20% (weight of ovalbumin / weight of product). The recombinant ovalbumin may be present in the baked product at a concentration of about 0.1% to about 5% (weight of ovalbumin / weight of product).

[0050] The recombinant ovalbumin may be suitable for gluten formation. The baked product may be selected from the group consisting of cakes, cookies, bagels, biscuits, breads, muffins, cup cakes, scones, pancakes, macarons, meringues, pie crusts and soufflés. The cake made using such raw materials may be a pound cake, a sponge cake, a yellow cake or an angel food cake. The composition may further comprise one or more components selected from the group consisting of sweeteners, gums, hydrocolloids, starches, fibers, plant proteins, algal proteins, colorants and flavor extracts.

[0051] The composition may provide one or more properties suitable for egg-like dishes, and the properties may be selected from the group consisting of foaming ability, coagulation, binding property, structure, texture, film formation, nutritional profile, cholesterol-free and protein fortification. In some embodiments, egg-like dishes comprising the raw material compositions described herein are provided herein. The egg-like dishes may be selected from the group consisting of scrambles, omelets, patties, soufflés, quiches and fritters. The egg-like dishes may be vegan, vegetarian, halal or kosher.

[0052] The composition may provide one or more properties suitable for processed meat products or meat-like products, and the properties may be selected from the group consisting of high protein content, binding property and sensory neutrality. In some embodiments, meat-like products comprising the raw material compositions provided herein are provided herein.

[0053] The meat-like products may be selected from the group consisting of hamburgers, patties, sausages, hot dogs, sliced deli meats, jerky, bacon, nuggets and ground meat-like mixtures or formed meat or meat-like compositions. The ovalbumin may be present in the meat-like product in an amount of about 0.1% to 30% (weight of ovalbumin / weight of product).

[0054] Recombinant ovalbumin may provide binding properties suitable for food coating adhesion. The food coating may contain the raw materials described herein. The food coating may be batter or breading. Recombinant ovalbumin may further provide the food coating with a crispy texture property when cooked, baked, or fried.

[0055] Recombinant ovalbumin may provide properties suitable for confectionery selected from the group consisting of odor neutrality, flavor, mouthfeel, texture, nutritional value, and protein fortification. The confectionery product may contain the raw material composition described herein. The confectionery may not contain eggs or egg whites. The confectionery may not contain any protein extracted from eggs or egg whites. Recombinant ovalbumin may provide the confectionery with a firm or chewy texture. Recombinant ovalbumin may be present in an amount of about 0.1% to 15% (weight of ovalbumin / weight of confectionery). The confectionery may be gummy, toffee, or nougat.

[0056] Recombinant ovalbumin may provide properties suitable for dairy-like beverages selected from the group consisting of odor neutrality, flavor, mouthfeel, foaming property, whipping property, texture, and nutritional value. The dairy-like beverage may contain the raw material composition described herein. The dairy-like beverage may not contain eggs or egg whites. The beverage may be selected from the group consisting of smoothies, milkshakes, "egg nog", and coffee beverages. Recombinant ovalbumin may be present in an amount of about 0.1% to 20% (weight of ovalbumin / volume of beverage).

[0057] Recombinant ovalbumin may provide suitable properties for dessert products selected from the group consisting of a creamy texture, low fat content, odor neutrality, flavor, mouthfeel, texture, binding property, and nutritional value. The dessert product may contain the raw material composition described herein. The dessert product may be selected from the group consisting of mousse, cheesecake, custard, pudding, ice candy with a stick, frozen confectionery, and ice cream. The dessert product may be vegan, vegetarian, or without dairy products. Recombinant ovalbumin may be present in an amount of about 0.1% to 10% (weight of ovalbumin / weight of dessert product).

[0058] Recombinant ovalbumin may provide suitable properties for a sauce or dressing selected from the group consisting of binding property, emulsifying ability, odor neutrality, and mouthfeel. The sauce or dressing may contain the raw material composition described herein. The sauce or dressing may be selected from the group consisting of salad dressing, mayonnaise, commercially available mayonnaise substitutes, Alfredo sauce, and Hollandaise sauce. The sauce or dressing may not contain eggs, egg whites, or any protein extracted from eggs.

[0059] Recombinant ovalbumin may provide suitable properties for snack foods selected from the group consisting of binding property, protein addition, flavor neutrality, odor neutrality, and mouthfeel. The snack food may contain the raw material composition described herein. The snack food may be a protein bar, a nutrition bar, or a granola bar. The raw material composition may further contain one or more additional components selected from the group consisting of sweeteners, gums, plant proteins, algal proteins, flavorings, colorants, thickeners, acidulants, and emulsifiers.

[0060] In some embodiments, a method for producing an egg white substitute is provided herein. The egg white substitute may include the step of providing recombinant ovalbumin and the step of mixing the recombinant ovalbumin with at least one additional component to form the egg white substitute. The recombinant ovalbumin may provide at least one egg white property selected from the group consisting of gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, creaminess and cohesiveness to the egg white substitute. The egg white substitute may not contain any eggs, egg whites, or proteins extracted or isolated from eggs. The at least one egg white property may be the same as or better than that of native eggs provided in the same amount or concentration (weight / volume).

[0061] The method may further include the step of producing recombinant ovalbumin in a heterologous host cell, and the host cell may be E. coli, yeast, filamentous fungus or Trichoderma. The yeast or filamentous fungus may be selected from the group consisting of Saccharomyces species and Pichia species. The recombinant ovalbumin may be secreted from the host cell. The recombinant ovalbumin may be glycosylated by the host cell, and the glycosylation of ovalbumin may not be the same as that of ovalbumin isolated from chicken eggs.

[0062] The method may further include the step of treating the secreted ovalbumin with a deglycosylation enzyme. The deglycosylation enzyme may be expressed by the host cell.

[0063] The host may contain a nucleic acid sequence encoding recombinant ovalbumin, and the recombinant ovalbumin may have an amino acid sequence of ovalbumin derived from an avian species. The host may contain a nucleic acid sequence encoding recombinant ovalbumin, and the recombinant ovalbumin may have an amino acid sequence of ovalbumin having at least 95% sequence identity with ovalbumin derived from an avian species. The avian species may be chicken, duck, goose, ostrich or quail.

[0064] The ovalbumin derived from avian species may be selected from the group consisting of SEQ ID NOs: 1 to 74.

[0065] In some embodiments, a recombinant protein composition for use as an egg white substitute is provided herein. The composition can include recombinant ovalbumin and at least one additional component. The recombinant ovalbumin may provide to the composition at least one egg white property selected from the group consisting of gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, creaminess, and cohesiveness. The composition may not contain any eggs, egg whites, or proteins extracted or isolated from eggs. The at least one egg white property may be the same as or better than native eggs when compared at the same amount or concentration (weight / volume).

[0066] The recombinant ovalbumin may have the amino acid sequence of ovalbumin derived from avian species. The recombinant ovalbumin may have the amino acid sequence of ovalbumin having at least 95% sequence identity with ovalbumin derived from avian species.

[0067] The avian species may be chicken, duck, goose, ostrich, or quail. The ovalbumin derived from avian species may be selected from the group consisting of SEQ ID NOs: 1 to 74.

[0068] The animal nutrition composition may include recombinant ovalbumin (rOVA). The rOVA may be in a form selected from whole cell extracts, fractionated cell extracts, and isolated proteins. The composition may be included in pet food, animal feed, chewy treats, bone broth, smoothies, or other liquids for animal nutrition and solid nutritional supplements suitable for animal food intake.

[0069] In addition, any composition, food product, ingredient, use, or method disclosed herein is applicable to any composition, food product, ingredient, use, or method disclosed herein. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein.

[0070] The novel features of the invention are particularly described in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments that utilize the principles of the invention, and the appended drawings are as follows.

Brief Description of the Drawings

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[0085] DETAILED DESCRIPTION OF THE INVENTION Various embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0086] Compositions for non - animal sources of proteins that provide nutritional and functional properties to food ingredients, methods of making such compositions, and ingestible products for oral ingestion by animals, including humans, for example, for daily meals, for human foods and snacks, and ingredients for human and animal nutrition are provided herein.

[0087] The compositions and methods provided herein contain recombinant - derived ovalbumin produced by recombinant technology, i.e., recombinant ovalbumin (rOVA). Compositions containing rOVA and methods for making compositions containing rOVA can increase the protein content of ingestible ingredients or food ingredients and also provide functional characteristics for use in the preparation of food ingredients and ingestible food products for oral ingestion by animals and humans.

[0088] In some embodiments, rOVA provides one or more functional properties such as gelling, foaming, whipping, fluffiness, binding, elasticity, air - miscibility, coating, film - forming, emulsifying, browning, thickening, texturizing, water - holding, clarifying, and aggregating. rOVA having such characteristics can be a food ingredient provided for the manufacture of egg - free or animal - free food ingredients or food products.

[0089] As used herein, "native" in the context of native egg white, native protein, native ovalbumin and native egg refers to egg white, protein, ovalbumin or whole egg produced by an animal or collected from an animal, particularly an egg-laying animal such as a bird, respectively. rOVA, and compositions containing rOVA, can be used in food ingredients and food products, such that the ingredient or product does not contain any native egg white, native protein, native ovalbumin and native egg. In some cases, the ingredient or food product made using rOVA does not contain any egg white protein other than rOVA. rOVA, and compositions containing rOVA, can be used in food ingredients and food products, such that the ingredient or product does not contain any animal product.

[0090] In some embodiments, rOVA can substitute for the use of whole egg or egg white in the manufacture of food products (either alone or with other ingredients). In some embodiments, the characteristics provided by rOVA are substantially the same as, or better than, the same characteristics provided by native egg white or native egg. For example, rOVA, and compositions containing rOVA, can have improved gelling, foaming, whipping, fluffiness, binding, elasticity, air incorporation, coating, film-forming, emulsifying, browning, thickening, texturizing, water retention (moisture-holding), clarification and agglomeration, and improved color, e.g., a whiter color, compared to compositions made with native egg white or native whole egg and native egg white. Food ingredients and food products having rOVA

[0091] The food ingredients and food products disclosed herein include a composition comprising, consisting essentially of, or consisting of rOVA, and the rOVA provides at least one functional characteristic to the composition, food ingredient, or food product. In some cases, at least one functional characteristic provided by the rOVA is of the same degree or substantially similar to that of native egg or egg white, or native OVA (nOVA). For example, it may provide any one of gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturization, water retention property (water holding capacity), clarification, and aggregability, which is of the same degree as that of whole egg, egg white, or nOVA composition. In some embodiments, at least one functional characteristic is provided or substantially provided by the inclusion of rOVA in the food ingredient or food product, for example, in the absence of any other whole egg protein or egg white protein.

[0092] Such a composition can comprise rOVA in an amount of from 0.1% to 25% on a weight / weight (w / w) or weight / volume (w / v) basis. The rOVA can be present at 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% on a weight / weight (w / w) or weight / volume (w / v) basis, or at least 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%. These concentrations can be based on the dry weight of the composition. Additionally or alternatively, the concentration of rOVA in such a composition is at most 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% on a w / w or w / v basis. In some embodiments, the rOVA in a food ingredient or food product can be in a concentration range of from 0.1% to 20%, 1% to 20%, 0.1% to 10%, 1% to 10%, 0.1% to 5%, 1% to 5%, 2 to 10%, 4 to 8%, 4 to 10%, 4 to 12%, 0.1% to 2%, 1% to 2% or 0.1 to 1%.

[0093] Provided herein are ingestible food compositions in which rOVA can capture at least one characteristic of whole eggs or egg whites, and methods of making such compositions. In some embodiments, rOVA is added to an ingestible food composition to increase, for example, the protein content with respect to the added nutrients. In some embodiments, rOVA is present in the ingestible food composition at about 1% to about 40% on a weight per weight (w / w) and / or weight per volume (w / v) basis per total weight of the composition. For example, in a 100 ml composition, rOVA is present at 30 g, and thus rOVA is at a concentration of 30% (w / v), or for example, in a 100 g composition, rOVA is present at 30 g, and thus rOVA is at a concentration of 30% (w / w). In some embodiments, the concentration of rOVA is 0.5%, 1%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% on a w / w and / or w / v basis of the composition, or about 0.5%, 1%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.In some embodiments, on a w / w and / or w / v basis, rOVA is present at a concentration of 0.5 - 1%, 1 - 5%, 2 - 8%, 4 - 8%, 2 - 12%, 4 - 12%, 5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, 25 - 30%, or about 0.5 - 1%, 1 - 5%, 2 - 8%, 4 - 8%, 2 - 12%, 4 - 12%, 5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, 25 - 30%, or rOVA is present at a concentration greater than 1%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.

[0094] An ingestible product can include one or more other proteins, such as a non-OVA protein or a non-recombinant protein. rOVA can increase the amount of protein content in the ingestible product and / or can provide one or more egg white-like characteristics. For example, an ingestible composition can include whey protein, pea protein, soybean protein, almond protein, kamut protein, flaxseed protein, vegetable protein, or egg white protein. The ingestible protein can include extruded plant protein or non-extruded plant protein. In some cases, one or more other proteins can include OVA having an amino acid sequence found naturally in birds or reptiles.

[0095] In some embodiments, the composition and the method for making the composition have egg white-like properties and increase the protein content in the composition. In some embodiments, the composition having egg white-like properties and the method for making the composition increase the protein content without adversely affecting the stability or one or more functional qualities of the composition.

[0096] In some embodiments, an ingestible food composition and a method for making an ingestible food composition include rOVA, and the addition of rOVA results in an egg white-like composition. The ingestible food composition may be a final product or a raw material for making a final product, such as a liquid or powdered rOVA composition.

[0097] The rOVA protein may be used by itself or in combination with other components to form a composition. In some embodiments, rOVA is used as a raw material to form a composition, and the rOVA raw material (or the added rOVA starting composition) may contain, by weight per total weight (w / w) and / or by weight per total volume (w / v), about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of rOVA. In some cases, the compositions described herein may contain, by w / w or w / v, up to about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of rOVA. In some embodiments, by weight per total weight (w / w) and / or by weight per total volume (w / v), about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the protein in the composition is rOVA. In some cases, by w / w or w / v, up to 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, or about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the protein in the composition is rOVA.

[0098] In some embodiments, the compositions described herein contain total protein at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 g, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 g of total protein per 100 mL of liquid (e.g., water). In some cases, the compositions described herein contain total protein at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g of total protein per 100 g of composition (e.g., powder).

[0099] In some embodiments, the compositions described herein contain rOVA at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 g, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 g per 100 mL of liquid (e.g., water). In some cases, the compositions described herein contain rOVA at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 13.2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 g of total protein per 100 g of composition (e.g., powder).

[0100] In some embodiments, the compositions described herein contain total protein at a concentration of about 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, or 5 g, or at least 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, or 5 g of total protein per 100 mL of liquid (e.g., water). In some cases, the compositions described herein contain total protein at a concentration of about 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, or 5 g, or at least 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, or 5 g of total protein per 100 g of composition (e.g., powder).

[0101] In some embodiments, the compositions described herein contain rOVA at a concentration of about 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7 or 5 g, or at least 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7 or 5 g per 100 mL of liquid (e.g., water). In some cases, the compositions described herein contain rOVA at a concentration of about 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7 or 5 g, or at least 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7 or 5 g per 100 g of composition (e.g., powder).

[0102] In some embodiments, the composition capable of being ingested with rOVA is a liquid composition. In such cases, the concentration of rOVA in the liquid composition may be from 0.1% to 90%. The concentration of rOVA in the liquid composition may be at least 0.1%. The concentration of rOVA in the liquid composition may be up to 90% at most. The concentration of rOVA in the liquid composition is by weight per total volume (w / v), and may be 0.1% - 1%, 0.1% - 5%, 0.1% - 10%, 0.1% - 15%, 0.1% - 20%, 0.1% - 25%, 0.1% - 30%, 0.1% - 35%, 0.1% - 40%, 1% - 5%, 1% - 10%, 1% - 15%, 1% - 20%, 1% - 25%, 1% - 30%, 1% - 35%, 1% - 40%, 5% - 10%, 5% - 15%, 5% - 20%, 5% - 25%, 5% - 30%, 5% - 35%, 5% - 40%, 10% - 15%, 10% - 20%, 10% - 25%, 10% - 30%, 10% - 35%, 10% - 40%, 15% - 20%, 15% - 25%, 15% - 30%, 15% - 35%, 15% - 40%, 20% - 25%, 20% - 30%, 20% - 35%, 20% - 40%, 25% - 30%, 25% - 35%, 25% - 40%, 30% - 35%, 30% - 40%, 35% - 40%, 40% - 45%, 45% - 50%, 50% - 55%, 55% - 60%, 60% - 65%, 65% - 70%, 70% - 75%, 75% - 80%, 80% - 85%, 85% - 90% or 90% - 95%. The concentration of rOVA in the liquid composition may be by w / v, about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. The concentration of rOVA in the liquid composition may be by w / v, at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. The concentration of rOVA in the liquid composition may be by w / v, up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% at most. In some embodiments, rOVA is the only protein in the liquid composition.In other embodiments, the liquid composition contains proteins other than rOVA.

[0103] In some embodiments, the ingestible composition of rOVA is a solid composition. In such cases, the concentration of rOVA in the solid composition may be from 0.1% to 70%. The concentration of rOVA in the solid composition may be at least 0.1%. The concentration of rOVA in the solid composition may be at most 70%. The concentration of rOVA in the solid composition is by weight per total weight (w / w) and / or weight per total volume (w / v), and may be 0.1% - 1%, 0.1% - 10%, 0.1% - 20%, 0.1% - 30%, 0.1% - 40%, 0.1% - 50%, 0.1% - 60%, 0.1% - 70%, 1% - 10%, 1% - 20%, 1% - 30%, 1% - 40%, 1% - 50%, 1% - 60%, 1% - 70%, 10% - 20%, 10% - 30%, 10% - 40%, 10% - 50%, 10% - 60%, 10% - 70%, 20% - 30%, 20% - 40%, 20% - 50%, 20% - 60%, 20% - 70%, 30% - 40%, 30% - 50%, 30% - 60%, 30% - 70%, 40% - 50%, 40% - 60%, 40% - 70%, 50% - 60%, 50% - 70% or 60% - 70%. The concentration of rOVA in the solid composition may be, by w / w or w / v, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70%. The concentration of rOVA in the solid composition may be, by w / w or w / v, at least 0.1%, 1%, 10%, 20%, 30%, 40%, 50% or 60%. The concentration of rOVA in the solid composition may be, by w / w or w / v, at most 1%, 10%, 20%, 30%, 40%, 50%, 60% or 70%.

[0104] In some embodiments, the composition capable of being ingested with rOVA is a powdered composition. In such cases, the concentration of rOVA in the powder composition may be 15% to 99% by weight (w / w) per total weight and / or by weight (w / v) per total volume. The concentration of rOVA in the powder composition may be at least 15% w / w or w / v. In an embodiment, the concentration of rOVA in the powder composition may be at most 99% w / w or w / v. The concentration of rOVA in the powder composition may be 15% - 30%, 15% - 45%, 15% - 60%, 15% - 75%, 15% - 80%, 15% - 85%, 15% - 90%, 15% - 95%, 15% - 99%, 30% - 45%, 30% - 60%, 30% - 75%, 30% - 80%, 30% - 85%, 30% - 90%, 30% - 95%, 30% - 99%, 45% - 60%, 45% - 75%, 45% - 80%, 45% - 85%, 45% - 90%, 45% - 95%, 45% - 99%, 60% - 75%, 60% - 80%, 60% - 85%, 60% - 90%, 60% - 95%, 60% - 99%, 75% - 80%, 75% - 85%, 75% - 90%, 75% - 95%, 75% - 99%, 80% - 85%, 80% - 90%, 80% - 95%, 80% - 99%, 85% - 90%, 85% - 95%, 85% - 99%, 90% - 95%, 90% - 99% or 95% - 99% w / w or w / v. The concentration of rOVA in the powder composition may be about 15%, 30%, 45%, 60%, 75%, 80%, 85%, 90%, 95% or 99% w / w or w / v. The concentration of rOVA in the powder composition may be at least 15%, 30%, 45%, 60%, 75%, 80%, 85%, 90% or 95% w / w or w / v. The concentration of rOVA in the powder composition may be at most 30%, 45%, 60%, 75%, 80%, 85%, 90%, 95% or 99% w / w or w / v. In some embodiments, rOVA is the only protein in the powder composition. In other embodiments, the powder composition contains proteins other than rOVA.

[0105] In some cases, the powder composition may be a concentrate containing at least 70% rOVA by w / w. In some cases, the powder composition may be a concentrate containing at least 80% rOVA by w / w. In some cases, the powder composition may be an isolate containing at least 90% rOVA by w / w. In some cases, the powder composition may be an isolate containing at least 95% rOVA by w / w.

[0106] In some embodiments, the ingestible composition of rOVA is a concentrated liquid composition. In such cases, the concentration of rOVA in the concentrated liquid composition may be 10% - 60% by weight per total weight (w / w) and / or weight per total volume (w / v). The concentration of rOVA in the concentrated liquid may be at least 10% by w / w or w / v. The concentration of rOVA in the concentrated liquid may be at most 60% by w / w or w / v. The concentration of rOVA in the concentrated liquid may be 10% - 20%, 10% - 30%, 10% - 40%, 10% - 50%, 10% - 60%, 20% - 30%, 20% - 40%, 20% - 50%, 20% - 60%, 30% - 40%, 30% - 50%, 30% - 60%, 40% - 50%, 40% - 60% or 50% - 60% by w / w or w / v. The concentration of rOVA in the concentrated liquid may be about 10%, 20%, 30%, 40%, 50% or 60% by w / w or w / v. The concentration of rOVA in the concentrated liquid may be at least 10%, 20%, 30%, 40% or 50% by w / w or w / v. The concentration of rOVA in the concentrated liquid may be at most 20%, 30%, 40%, 50% or 60% by w / w or w / v. The liquid may contain any ingestible solvent, such as water, dairy products, oils or other culinary bases.

[0107] In some embodiments, a composition ingestible with rOVA is, for example, a cooked food such as a baked good, salad dressing, egg-like dish (such as an egg patty or scramble), dessert or dairy-like product, or meat analog (such as a vegan meat patty, sausage or hot dog). Such a composition can contain rOVA in an amount of 0.1% to 20% on a weight / weight (w / w) or weight / volume (w / v) basis. rOVA can be present at 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, or at least 0.1%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on a weight / weight (w / w) or weight / volume (w / v) basis. Additionally, or alternatively, the concentration of rOVA in such a composition is at most 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% on a w / w or w / v basis. In some embodiments, rOVA in a food ingredient or food product can be in a concentration range of 0.1% to 20%, 1% to 20%, 0.1% to 10%, 1% to 10%, 0.1% to 5%, 1% to 5%, 0.1% to 2%, 1% to 2% or 0.1 to 1%. Features and properties of rOVA compositions, food ingredients and food products containing rOVA

[0108] The rOVA-containing compositions of the present specification can provide one or more functional characteristics to food ingredients and food products. In some embodiments, rOVA provides nutritional characteristics such as protein content, protein fortification, and amino acid content to a food ingredient or food product. The nutritional characteristics provided by rOVA in the composition may be comparable or substantially similar to those of eggs, egg whites, or native OVA (nOVA). The nutritional characteristics provided by rOVA in the composition may be better than those provided by native whole eggs or native egg whites. In some cases, rOVA provides one or more functional characteristics of egg white in the absence of any other egg white proteins.

[0109] The rOVA compositions disclosed herein can provide foaming properties and foam volume to the composition. For example, rOVA can be used to form foams and can be used in baked goods such as cakes, meringues, and other foods where rOVA can provide foam volume in place of egg white. In some cases, rOVA provides the foaming properties and foam volume of egg white in the absence of any other egg white proteins.

[0110] The composition containing rOVA may have a foam height higher than that of the composition containing egg white or nOVA. In some cases, the composition containing rOVA may have a foam height that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher than that of the egg white, nOVA composition or alternative egg white, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher. In some cases, the composition containing rOVA may have a foam height that is up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher than that of the egg white, nOVA composition or alternative egg white. Alternative egg whites may include products such as aquafaba, chia seeds, flax seeds, starch; apple sauce, banana puree; condensed milk, etc., which are commonly used as egg white substitutes.

[0111] Compositions containing rOVA may have a foam stability that is higher than that of egg white, nOVA compositions or alternative egg white foams. In some cases, the composition containing rOVA may have a foam stability that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500%, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher than that of egg white or alternative egg white. In some cases, the composition containing rOVA may have a foam stability that is up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher than that of egg white. Foam stability may be calculated by measuring the drainage of the foamed solution. Drainage may be measured every 10 minutes for 30 minutes to accumulate data on foam stability. The volume of liquid drained after 30 minutes may be compared to the initial volume of liquid (5 mL), for example, foam stability (%) = (initial volume - volume drained) / initial volume × 100.

[0112] The composition containing rOVA may have a foam volume higher than that of egg white, nOVA composition or alternative egg white foams. In some cases, the composition containing rOVA may have a foam volume that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher, compared to egg white, nOVA composition or alternative egg white. In some cases, the composition containing rOVA may have a foam volume that is up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450% or 500% higher, compared to egg white, nOVA composition or alternative egg white. The foam volume may be determined by measuring the initial volume of the foam after whipping and comparing it to an initial volume of 5 mL. Foam volume (%) = (foam volume / initial volume) × 100.

[0113] The liquid composition may foam faster than compositions containing egg white, nOVA, or alternative egg white. In some cases, the rOVA composition foams at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% faster than egg white, nOVA, or alternative egg white compositions. In some cases, the rOVA composition foams up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% faster than egg white, nOVA, or alternative egg white compositions.

[0114] The composition containing rOVA may have a gel strength higher than that of the gel of egg white, nOVA composition or egg white substitute. In some cases, the rOVA composition may have a gel strength within the range of 100 g to 1500 g, 500 g to 1500 g or 700 g to 1500 g. In some cases, the rOVA composition has a gel strength of about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 or 1500 g, or at least 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 or 1500 g. In some cases, the rOVA composition has a gel strength of up to 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 or 1500 g. In some cases, the rOVA composition has a gel strength of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to egg white, nOVA or egg white substitute. In some cases, the rOVA composition has a gel strength of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to egg white, nOVA or egg white substitute.

[0115] The rOVA compositions disclosed herein can provide a structure, texture, or a combination of structure and texture. In some embodiments, rOVA is added to a food ingredient or food product for baked goods, and the rOVA provides a structure, texture, or a combination of structure and texture to the baked good product. The rOVA can be used in such baked good products in place of native egg white, native egg, or native egg protein. The addition of rOVA to the baked good product can also provide protein fortification and improve the nutritional content. In some embodiments, the rOVA is used in an amount of from 0.1% to 25% by weight / weight or weight / volume in the baked good product. In some embodiments, the rOVA is used in an amount of from 0.1% to 5% in the baked good product. In some cases, the rOVA provides the structure and / or texture of egg white in the absence of any other egg white protein.

[0116] The rOVA compositions disclosed herein can be compatible with gluten formation, such that the rOVA can be used when gluten formation provides structure, texture, and / or foam to a food ingredient or food product.

[0117] Exemplary baked goods products that can use rOVA as a raw material include, but are not limited to, cakes, cookies, breads, bagels, biscuits, muffins, cup cakes, scones, pancakes, macarons, pie crusts, meringues, and soufflés. For example, rOVA can be used as a raw material to make cakes such as pound cake, sponge cake, yellow cake, or angel food cake, and such cakes do not contain any native egg white, native whole egg, and native egg protein. Together with rOVA, the baked goods product may contain additional raw materials such as flour, sweeteners, gums, hydrocolloids, starches, fibers, flavorings (such as flavor extracts), and other protein sources. In some embodiments, the baked goods product may include rOVA, as well as at least one fat or oil, at least one cereal starch, and optionally at least one sweetener. Cereal starches for use in such compositions include flours such as wheat flour, rice flour, corn flour, multigrain flour, spelt flour, and rye flour, and starches derived from corn, potato, sorghum, and kudzu root. Oils and fats for use in such compositions include plant-derived oils and fats such as olive oil, corn oil, avocado oil, nut oils (such as almond, walnut, and peanut), and safflower oil. rOVA may provide such baked goods having at least one property of egg white, such as binding, elasticity, air incorporation, browning, texture, water retention, and cohesiveness. In some cases, the baked goods product does not contain any natural egg white and natural egg, and / or does not contain any protein derived from any other egg white except rOVA. In some cases, rOVA is provided to the baked goods composition as a raw material starting from a concentrate, isolate, or powder form of rOVA, for example. In some cases, the rOVA provided as a raw material for the baked goods product has a pH range of about 3.5 to 7.0. In some cases, sweeteners such as sugar, syrup, honey, or sugar substitutes are included in the baked goods product.

[0118] The rOVA compositions disclosed herein can also be used to prepare egg-free food products such as food products made when native whole eggs or native egg whites are basic or characteristic ingredients such as in scrambled eggs, omelets, patties, soufflés, quiches, and fritattas. In some embodiments, rOVA provides one or more functional characteristics to the cooked product, including foaming, coagulation, binding, structure, texture, film formation, nutritional profile, absence of cholesterol (i.e., cholesterol-free), and protein fortification. Such egg-free cooked products can be vegan, vegetarian, halal or kosher, or combinations thereof. The egg-free cooked product (also referred to as an egg white substitute) may include rOVA, as well as at least one fat or oil, a polysaccharide or polysaccharide-containing ingredient, and starch. In some cases, the egg-free cooked product may also include flavorants (such as to provide a salty, sulfurous or umami flavor) and / or colorants (such as to provide a yellowish or off-white color to a baked product). In some cases, the inclusion or rOVA in the egg-free cooked product provides the properties of native egg white such as hardness, adhesiveness, crushability, cohesiveness, stickiness, and chewiness when the composition is heated or cooked. Exemplary polysaccharides or polysaccharide-containing ingredients for such compositions include gellan gum, sodium alginate, and psyllium. Oils and fats for use in such compositions include plant-derived oils and fats such as olive oil, corn oil, avocado oil, and safflower oil.

[0119] The rOVA compositions disclosed herein can be used for processed meat products or meat-like products, or for fish-like or crustacean-like products. In such products, rOVA can provide one or more functional properties such as protein content and protein addition, as well as binding properties, texturizing properties. Exemplary meat and meat-like products include hamburgers, patties, sausages, hot dogs, sliced deli meats, jerky, bacon, nuggets and ground meat-like mixtures. Meat-like products can resemble meat from cows, pigs, chickens, lambs and other edible animals, as well as meat consumed by humans and other animals. Fish-like and crustacean-like products can resemble, for example, fish cakes, crab cakes, shrimp, shrimp balls, fish sticks, seafood meat, crab meat, fish fillets and clam meats. In some embodiments, rOVA is present in meat or meat-like products in an amount of about 0.1% to 30% w / w or w / v. In some embodiments, rOVA is used for meat-like products (also referred to as meat analogs, containing at least one fat or oil and a plant-derived protein). Oils and fats for use in such compositions include plant-derived oils and fats such as olive oil, corn oil, avocado oil and safflower oil. Plant-derived proteins for use in meat analogs include soybean protein, nut protein, pea protein, lentil and other legume proteins, and whey protein. In some cases, such plant proteins are extruded, and in other cases, such plant proteins are non-extruded proteins. In some cases, the meat analog contains about 2% to 15% (w / w) rOVA. In some cases for meat analog compositions, rOVA acts as a binder, a gelling agent, or a combination of a binder and a gelling agent for such compositions.

[0120] The rOVA compositions disclosed herein can be used in coatings for food products. For example, rOVA can provide binding or adhesion properties to adhere batter or breading to another food ingredient. rOVA can be used as an "egg-free egg white", and the rOVA protein provides appearance, color, and texture when coated on other food ingredients or food products, such as baked goods. In one example, an "egg-free egg white" may be used to coat a baked good, and as a result, the baked good adheres to the coating (e.g., seeds, salt, spices, and herbs). Addition of rOVA as a coating to a food product can provide a crispy texture or increase the hardness of the outside of the food product, such as when the product is cooked, baked, or fried.

[0121] The rOVA compositions disclosed herein are included in sources and dressings such as egg-free mayonnaise, commercially available mayonnaise substitutes, gravy, sandwich spreads, salad dressings or food sauces. Inclusion of rOVA into sources or dressings such as these can provide one or more properties such as binding, emulsifying, odor neutrality and mouthfeel. In some embodiments, rOVA is present in such sources and dressings in an amount of 0.1% to 3% or about 3% to about 5% w / w or w / v. In some cases, the amount of rOVA in the source or dressing may be substantially similar to the amount of whole egg, egg white or nOVA used in commercially available or commonly used recipes. Exemplary sources and dressings include mayonnaise, commercially available mayonnaise substitutes, Alfredo sauce and Hollandaise sauce. In some embodiments, the source or dressing containing rOVA does not contain whole egg, egg white, or any other protein extracted from eggs. In some cases, sources, dressings or other emulsified products made with rOVA contain at least one fat or oil, and water. Exemplary fats and oils for such compositions include corn oil, safflower oil, nut oils and avocado oil.

[0122] Using the rOVA composition, it is possible to cook egg-free, animal-free, vegetarian and vegan confectioneries such as confectionery. rOVA can provide one or more functional characteristics including odor neutrality, flavor, mouthfeel, texture, gelling property, cohesiveness, foaming property, whipping property, nutritional value and protein fortification to the confectionery. In some embodiments, the cooked confectionery containing rOVA does not contain any native egg protein and native egg white. The rOVA in such confectionery can provide a firm or chewy texture. In some embodiments, rOVA is present in the confectionery at about 0.1% to 15%. Exemplary confectioneries include gummies, taffies, rock candies, meringues, marshmallows and nougats. In some embodiments, the confectionery comprises rOVA, at least one sweetener, and optionally an ingestible liquid. Exemplary sweeteners include sugar, honey, sugar substitutes and plant-derived syrups. In some cases, rOVA is provided at a pH of about 3.5 to about 7.0 as a raw material for making confectionery. In some cases, rOVA is present in the confectionery composition at about 2% to about 15% (w / v). In some embodiments, the confectionery is a food product such as a meringue, a whipped dessert or a whipped topping. In some embodiments, the rOVA in the confectionery provides foaming property, whipping property, fluffiness or air miscibility to the food product and / or provides gelling. In some cases, the confectionery is a liquid such as a foaming drink. In some cases, the liquid may contain an ingestible alcohol (such as a sweet cocktail or an after-dinner drink).

[0123] The rOVA composition of the present specification can be used in dairy products, dairy-like products or dairy-containing products. For example, rOVA can be used in the preparation of beverages such as smoothies, milkshakes, "eggnog" and coffee beverages. In some embodiments, rOVA is added to additional ingredients, and at least one ingredient is a dairy ingredient or a dairy-derived ingredient (such as milk, cream, whey and butter). In some embodiments, rOVA is added to additional ingredients to create a beverage that does not contain any native egg protein, native egg white and native egg. In some embodiments, rOVA is an ingredient in a beverage that does not contain animal-derived ingredients such as those that do not contain any native egg-derived ingredients and any dairy-derived ingredients. Examples of such non-dairy-derived drinks include soy milk or nut milk such as almond milk. rOVA can also be used to create beverage additives such as creamers or "milks" to provide protein, flavor, texture and mouthfeel to beverages such as coffee, tea, alcoholic beverages or cocoa. In some embodiments, rOVA is present in the beverage ingredient or beverage additive in an amount of about 0.1% to 20% w / w or w / v.

[0124] In some embodiments of the present specification, rOVA can be used to prepare dairy-like products such as yogurt, cheese or butter. Dairy products having rOVA can contain other animal-based milk components or proteins. In some embodiments, the dairy products prepared using rOVA do not contain any animal-based ingredients.

[0125] The culinary preparations of dessert products can be prepared using rOVA. In dessert products, rOVA can provide one or more properties such as a creamy texture, low fat content, odor neutrality, flavor, mouthfeel, texture, binding properties, and nutritional value. rOVA may be present in the raw materials or set of raw materials used to prepare the dessert product. Exemplary dessert products suitable for preparation using rOVA include mousse, cheesecake, custard, pudding, ice candy with a stick, and ice cream. In some embodiments, dessert products prepared to contain rOVA are vegan, vegetarian, or without dairy products. Dessert products containing rOVA can have an amount of rOVA that is from about 0.1% to about 10% rOVA, by w / w or w / v.

[0126] rOVA can be used to prepare snack foods such as protein bars, energy bars, nutritional bars, or granola bars. rOVA can provide properties to snack foods that include one or more of binding properties, protein addition, flavor neutrality, odor neutrality, coating, and mouthfeel. In some embodiments, rOVA is added to the culinary preparation of the snack food in an amount of from about 0.1% to 30%, by w / w or w / v.

[0127] rOVA can be used for nutritional supplements such as parenteral nutrition, protein drink supplements, protein shakes, etc., where rOVA provides high protein addition. In some embodiments, rOVA can be added to such compositions in an amount of from about 10% to 30%, by w / w or w / v.

[0128] In some embodiments, the rOVA composition can be used as an egg substitute and an egg white substitute. The rOVA can be mixed or combined with at least one additional ingredient to form an egg white substitute. The rOVA can provide one or more properties to the egg substitute or egg white substitute, such as gelling, foaming, whipping, fluffiness, binding, elasticity, air miscibility, creaminess, and cohesiveness. In some embodiments, the properties are the same as or better than those of native eggs or native egg whites provided at the same amount or concentration (w / w or w / v). In some embodiments, the egg substitute or egg white substitute does not contain any eggs, egg whites, or proteins extracted or isolated from eggs.

[0129] Food ingredients and food products containing rOVA, such as those described herein, can contain additional ingredients or components. For example, the rOVA composition can be prepared using additional ingredients such as one or more of sweeteners, gums, flavorings, thickeners, acidulants, and emulsifiers. Other ingredients such as flours, grains, oils and fats, fibers, fruits, and vegetables can be combined with the rOVA. Such rOVA compositions can be vegan, vegetarian, halal, kosher, and animal-free, or combinations thereof. In some embodiments, the rOVA can be a food ingredient or can be prepared for food products that are normally animal-based or that normally contain animal-derived components such as meat, dairy products, or eggs.

[0130] Compositions comprising rOVA, including food ingredients and food products, may be compatible with one or more steps of ingestible preparations such as heating, baking, grilling, roasting, steaming, microwaving, direct-fire roasting, boiling, steaming, extruding, deep-frying in abundant oil, or pan-frying, or may be processed using ohmic heating, sous vide cooking, freezing, chilling, blanching, packaging, canning, bleaching, concentrating, drying, pressing, grinding, mixing, par-cooking, cooking, fermenting dough, marinating, cutting, slicing, dicing, breaking, mincing, shredding, whisking, coring, spiraling, rolling, juicing, straining, rubbing, kneading, foaming, stirring, whipping, mashing, stuffing, peeling, smoking, curing, salting, preserving, pickling, fermenting, homogenizing, pasteurizing, sterilizing, irradiating, cold plasma treating, high-pressure treating, pulsed electric field treating, microwave-assisted thermal sterilization, stabilizing, blending, puréeing, enhancing nutritional value, purifying, hydrogenating, aging, extending shelf life, or adding enzymes.

[0131] Food ingredients and food products prepared using rOVA can be essentially free of any microbial cells or microbial cell debris. For example, rOVA may be secreted from a microbial host cell and / or isolated from microbial cells, culture medium, and / or microbial cell debris.

[0132] In some embodiments, rOVA may be prepared as a whole cell extract or a fractionated extract, such that the rOVA composition contains microbial cells and / or microbial cell components.

[0133] In one embodiment, the rOVA composition is prepared for animal food intake, the rOVA is present in whole cell extracts or fractionated extracts, and as a result, the rOVA composition contains microbial cells and / or microbial cell components. In some embodiments, the rOVA composition is prepared for animal food intake, and the rOVA is isolated from microbial cells, culture medium, and microbial cell debris. Exemplary compositions for animal food intake can include pet food, animal feed, crunchy snacks, bone broth, smoothies, or other liquids for animal nutrition and solid nutritional supplements suitable for animal food intake. In these cases, the microbial cell extracts or microbial cell debris may provide additional nutritional value.

[0134] Animals that can ingest the rOVA composition can include companion animals (e.g., dogs, cats, horses), livestock, exotic animals (lions, tigers, zebras), and domestic animals (such as cows, pigs, sheep, goats, etc.). The rOVA compositions described herein can also be used for aquaculture (such as for fish and crustaceans) and avian nutrition (such as for pet birds, zoo birds, wild birds, poultry, and birds raised for human and animal food).

[0135] In some embodiments of the ingestible food compositions described herein, the composition is essentially free of animal-derived components, whey protein, casein salts, fat, lactose, hydrolyzed lactose, soy protein, collagen, hydrolyzed collagen or gelatin, or any combination thereof. The compositions described herein may also be essentially free of cholesterol, glucose, fat, saturated fat, trans fat or any combination thereof. In some cases, the compositions described herein contain less than 10%, 5%, 4%, 3%, 2%, 1% or 0.5% by dry weight. In some embodiments, the composition may contain fat (e.g., mayonnaise and commercially available mayonnaise substitutes), such compositions may contain up to about 60% fat or may be low-fat compositions (e.g., low-fat mayonnaise and commercially available mayonnaise substitutes), and such compositions may contain lower percentages of fat. Compositions free of animal-derived components can be considered vegetarian and / or vegan.

[0136] In some embodiments, the rOVA powder composition contains less than 5% ash. The term "ash" is a term known in the art and represents one or more inorganic substances such as ions, elements, minerals and / or compounds. In some cases, the rOVA powder composition contains less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25% or 0.1% ash by weight per total weight (w / w) and / or weight per total volume (w / v).

[0137] In some embodiments, the water content of the rOVA powder composition may be less than 15%. The rOVA powder composition may have less than 15%, 12%, 10%, 8%, 6%, 5%, 3%, 2% or 1% water by weight (w / w) and / or by weight per total volume (w / v). In some embodiments, the carbohydrate content of the rOVA powder composition may be less than 30%. The rOVA powder composition may have less than 30%, 27%, 25%, 22%, 20%, 17%, 15%, 12%, 10%, 8%, 5%, 3% or 1% carbohydrate by w / w or w / v. Functional neutrality and improved functional appeal

[0138] In some embodiments, in addition to egg white-like properties, the addition of rOVA to an ingestible food composition provides increased protein nutritional content, functional neutrality, or improved functional appeal compared to other proteins in such a composition. As used herein, "functional neutrality" refers to the absence of strong or distinctive tastes, odors (smells), or combinations of tastes and odors, as well as texture, mouthfeel, aftertaste, and color. A sensory panel, such as that described in Kemp et al. 2009, may be used by trained sensory analysts. Functional neutrality may provide improved functional appeal to testers, such as food testers or consumers, when an ingestible food composition containing rOVA is compared to another such as a composition having different proteins, such as nOVA, whey protein, pea protein, soy protein, whole egg, or egg white protein, at the same concentration.

[0139] In some embodiments, rOVA, when added to an ingestible food composition, is substantially odorless as measured by trained sensory analysts in comparison to different solutions / products having different protein components present at the same concentration as the solution / product containing rOVA. For example, in comparisons where the comparison is whey, soy, collagen, pea, egg white solids isolate, and / or nOVA, etc. In some embodiments of the rOVA compositions described herein, such compositions have a protein concentration of rOVA of about 0.5 - 1%, 1% - 5%, 5 - 10%, 10 - 15%, 15 - 20%, 20 - 25%, 25 - 30% by weight per total weight (w / w) and / or weight per total volume (w / v), or per 100 mL of solution (e.g., per 100 mL of water), about 0.1, 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g of total rOVA protein and is substantially odorless.

[0140] In some embodiments, the addition of rOVA to an ingestible food composition provides a neutral flavor in addition to properties such as egg white-like properties and increased protein nutrient content. The neutral flavor can be measured, for example, by trained sensory analysts in comparison to solutions containing different proteins present at the same concentration as rOVA, such as whey, soy, collagen, pea, whole egg, and egg white solids isolate (including native OVA).

[0141] In some embodiments, the addition of rOVA provides a reduction in certain odors and / or flavors associated with other proteins or egg white. For example, the addition of rOVA results in less of an "egg-like" odor or flavor compared to the addition of rOVA to an ingestible food composition of whole egg, fractionated egg, or egg white. In some embodiments, the addition of rOVA results in less of a metallic odor or flavor compared to other protein sources.

[0142] In some embodiments, the addition of rOVA has an improved mouthfeel compared to the addition of other protein sources used to create egg white-like properties. For example, the addition of rOVA has less coarseness or fewer precipitates or solids compared to other protein sources.

[0143] In some embodiments, the addition of rOVA has an improved texture, for example, compared to other available auxiliary protein sources.

[0144] An ingestible composition having rOVA may also have an improved sensory appeal compared to a composition without rOVA or a composition having a different protein present at an equal concentration to rOVA. Such improved sensory appeal may relate to taste and / or odor. Taste and odor can be measured, for example, by trained sensory analysts. In some examples, the sensory analyst compares an ingestible composition having rOVA to one without rOVA or one having an equal amount of a different protein or protein source.

[0145] As described herein, the ingestible compositions herein can be in liquid form. The liquid form can be an intermediate product such as a soluble rOVA solution. In some cases, the liquid form can be a final product such as a beverage containing rOVA. Examples of different types of beverages contemplated herein include juices, sodas, soft drinks, flavored waters, protein waters, enhanced waters, carbonated waters, nutritional drinks, energy drinks, sports drinks, recovery drinks, alcoholic drinks, warm drinks, coffee-based drinks, tea-based drinks, plant-based milks, nut milks, milk-based drinks, non-dairy products, plant-based mild drinks, infant formula drinks, and meal replacement drinks. The pH of the composition

[0146] The pH of the rOVA composition may be 3.5 to 8. The pH of the rOVA composition may be at least 3.5. The pH of the rOVA composition may be at most 8. The pH of the rOVA composition may be 3.5 to 4, 3.5 to 4.5, 3.5 to 5, 3.5 to 5.5, 3.5 to 6, 3.5 to 6.5, 3.5 to 7, 3.5 to 7.5, 3.5 to 8, 4 to 4.5, 4 to 5, 4 to 5.5, 4 to 6, 4 to 6.5, 4 to 7, 4 to 7.5, 4 to 8, 4.5 to 5, 4.5 to 5.5, 4.5 to 6, 4.5 to 6.5, 4.5 to 7, 4.5 to 7.5, 4.5 to 8, 5 to 5.5, 5 to 6, 5 to 6.5, 5 to 7, 5 to 7.5, 5 to 8, 5.5 to 6, 5.5 to 6.5, 5.5 to 7, 5.5 to 7.5, 5.5 to 8, 6 to 6.5, 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 7.5, 7 to 8 or 7.5 to 8. The pH of the rOVA composition may be 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8. The rOVA composition having a pH of 3.5 to 7 may have one or more improved functionalities as compared to nOVA, egg white, and egg white substitute compositions.

[0147] The pH of the rOVA composition may be 2 to 3.5. The pH of the rOVA composition may be at least 2. The pH of the rOVA composition may be at most 3.5. The pH of the rOVA composition may be 2 to 2.5, 2 to 3, 2 to 3.5, 2.5 to 3, 2.5 to 3.5 or 3 to 3.5. The pH of the rOVA composition may be 2, 2.5, 3 or 3.5.

[0148] The pH of the rOVA composition may be from 7 to 12. The pH of the rOVA composition may be at least 7. The pH of the rOVA composition may be at most 12. The pH in the rOVA composition may be 7 - 7.5, 7 - 8, 7 - 8.5, 7 - 9, 7 - 9.5, 7 - 10, 7 - 10.5, 7 - 11, 7 - 11.5, 7 - 12, 7.5 - 8, 7.5 - 8.5, 7.5 - 9, 7.5 - 9.5, 7.5 - 10, 7.5 - 10.5, 7.5 - 11, 7.5 - 11.5, 7.5 - 12, 8 - 8.5, 8 - 9, 8 - 9.5, 8 - 10, 8 - 10.5, 8 - 11, 8 - 11.5, 8 - 12, 8.5 - 9, 8.5 - 9.5, 8.5 - 10, 8.5 - 10.5, 8.5 - 11, 8.5 - 11.5, 8.5 - 12, 9 - 9.5, 9 - 10, 9 - 10.5, 9 - 11, 9 - 11.5, 9 - 12, 9.5 - 10, 9.5 - 10.5, 9.5 - 11, 9.5 - 11.5, 9.5 - 12, 10 - 10.5, 10 - 11, 10 - 11.5, 10 - 12, 10.5 - 11, 10.5 - 11.5, 10.5 - 12, 11 - 11.5, 11 - 12 or 11.5 - 12. The pH of the rOVA composition may be 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.

[0149] In some embodiments, the pH of rOVA may be adjusted before its inclusion in the composition or before its use as a raw material. In some embodiments, the pH of rOVA is adjusted during the purification and / or isolation process. In some embodiments, the pH of rOVA in the raw material or for use in the manufacture of a food product composition is adjusted to about 3.5 to about 7.0. In some cases, the pH of rOVA may be adjusted to a pH higher than 1 during the manufacturing process. For example, rOVA may be expressed in a host cell such as a microbial cell, and in some cases, rOVA is secreted by the host cell into a growth medium (e.g., a liquid medium). rOVA is separated from the host cell, and such separation steps may be performed at a selected pH, e.g., a pH of about 3.5. In some cases, rOVA at such a separation pH may not be soluble or may not be completely soluble, and the pH is adjusted to a higher pH, e.g., about pH 12. Then, rOVA may be adjusted to a final pH of about 3.5 to about 7.0. The separation of rOVA from other components of the host cell or other components of the liquid medium can include one or more of ion exchange chromatography such as cation exchange chromatography and / or anion exchange chromatography, filtration, and ammonium sulfate precipitation. Additional components of the composition

[0150] The ingestible food compositions containing rOVA disclosed in this specification and methods for making such compositions may include the step of adding rOVA to one or more raw materials or the step of mixing rOVA with one or more raw materials. For example, food additives may be added to or mixed with the composition. The food additives can add volume and / or mass to the composition. The food additives may improve functional performance and / or physical properties. For example, the food additives may prevent gelation or an increase in viscosity caused by the lipid portion of lipoproteins in freeze-thaw cycles. An anti-coagulant may be added to produce a free-flowing composition. Carbohydrates can be added to increase resistance to heat damage, such as a reduction in protein denaturation during drying, and can improve the stability and fluidity of the dry composition. Food additives include, but are not limited to, food colorants, pH adjusters, natural flavorings, artificial flavorings, flavor enhancers, batch markers, edible acids, fillers, anti-coagulants (e.g., sodium aluminosilicate), anti-chlorination agents (e.g., citric acid), food stabilizers, foam stabilizers or binders, antioxidants, acid regulators, extenders, color retention agents, whipping agents (e.g., ester-type whipping agents, triethyl citrate, sodium lauryl sulfate), emulsifiers (e.g., lecithin), water retention agents, thickeners, excipients, solid diluents, salts, nutrients, sweeteners, brighteners, preservatives, vitamins, food elements, carbohydrates, polyols, gums, starches, flours, oils or bran.

[0151] Food colorants include, but are not limited to, FD&C Yellow #5, FD&C Yellow #6, FD&C Red #40, FD&C Red #3, FD&C Blue No.1, FD&C Blue No.2, FD&C Green No.3, carotenoids (e.g., saffron, β-carotene), anthocyanins, annatto, betanin, fava bean, caramel color, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, Ponceau 4R, Patent Blue V and Green S.

[0152] Examples of raw materials for pH adjustment include, but are not limited to, Tris buffer, potassium phosphate, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, sodium bicarbonate, and hydrochloric acid.

[0153] Examples of salts include, but are not limited to, acid salts, alkali salts, organic salts, inorganic salts, phosphates, chloride salts, sodium salts, sodium chloride, potassium salts, potassium chloride, magnesium salts, magnesium chloride, magnesium perchlorate, calcium salts, calcium chloride, ammonium chloride, iron salts, iron chloride, zinc salts, and zinc chloride.

[0154] Examples of nutrients include, but are not limited to, macronutrients, micronutrients, essential nutrients, non-essential nutrients, dietary fiber, amino acids, essential fatty acids, omega-3 fatty acids, and conjugated linoleic acid.

[0155] Examples of sweeteners include, but are not limited to, sugar substitutes, artificial sweeteners, acesulfame potassium, advantame, alitame, aspartame, sodium cyclamate, dulcin, glycine (glucin), neohesperidin dihydrochalcone, neotame, P-4000, saccharin, aspartame-acesulfame salt, sucralose, brazzein, curculin, glycyrrhizin, glycerol, inulin, mogroside, mabinlin, maltooligosaccharide, mannitol, miraculin, monatin, monellin, osladin, pentadin, stevia, trilobatin, and thaumatin.

[0156] Examples of carbohydrates include, but are not limited to, sugar, sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allose, tagatose, xylose, arabinose, high fructose corn syrup, high maltose corn syrup, corn syrup (e.g., glucose-free corn syrup), sialic acid, monosaccharides, disaccharides, polysaccharides (e.g., polydextrose, maltodextrin), and starch.

[0157] Examples of polyols include, but are not limited to, xylitol, maltitol, erythritol, sorbitol, trehalose, arabinitol, hydrolyzed hydrogenated starch, isomalt, lactitol, mannitol, and galactitol (dulcitol).

[0158] Examples of gums include, but are not limited to, gum arabic, gellan gum, guar gum, locust bean gum, acacia gum, cellulose gum, and xanthan gum.

[0159] Examples of vitamins include, but are not limited to, niacin, riboflavin, pantothenic acid, thiamine, folic acid, vitamin A, vitamin B6, vitamin B12, vitamin D, vitamin E, lutein, zeaxanthin, choline, inositol, and biotin.

[0160] Examples of food elements include, but are not limited to, calcium, iron, magnesium, phosphorus, potassium, sodium, zinc, copper, manganese, selenium, chlorine, iodine, sulfur, cobalt, molybdenum, nickel, and bromine. rOVA Protein and Production of rOVA Protein

[0161] rOVA can have an amino acid sequence derived from any species. For example, rOVA can have an amino acid sequence of OVA derived from birds or reptiles, or other egg-laying species. rOVA having an amino acid sequence derived from birds can be selected from the group consisting of domesticated birds, poultry, waterfowl, game birds, chickens, quails, turkeys, ducks, ostriches, geese, seagulls, pigeons, pheasants, emus, and any combination thereof. rOVA can have an amino acid sequence derived from a single species, for example, Gallus gallus domesticus. Alternatively, rOVA can have an amino acid sequence derived from two or more species and can be, for example, a hybrid.

[0162] Exemplary OVA amino acid sequences contemplated in this specification are provided in Table 1 below as SEQ ID NOs: 1 to 74. Table 1. OVA sequence

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

[0163] Expression of rOVA in host cells, such as Pichia species, Saccharomyces species, Trichoderma species, Pseudomonas species, may result in the addition of one or more amino acids to the OVA sequence as part of post-transcriptional or post-translational modifications. Such amino acids may not be part of the native OVA sequence. For example, expression of the OVA sequence in Pichia species, such as Komagataella phaffii and Komagataella pastoris, may result in the addition of one or more amino acids at the N-terminus or C-terminus. In some cases, the 4-amino acid EAEA (SEQ ID NO: 75) is added to the N-terminus of the OVA sequence upon expression in the host cell, as shown in SEQ ID NO: 1. For example, chicken rOVA encoding SEQ ID NO: 1 may be provided, and after expression and secretion, rOVA has the amino acid sequence of SEQ ID NO: 2.

[0164] rOVA can be a non-naturally occurring variant of OVA. Such variants can include one or more amino acid insertions, deletions, or substitutions compared to the native OVA sequence.

[0165] Such variants can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NOs: 1-74. As used herein, the term "sequence identity" in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a selected sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity, with any conservative substitutions not being considered part of the sequence identity. Alignments for the purpose of determining the percent amino acid sequence identity can be achieved by various methods within the skill in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the preferred alignment algorithm. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0166] Depending on the host organism used to express rOVA, rOVA can have a pattern of glycosylation, acetylation or phosphorylation that is different from wild-type OVA. For example, the rOVA of the present specification may or may not be glycosylated, acetylated or phosphorylated. rOVA may have a pattern of glycosylation, acetylation or phosphorylation of avian, non-avian, microbial, non-microbial, mammalian or non-mammalian origin.

[0167] In some cases, rOVA may be deglycosylated (e.g., chemically, enzymatically, Endo-H, PNGase F, O-glycosidase, neuraminidase, β1-4 galactosidase, β-N-acetylglucosaminidase), deacetylated (e.g., protein deacetylase, histone deacetylase, sirtuin), or dephosphorylated (e.g., acid phosphatase, lambda protein phosphatase, calf intestinal phosphatase, alkaline phosphatase). Deglycosylation, deacetylation or dephosphorylation can produce proteins that can produce a more uniform or less variable composition.

[0168] rOVA is recombinantly expressed in a host cell. As used herein, "host" or "host cell" herein refers to any protein-producing host that has been selected or genetically modified to produce the desired product. Exemplary hosts include fungi such as filamentous fungi, as well as bacteria, yeast, plants, insects, and mammalian cells. Host cells include Arxula spp., Arxula adeninivorans, Kluyveromyces spp., Kluyveromyces lactis, Komagataella phaffii, Pichia spp., Pichia angusta, Pichia pastoris, Saccharomyces spp., Saccharomyces cerevisiae, Schizosaccharomyces spp., Schizosaccharomyces pombe, Yarrowia spp., Yarrowia lipolytica, Agaricus spp., Agaricus bisporus, Aspergillus spp., Aspergillus awamori, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Colletotrichum spp., Colletotrichum gloeosporiodes, Endothia spp., Endothia parasitica, Escherichia coli, Fusarium spp., Fusarium graminearum, Fusarium solani, Mucor spp., Mucor miehei, Mucor pusillus, Myceliophthora spp., Myceliophthora thermophila, Neurospora spp., Neurospora crassa, Penicillium spp., it may also be Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicillium funiculosum, Penicillium purpurogenum, Penicillium roqueforti, Pleurotus spp., Pleurotus ostreatus, Rhizomucor spp., Rhizomucor miehei, Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma spp., Trichoderma altroviride, Trichoderma reesei or Trichoderma vireus. The host cell can be an organism approved as generally safe by the US Food and Drug Administration.

[0169] The rOVA protein can be recombinantly expressed in yeast, filamentous fungi or bacteria. In some embodiments, the rOVA protein is recombinantly expressed in Pichia species (Komagataella phaffii and Komagataella pastoris), Saccharomyces species, Trichoderma species, Pseudomonas species or E. coli species.

[0170] Expression of rOVA can be provided by an expression vector, plasmid, nucleic acid integrated into the host genome, or other means. For example, the vector for expression can include (a) a promoter element, (b) a signal peptide, (c) an OVA sequence heterologous to the host cell, and (d) a terminator element.

[0171] Expression vectors that can be used for the expression of OVA include those containing an expression cassette having elements (a), (b), (c) and (d). In some embodiments, the signal peptide (b) need not be included in the vector. Generally, the expression cassette is designed to mediate the transcription of the transgene when integrated into the genome of a cognate host microorganism.

[0172] For assistance in the amplification of the vector prior to transformation of the host microorganism, an origin of replication (e) may be contained in the vector (such as PUC_ORIC and PUC (DNA2.0)). For assistance in the selection of microorganisms stably transformed with the expression vector, the vector may also contain a selection marker (f) such as the URA3 gene and the zeocin resistance gene (ZeoR). The expression vector may also contain a restriction enzyme site (g) that allows for the linearization of the expression vector prior to transformation of the host microorganism to facilitate stable integration of the expression vector into the host genome. In some embodiments, the expression vector may contain any subset of elements (b), (e), (f) and (g), including without elements (b), (e), (f) and (g). Other expression elements and vector elements known to those skilled in the art can be used in combination with or in place of the elements described herein.

[0173] Exemplary promoter elements (a) may include, but are not limited to, constitutive promoters, inducible promoters, and hybrid promoters. Promoters may include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, α-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cell filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), G1, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, α-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, β-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxine 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PHO89, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserine aminotransferase (SER1), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, and any combination thereof.

[0174] A signal peptide (b), also known as a signal sequence, target signal, localization signal, localization sequence, signal peptide, transport peptide, leader sequence or leader peptide, can assist in the secretion of a protein or polynucleotide. Extracellular secretion of a recombinant or heterologous expressed protein from a host cell can facilitate protein purification. The signal peptide may be derived from a protein precursor (e.g., prepropeptide, preprotein). The signal peptide can be derived from a protein precursor other than the signal peptide in native OVA. An example of a secreted protein is the S. cerevisiae alpha factor prepro sequence shown in bold and underlined in SEQ ID NO: 1.

[0175] Any nucleic acid sequence encoding OVA can be used as (c). Preferably, such a sequence is codon-optimized for the host cell.

[0176] Exemplary transcriptional terminator elements include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, α-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cell filament polypeptide (cfp), cpc-2, ctr4+, CUP1, dihydroxyacetone synthase (DAS), enolase (ENO, ENO1), formaldehyde dehydrogenase (FLD1), FMD, formate dehydrogenase (FMDH), G1, G6, GAA, GAL1, GAL2, GAL3, GAL4, GAL5, GAL6, GAL7, GAL8, GAL9, GAL10, GCW14, gdhA, gla-1, α-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUT1), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, β-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxine 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PHO89, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserine aminotransferase (SER1), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THI11, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, and any combination thereof.

[0177] Exemplary selectable markers (f) include, but are not limited to, antibiotic resistance genes (e.g., zeocin, ampicillin, blasticidin, kanamycin, nourseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), auxotrophic markers (e.g., ade1, arg4, his4, ura3, met2, and any combination thereof).

[0178] In one example, a vector for expression in Pichia sp. can include an AOX1 promoter operably linked to a signal peptide (alpha mating factor) fused in-frame with a nucleic acid sequence encoding OVA, and a terminator element (AOX1 terminator) immediately downstream of the nucleic acid sequence encoding OVA.

[0179] In another example, a vector containing a DAS1 promoter is operably linked to a signal peptide (alpha mating factor) fused in-frame with a nucleic acid sequence encoding OVA, and a terminator element (AOX1 terminator) immediately downstream of OVA.

[0180] The recombinant proteins described herein may be secreted from one or more host cells. In some embodiments, the rOVA protein is secreted from the host cell. The secreted rOVA may be isolated and purified by methods such as centrifugation, fractionation, filtration, ion exchange chromatography, affinity purification, and other methods for separating proteins from cell, liquid and solid media components, and other cell products and by-products. In some embodiments, rOVA is produced in Pichia sp. and secreted from the host cell into the culture medium. The secreted rOVA is then separated from other medium components for further use.

[0181] The present disclosure contemplates modifying the glycosylation of recombinant OVA in order to change or enhance one or more functional properties of the protein and / or its production. In some embodiments, the changes in rOVA glycosylation can result from the host cell that glycosylates the rOVA. In some embodiments, rOVA has a glycosylation pattern that is not identical to native ovalbumin (nOVA), e.g., nOVA from chicken eggs. In some embodiments, rOVA is treated with a deglycosylating enzyme before it is used as a starting material in an rOVA composition or when the rOVA is present in the composition. In some embodiments, the glycosylation of rOVA is modified or removed by expressing one or more enzymes in a host cell and exposing the rOVA to the one or more enzymes. In some embodiments, the rOVA, and the one or more enzymes for modification or removal of glycosylation are co-expressed in the same host cell.

[0182] For example, native ovalbumin (nOVA) isolated from chicken or other avian eggs has a very complex branched pattern of glycosylation. The glycosylation pattern includes N-linked glycan structures such as N-acetylglucosamine units, galactose, and N-linked mannose units. See, for example, FIG. 1A. In some cases, rOVA produced using the methods described herein for use in ingestible compositions disclosed herein has a glycosylation pattern different from that of nOVA. For example, when rOVA is produced in Pichia sp., the protein may be glycosylated differently from nOVA and may lack galactose units in N-linked glycosylation. FIG. 1B illustrates the glycosylation pattern of rOVA produced by P. pastoris, which shows a complex branched glycosylation pattern. In some embodiments of the compositions and methods disclosed herein, rOVA is processed such that the glycosylation pattern is modified from that of nOVA and is also modified compared to rOVA produced by Pichia sp. without such processing. In some cases, rOVA lacks glycosylation.

[0183] The molecular weight of rOVA may be different compared to nOVA. The molecular weight of the protein may be less than that of nOVA or less than that of rOVA produced by a host cell in which the glycosylation of rOVA has not been modified. In embodiments, the molecular weight of rOVA may be from 40 kDa to 55 kDa. In some cases, rOVA having modified glycosylation has a different molecular weight compared to, for example, native OVA (produced by an avian host species), or compared to a host cell that glycosylates rOVA, such as when rOVA includes N-linked mannosylation. In some cases, the molecular weight of rOVA is greater than that of rOVA in which post-translational modifications have been completely avoided or rOVA that lacks all forms of N-linked glycosylation. Definitions

[0184] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

[0185] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0186] The terms "including", "includes", "having", "has", "with" or their variants are used in either the detailed description and / or the claims, and such terms are intended to be construed in a manner similar to the term "comprising".

[0187] Ranges can be expressed herein as from a particular value with "about" or "approximately" and / or to another particular value with "about" or "approximately". When such a range is expressed, another case includes from a particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about" or "approximately", it is understood that the particular value forms another case. It is further understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. The term "about" or "approximately" as used herein refers to a range of plus or minus 15% from the numerical value mentioned within the context of a particular usage. For example, about 10 would include the range of 8.5 to 11.5. The term "about" or "approximately" also consists of typical errors or inaccuracies in the measurement of a value.

[0188] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein.

Examples

[0189] (Example 1) Preparation of Recombinant Ovalbumin The Gallus gallus OVA coding sequence was fused in-frame with the alpha mating factor signal sequence downstream of the promoter sequence (SEQ ID NO: 1). The promoter was placed upstream of the signal sequence OVA coding sequence, and the transcription terminator was placed downstream of the OVA sequence. The expression construct was placed in the Kpas-URA 3 vector.

[0190] The expression construct was transformed into Pichia pastoris. Successful integration was confirmed by genomic sequencing.

[0191] Fermentation: Recombinant OVA was produced in a bioreactor under ambient conditions. A seed train for the fermentation process was initiated by inoculating a shake flask with liquid growth broth using a 2 ml cryovial of Pichia pastoris stored at -80 °C and thawed at room temperature before inoculation.

[0192] The inoculated shake flask was kept at 30 °C for 24 hours in a shaker, and then the grown Pichia pastoris was transferred to a production-scale reactor.

[0193] The culture was grown at 30 °C at a set pH and dissolved oxygen (DO). A carbon source was supplied to the culture. At the end of fermentation, the target OVA protein was recovered from the supernatant.

[0194] Cell debris was removed, the protein was purified, and lyophilized to a dry powder. The produced OVA was used in the examples described below. (Example 2) Cooking of Egg-Free Cake Using Recombinant Ovalbumin

[0195] An eggless pound cake can be prepared using the following ingredients. A first ingredient composition was prepared by mixing 2% - 5% recombinant ovalbumin and 0.05% - 0.5% sunflower lecithin. To prepare the pound cake, up to 4% of the dried first ingredient composition was added to 22 - 26% unsalted butter, 20 - 25% all-purpose flour, 18 - 26% water, 20 - 25% sugar, 4 - 6% sour cream, 1.2% baking powder, 0.4% vanilla flavor, 0.05 - 1.5% gums and starches, and 0.18% salt, and then all the ingredients were mixed to make a batter. For this recipe, recombinant ovalbumin can be used at 2 - 5% and sunflower lecithin at 0.05 - 0.5%.

[0196] In one example, pound cakes using rOVA and whole eggs (for comparison) were made as follows. [Table 2] [Table 3] [Table 4]

[0197] For each recipe, the batter was baked at 325°F until cooked to the point where nothing sticks when a toothpick is inserted into the center of the cake. [Table 5]

[0198] Textural qualities such as cohesiveness, resilience, hardness, chewiness, and springiness were measured using a Brookfield CT3 Texture Analyzer with a 1500 g load cell. No significant differences were observed between the control egg cakes and those made with rOVA with respect to the nature of the texture and the height of the cake. The sensory properties were equivalent to those of the control cakes made with whole eggs. .

[0199] rOVA in pound cake demonstrated several functional characteristics, along with its usefulness in baked goods and other food products and ingredients. The results are shown in Figure 2.

Table 6

[0200] This example investigated the feasibility of making meringue using rOVA in a recipe without cream of tartar. using to make meringue.

[0201] Materials: rOVA (as is, pH: 4.12), nOVA (as is, pH: 6.06), fresh egg white (as is, pH: 9), xanthan gum, sodium lauryl sulfate (SLS), cream of tartar, granulated sugar, flavor.

[0202] Equipment: Kitchen Aid, Classic Plus, Breville BOV800XL Smart Electric Oven

[0203] Method: At refrigerated temperature, carefully separate the egg white from the egg yolk, then bring the egg white to room temperature and whip it. Control meringue samples were made using the egg white. Test samples were made using nOVA or rOVA. The egg white, or nOVA or rOVA solution (10% solution) was transferred to a mixing bowl and whipped at medium speed for 30 seconds (to obtain a homogeneous solution), then cream of tartar was added (for egg white only) and mixed at high speed until soft peaks formed. While stirring constantly, sugar was gradually added and stirred at high speed after each addition until the sugar was completely dissolved. Mixing was continued until shiny, stable peaks formed, and finally, flavor was added. The soft meringue mix was transferred to a pan. The oven was preheated to 250°F and the meringue was baked for 50 minutes (or until an internal temperature of 160°F). After cooling, the meringue was stored in an airtight container.

[0204] Exemplary meringue recipes using rOVA may contain 5 - 10% rOVA, about 26 - 32% sugar, flavorings (e.g., 1 - 4%), about 59 - 64% water, about 0.01 - 0.5% xanthan gum, and about 0.01 - 0.1% sodium lauryl sulfate (all w / w). One such exemplary recipe, and comparative recipes with fresh egg white or native OVA or rOVA, were constructed as shown below. [Table 7] [Table 8]

[0205] Conclusion: The lowest weight loss was observed in the meringue using rOVA. Furthermore, the rOVA meringue showed the highest fluffiness compared to the egg control and nOVA. The results are presented in Figure 3.

[0206] The use of rOVA in meringue demonstrates some functional characteristics of rOVA. [Table 9] (Example 4) Comparison of Foam Volume and Foam Stability

[0207] This example evaluated the foam volume / stability and coagulation properties of rOVA and compared it with fresh whole eggs, egg white, and nOVA.

[0208] Materials: Commercially available eggs, nOVA (Bioceutica), rOVA

[0209] Method: A stock solution of OVA (nOVA or rOVA) was prepared by mixing 0.7 g of OVA in 9.3 g of distilled water (total volume 10 ml). The pH was adjusted using tartaric acid (see Table 10 below). Bubbles were created using a Dremel at speed 3. The whipping time was recorded. The gel was prepared by heating 1 ml of the sample at 72 °C for 10 minutes using a heat block.

Table 10

[0210] The results of the bubble volume and stability are shown in Table 11 below. In this setting, the pH was not adjusted.

[0211] * Bubble volume % = [(Initial liquid volume (ml) / Bubble volume (ml))] × 100

[0212] ** Bubble stability % = [((Initial liquid volume (ml) - Liquid drainage volume at 30 minutes (ml)) / Initial liquid volume (ml))] × 100

Table 11

[0213] Conclusion: nOVA at pH 6 showed the highest bubble volume compared to egg white, however, its bubble stability was lower than that of egg white. The results are presented in Figure 4.

[0214] The experiment was repeated using tartaric acid to prepare the pH.

Table 12

[0215] Conclusion: The foam volume of nOVA after pH reduction was still higher than that of egg white. The foam volume of rOVA was higher compared to that of fresh egg white. The whipping time for rOVA was half the time required for fresh egg white. The results are shown in Figure 5. (Example 5) Preparation of recombinant chicken ovalbumin-expressing strain

[0216] Expression construct. Seven expression cassettes were created for the expression of Gallus gallus OVA (SEQ ID NO: 2) in Pichia pastoris. [Table 13]

[0217] The first three cassettes were made to express chicken OVA containing the amino acid sequence of chicken OVA (SEQ ID NO: 2) fused in-frame with the nucleic acid encoding the secretion signal sequence, and the expressed fusion protein has the amino acid sequence of (SEQ ID NO: 1). In each of the three cassettes, the alcohol oxidase 1 (AOX1) promoter was placed upstream of the secretion signal sequence, and the K phaffii AOX1 transcription terminator was placed downstream of the OVA coding sequence. These cassettes were labeled GgOVA-A1, GgOVA-A2, and GgOVA-A3 and combined into the first plasmid.

[0218] The fourth cassette contained the chicken OVA coding sequence (encoding SEQ ID NO: 2) fused in-frame with the nucleic acid encoding the secretion signal sequence (thereby encoding SEQ ID NO: 1), but the glyceraldehyde 3-phosphate dehydrogenase (DAS2) promoter was placed upstream of the secretion signal sequence, and the K phaffii AOX1 transcription terminator was placed downstream of the OVA coding sequence. This construct was labeled GgOVA-D1.

[0219] Cassettes 5 and 6 were in-frame and contained the chicken OVA coding sequence (encoding SEQ ID NO: 2) fused to a nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 1), with the formaldehyde dehydrogenase (FLD) promoter placed upstream of the secretion signal sequence and the K. phaffii AOX1 transcription terminator placed downstream of the OVA coding sequence. These cassettes were labeled GgOVA-F1 and GgOVA-F2 and combined with GgOVA-D1 in the second plasmid.

[0220] Cassette 7 contained the peroxisome biogenesis (PEX11) promoter placed upstream of the helper factor protein HAC1 coding sequence and the K. phaffii AOX1 transcription terminator placed downstream of the helper factor sequence. This cassette was labeled HF-1 and transformed into the third plasmid.

[0221] The three plasmids were transformed stepwise into a strain in the background of Pichia pastoris. Genome sequencing confirmed the integration of the expression constructs, and the copy numbers of each construct are shown in Table 14 below.

Table 14

[0222] Expression constructs: One cassette for the expression of Anas platyrhynchos (duck) OVA and one cassette for the expression of Struthio camelus (ostrich) OVA were created for expression in Pichia pastoris.

Table 15

[0223] One expression cassette was created for the expression of ostrich OVA. The nucleic acid encoding Struthio camelus OVA (SEQ ID NO: 71) was fused in-frame with the nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 72). The ostrich construct contained the alcohol oxidase 1 (AOX1) promoter located upstream of the secretion signal sequence and the K phaffii AOX1 transcription terminator located downstream of the OVA sequence. This expression cassette, called ScOVA, was transformed into Pichia pastoris. Successful integration of four copies of the ostrich OVA construct was confirmed by genomic sequencing. See Table 15.

[0224] One expression cassette was created for the expression of duck OVA. The nucleic acid encoding Anas platyrhynchos OVA (SEQ ID NO: 73) was fused in-frame with the nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 74). The duck cassette contained the alcohol oxidase 1 (AOX1) promoter located upstream of the secretion signal sequence and the K phaffii AOX1 transcription terminator located downstream of the OVA sequence. This expression cassette, called ApdOVA, was transformed into Pichia pastoris. Successful integration of two copies of the duck OVA construct was confirmed by genomic sequencing. See Table 16. [Table 16] (Example 7) Fermentation and production of rOVA

[0225] Fermentation: Strains for fermenting recombinant OVA (rOVA) were each cultured in a bioreactor under ambient conditions. A seed train for the fermentation process was initiated by inoculating a shake flask with liquid growth broth. The inoculated shake flask was kept in a shaker and then the grown P. pastoris was transferred to a production-scale reactor.

[0226] To expand production, the seed vials of the rOVA P. pastoris strain were taken out of cryopreservation and thawed to room temperature. The contents of the thawed seed vials were used to inoculate the liquid seed culture medium in baffled flasks grown at 30 °C in an orbital incubator. These seed flasks were then transferred to a series of even larger seed fermenters (the number varies depending on the scale) containing basal salt medium, trace metals and glucose and grown. The temperature in the seed reactors was controlled at 30 °C, pH 5 and dissolved oxygen (DO) 30%. The pH was maintained by supplying ammonia hydroxide which also acts as a nitrogen source. Once sufficient cell mass was reached, the grown rO VA P. pastoris was inoculated into a production-scale reactor containing basal salt medium, trace metals and glucose.

[0227] As in the seed tank, the culture was also controlled at 30 °C, pH 5 and 30% DO throughout the process. The pH was maintained by supplying ammonia hydroxide again. During the initial batch glucose stage, the culture was left to consume all the glucose and the ethanol produced thereafter. Once the target cell density was achieved and the glucose and ethanol concentrations were confirmed to be zero, the glucose fed-batch growth stage was initiated. In this stage, glucose was supplied until the culture reached the target cell density. Glucose was supplied at a restricted rate to prevent ethanol build-up in the presence of non-zero glucose concentrations. In the final induction stage, the culture was co-fed with glucose and methanol and induced to produce rOVA via the pAOX promoter. Glucose was supplied in an amount to yield the desired growth rate, while methanol was supplied to maintain a 1% methanol concentration to ensure that expression was constantly induced. Periodic samples were taken throughout the fermentation process for analysis of specific process parameters (e.g., cell density, glucose / methanol concentration, productivity titer and quality). After a specified amount of fermentation time, the secreted rOVA was collected and transferred to downstream processing.

[0228] The fermentation broth containing the secreted rOVA was subjected to centrifugation at 12,000 rpm. The supernatant was clarified using microfiltration. Ultrafiltration at room temperature was used to concentrate the protein and remove excess water. A filter of appropriate size was used to retain the target rOVA while allowing compounds, salts, and water smaller than rOVA to pass through the filter. To reduce the final salt content and conductivity in the preparation for chromatography, the concentrated rOVA residue was dialyzed at pH 3.5 to a final conductivity of 1.7 mS / cm. Bulk purification was performed at pH 3.5 using cation exchange chromatography. A citrate buffer containing a high salt concentration of sodium chloride was used to elute the rOVA bound to the resin. To remove the excess salt, the eluate was subjected to final dialysis to produce a final protein solution containing approximately 5 - 10% protein and 85 - 95% water. The final solution was sterilized by passing it through a 0.2 µm bioburden filter. Water was evaporated at an appropriate temperature using a spray dryer / freeze dryer to produce a final powder containing approximately 80% protein. (Example 8) Preparation of solubilized rOVA

[0229] In this example, the hydrophobic recombinant chicken rOVA was solubilized and passed through a 0.2 µm filter.

[0230] Recombinant rOVA was purified by ion exchange chromatography at pH 3.5 and found to be insoluble. Sodium hydroxide was added to the solution to change the pH to 12.5 to solubilize the rOVA. The rOVA solution at pH 12.5 was passed through a 0.2 µm filter. After filtration, the pH was returned to 6.5 using hydrochloric acid and the rOVA was spray dried or freeze dried. This dried chicken rOVA was then used in the following examples. (Example 9) Glycosylation of Gallus gallus rOVA

[0231] In this example, Pichia - secreted rOVA was analyzed for its glycosylation pattern.

[0232] Native ovalbumin (nOVA) has two potential N - linked glycosylation sites (Figure 1A). A single site of glycosylation at Asn - 292 is found in egg white. MALDI - TOF analysis showed that the typical glycan in native OVA was organized as (Man)5(GlcNAc)5(Gal)1 (Figure 1A) (Harvey et al., 2000). Analysis of the glycans in rOVA showed the typical glycosylation pattern shown (Figure 1B).

[0233] Pichia - secreted chicken rOVA from the above example was analyzed by migration on gel electrophoresis, and three different forms (the three white arrows point to rOVA under the "input" lane, a) non - glycosylated, b) monoglycosylated, and c) diglycosylated) were observed. Both mono - and diglycosylated glycan chains were cleaved from the mature rOVA protein using either EndoH or PNGaseF of endoglycosidase. Both "denaturing" or "native" deglycosylation protocols were used (as described in the NEB catalog). The green arrow indicates exogenous EndoH, and the purple arrow indicates exogenous PNGaseF added to the in vitro reaction (Figure 6A).

[0234] Pichia - secreted chicken OVA was subjected to standard analysis using mass spectrometry. It was found to have five versions of N - linked glycans (ManGlcNAc): high - mannose glycans of the Man9 (about 40%), Man10 (about 47%), or Man11 (about 13%) type of N - glycan structure (Figure 6B). (Example 10) Comparison of the foaming functionality of rOVA from different species

[0235] In this example, chicken rOVA, duck rOVA, and ostrich rOVA were evaluated for their foaming ability and foam retention properties.

[0236] rOVA from ostrich and duck were produced, purified, and lyophilized using a method similar to the method described in Examples 5 - 7. The ostrich rOVA and duck rOVA remained near the acidic pH used for purification. Chicken rOVA was produced as described in Example 5, solubilized at pH 12, then the bioburden was removed, and after returning to pH 6 as described in Example 7, it was dried.

[0237] The lyophilized rOVA samples were blended into distilled water. Then, the clarity and solubility of the rOVA solutions were visually evaluated. All samples were compared to chicken nOVA and chicken rOVA.

[0238] 11 mL of solution (7% w / v protein) was made for each of ostrich rOVA, chicken rOVA, and chicken nOVA. 6 mL of solution (7% w / v protein) was made for duck rOVA due to sample availability limitations. The percent protein of the powder was used in the calculation to determine the amount needed for a 7% solution. 1 mL of each solution was taken out for later use in testing gelation and verified in microtubes before use. The samples were divided into 5 mL aliquots and tested for foam volume and stability.

[0239] Each 5 mL aliquot was pipetted into a beaker and whipped using a Dremel at speed 3. After achieving fluffy foam, the whipping time and the initial volume of the foam were recorded. The foam volume was determined by measuring the initial volume of the foam after whipping and comparing it to the initial volume of 5 mL. Foam volume (%) = (foam volume / initial volume) × 100.

[0240] Drainage was measured for 30 minutes every 10 minutes to accumulate data on the stability of the foam. The volume of liquid drained after 30 minutes was compared with the initial liquid volume (5 mL). Foam stability (%): (Initial volume - Drained volume) / Initial volume × 100.

[0241] Chicken rOVA and ostrich rOVA were adjusted to pH 6 and tested again to elucidate the effect of pH.

[0242] Chicken nOVA quickly formed fluffy white foam. Ostrich rOVA foamed after 15 seconds. Duck rOVA foamed after 20 seconds.

Table 17

[0243] Table 17 shows the results for foaming time, foam volume, and foam stability for chicken nOVA at pH 5.87, chicken rOVA at pH 6.49 and pH 6.08, ostrich rOVA at pH 3.7 and pH 5.73, duck rOVA at pH 4.3, and egg white OVA at pH 9.0. Recombinant OVAs from chicken, duck, and ostrich generally have similar or improved foam volume and foam stability compared to egg white, and these recombinant OVA proteins provided foam volume and foam stability at least from pH 3.5 to 6.5. The foam volume and foam stability of rOVA provide utility in compositions such as baked goods compositions. (Example 11) Comparison of Gelation of Various rOVA Species

[0244] In this example, the rOVA proteins of chicken, duck, and ostrich were evaluated for their gelation properties. The gelation properties provide utility in applications such as cooked egg compositions.

[0245] 1 mL of each OVA solution was removed for use in testing gelation. After the Dremel procedure and the foaming test in Example 10 were completed, another 1 mL sample was extracted from the drained liquid (containing OVA) and pipetted into another microtube. Both the fractions collected before and after foaming were placed in a water bath and heated to 72 °C for 10 minutes. The samples were observed for gel formation.

[0246] Figure 7 shows the results for gelation before and after foaming for chicken nOVA at pH 5.87, chicken rOVA at pH 6.49 and pH 6.08, turkey rOVA at pH 3.7 and pH 5.73, duck rOVA at pH 4.3, and egg white OVA at pH 9.0. Duck rOVA showed better gelation properties compared to chicken rOVA. Duck rOVA had a gelation functionality similar to that of natural egg white.

[0247] These data showed that the favorable properties disclosed above for recombinant chicken OVA (see Example 10) could also be obtained with recombinant OVA from other species. (Example 12) Comparison of foaming of rOVA solutions

[0248] In this example, an rOVA (chicken) solution was compared with fresh egg white for its foaming ability and foam retention properties.

[0249] Lyophilized samples were blended into aqueous solutions (distilled water) of different concentrations and pH. The clarity and solubility of the solutions were then visually evaluated for foaming ability and foam retention.

[0250] Protein solutions were prepared for 4% rOVA, 7% rOVA, fresh egg white (12% protein), and 12% rOVA, respectively. The powdered protein percentage was used in the calculation to determine the amount required for each solution. 1 mL of each solution was aliquoted for later use in testing gelation and verified in microtubes before use. The samples were divided into 5 mL aliquots and tested for foam volume and stability.

[0251] Each 5 mL aliquot was pipetted into a beaker and whipped using a Dremel at speed 3. After achieving fluffy foam, the whipping time and the initial volume of the foam were recorded. The foam volume was determined by measuring the initial volume of the foam after whipping and compared to the initial volume of 5 mL. Foam volume (%) = (foam volume / initial volume) × 100.

[0252] Drainage was measured every 10 minutes for 30 minutes to accumulate data on foam stability. The volume drained after 30 minutes was compared to the initial liquid volume (5 mL). Foam stability (%) = (initial volume - volume drained) / initial volume × 100.

Table 18

[0253] 4%, 7%, and 12% rOVA have higher foam volume, higher foaming stability, and form foam more rapidly than fresh egg white. (Example 13) Browning and gloss properties of rOVA

[0254] In this example, the properties of browning and gloss film formation were evaluated for the functionality of rOVA in bread applications. The functionality of rOVA for film formation was evaluated in terms of the visual (sensory) properties of bread.

[0255] Description of baking bread: Yeast, sugar, and warm water were mixed together in a small bowl and left for 5 minutes. Flour was mixed into the yeast solution (for 30 seconds) until a stable dough was formed (mixed at speed 3 for 2 minutes). The dough was kneaded on a floured board and placed in an oiled bowl and fermented at 80°F for 45 minutes. The dough was kneaded again, shaped into 25g mini-loaves, and placed on an oiled baking sheet. The mini-loaves were covered and fermented at room temperature for 30 minutes. An appropriate liquid of 0.75g volume was applied to the top of the dough balls. The mini-loaves were baked at 350°F for 8 minutes or until golden brown. The location of the bread loaves was alternated in the oven for 4 minutes to achieve uniform baking for all samples.

[0256] The list of raw materials used in the control bread and other samples and their proportions are shown in Table 19 below.

Table 19

Table 20

[0257] Colorimetric analysis assay: Photographs of individual samples were analyzed for color data in the RGB spectrum using the Colorgrab application (Loomatix). Sample values were created using a 2×2 cm cross-section taken from the center of the bread surface. The RGB data was then converted to the CIELAB system using the online software www.colormine.org. The CIELAB model is a color space system that represents color with three values: L * ranges from black (0) to white (100), a * ranges from green (-) to red (+), b * is the lightness ranging from blue (-) to yellow (+).

Table 21

[0258] The rOVA and egg white protein samples had higher L * values and suggested higher lightness or brightness. The control (no egg solution), commercially available egg solution substitutes, and egg white protein samples had low a * values and suggested lower redness or brownness compared to the whole egg and rOVA samples. The 8% egg white protein and rOVA samples also had similar b * values and suggested a similar yellow hue compared to the other samples.

[0259] Visual inspection: The control sample appeared pale, wrinkled, and dull. The samples using whole eggs had good browning, high gloss, and a smooth surface. The use of commercially available egg solution substitutes had a smooth surface and a slight noticeable gloss but lacked browning. The nOVA samples had good brown color and a smooth texture but lacked shine / gloss. Similarly, for the rOVA samples, these had good browning and a smooth texture but lacked shine / gloss. Photographs of the samples are shown in Figure 8. As a conclusion, rOVA was able to form a film to the same extent as commercially available egg solution substitutes and nOVA. (Example 14) Adhesive properties of rOVA

[0260] In this example, rOVA was evaluated for its functional film-forming property of adhesiveness in the application of bread to create a uniform film and assist in the addition of toppings (e.g., sesame seeds).

[0261] Retention of sesame seeds: The retention of any topping on cakes, bread, bagels, or other baked goods is an intended result of egg solution. Sesame seeds were used to evaluate the topping retention function of each film-forming agent after baking.

[0262] Dough balls and the target protein were cooked as in Example 13. Ten sesame seeds were applied to each dough ball after the application of the liquid and before baking. The retention of these sesame seeds was calculated based on the amount of seeds stuck to the bread after baking.

[0263] The following results were obtained. The control sample without egg solution had no binding ability for sesame seeds, and zero sesame seeds were retained on the surface after baking. All other film-forming agents retained all 10 seeds after baking, suggesting a 100% retention rate for the topping. [Table 22] (Example 15) Combined protein rOVA emulsion

[0264] In this example, the functionality of the emulsifying action of the recombinant proteins, individually and in combination, was observed in salad dressing application.

[0265] A list of the raw materials used in the control dressing and other samples and their proportions are presented in Table 23 below. [Table 23]

[0266] Water, acetic acid, and the protein of interest were mixed with a mixer for 30 seconds. Oil was added gradually for 30 seconds and further mixed for 2.5 minutes. The samples were cooked without acetic acid to test the emulsifying ability of the protein at neutral pH. The pH of the solution was adjusted using 1N sodium hydroxide. The emulsion was homogenized at ambient temperature for 9 minutes at 4000 rpm using an L5M-A homogenizer (Silverson) Square Hole share head mixer.

[0267] All emulsion samples were transferred to glass tubes, sealed with plastic caps, and stored at 4 °C or ambient temperature for 3 days. The stability of the samples was evaluated by visually monitoring the height of the visible serum separation in the lower phase over storage time. Physical stability was monitored for 3 days under both ambient and refrigerated conditions. The stability of the emulsion was expressed as the creaming index (CI) = (Ht / H0) × 100, where (H0) represents the initial height of the emulsion and (Ht) represents the height of the visible serum separation layer.

[0268] The list of raw materials used in the control and other salad dressing samples having the specific protein of interest, along with their percentages, is presented in Table 25 below.

Table 24

Table 25

[0269] Results at acidic pH: At day 0, all samples except the negative control showed good emulsifying properties. Subsequently, the samples were stored at ambient or refrigerated temperature and stability was monitored. Samples with egg white protein (EWP) had a slightly yellow appearance and separated on day 1 under both storage conditions. The control sample separated immediately on day 1 under both storage conditions. 8 percent nOVA also showed emulsion breakdown on day 1, however, recombinant OVA showed good emulsion properties with only minimal noticeable separation. The emulsion remained equally stable until day 3 without any further separation being observed. Overall, 8% rOVA worked significantly better than 8% nOVA. rOVA also showed better emulsion stability than EWP. Photographs of the samples are shown in Figure 9A.

[0270] Results at neutral pH: The stability of the rOVA emulsion was comparable to that of egg white protein on day 0 and day 3. Neither rOVA nor egg white protein could maintain the stability of the emulsion over 3 days, either in refrigerated form or at ambient temperature. Photographs of the samples are shown in Fig. 9B. (Example 16) Foaming functionality

[0271] In this example, the foaming functionality of rOVA was observed in alcoholic beverages (e.g., whiskey sours containing a foaming agent, etc.).

[0272] Bourbon whiskey, fresh lemon juice, simple syrup and the protein of interest were mixed in a cocktail shaker and shaken for 15 seconds. Ice was added to the cocktail shaker and the mixture was shaken for an additional 15 seconds. The shaken mixture was poured into a glass and observed.

[0273] Formulation: The control formulation contained natural egg white. The negative formulation was prepared without any egg white. [Table 26] Using the protein of interest to replace the natural egg white protein, the following formulations were used. [Table 27]

[0274] The pH of the rOVA solution was adjusted to pH 6 (with 1 M NaOH) to provide optimal foaming performance.

[0275] The original recipe using 0.5 oz of egg white and the same ratio was used for the recombinant protein test. 7% and 12% rOVA foamed well, but no significant difference was observed between the two levels.

[0276] Photographs of the craft cocktails prepared with the samples are shown in Fig. 10. (Example 17) Cohesiveness of the Hamburger

[0277] In this example, texture analysis was used to observe the hardness characteristics of both raw and cooked vegan hamburgers made with rOVA and other binders, along with cohesiveness, elasticity, and chewiness.

[0278] The purpose of this example was to evaluate the functionality of the binding property of rOVA. Parameters such as aspects of texture such as appearance (how well the hamburger holds together), cohesiveness, elasticity, chewiness, and hardness were evaluated and compared against egg white, nOVA, and commercially available non - protein binders being used.

[0279] Materials: Dry ingredients: Extruded soy protein 1 (Arcon T U172 (158172)), Extruded soy protein 2 (Arcon T Caramel Crumble 240 (158225)), Extruded soy protein 3 (Arcon T U - 118 (158118)), binder / protein of interest. Wet ingredients: Canola oil, coconut oil, water. Binders of interest to be tested: Natural egg white protein (“NEW”), Methylcellulose (“MC”), nOVA 90% protein content, rOVA (chicken) 92% protein content.

[0280] Mixing: Extruded soy protein 1 was mixed with 1 / 3 amount of water for 2.5 minutes. The remaining extruded samples and water were combined with the previous mixture for an additional 7.5 minutes. The blend was chilled in the freezer for 10 minutes. The binder was added and mixed for 30 seconds. A blend of canola oil and coconut oil was added and mixed for 30 seconds. The mixture was chilled in the freezer for 5 minutes, then formed into 5 - g hamburger shapes and frozen.

[0281] Cooking: The frozen hamburger samples were thawed in the refrigerator to an internal temperature of 4°C. The samples were cooked on a griddle set at 350°F for 5 - 6 minutes until they reached an internal temperature of 165°F.

[0282] Formulation: The list of raw materials used in the control and other experimental burger samples having the specific proteins of interest and their proportions are presented in Table 28 below. [Table 28]

[0283] Texture analysis: Texture analysis was performed to analyze the characteristics of the vegan burger relative to the control. Texture analysis was used to quantify the hardness characteristic along with cohesiveness, elasticity, and chewiness.

[0284] The texture properties of the vegan burger were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). The test parameters used are presented in Table 29. [Table 29]

[0285] Frozen samples were thawed in a refrigerator to an internal temperature of 4 °C and tested for raw cohesiveness. The thawed samples were also cooked and used to measure the cooked cohesiveness values.

[0286] Findings on raw cohesiveness: With respect to hardness, rOVA was significantly higher than methylcellulose and native egg white, and no difference was observed between nOVA and rOVA. All samples were similar with respect to cohesiveness and elasticity. rOVA showed significantly better chewiness than methylcellulose and native egg white. The results are presented in Table 30.

[0287] Table 30: Texture profile analysis (TPA) results for raw cohesiveness with respect to hardness, cohesiveness, elasticity, and chewiness. Data not sharing the same letter within a specific characteristic are significantly different from each other (p < 0.05). Results were averaged over n = 3. [Table 30]

[0288] Findings on cooked cohesiveness: rOVA showed significantly higher hardness values than methylcellulose and native egg white. All samples were similar to each other with respect to cohesiveness. For elasticity, the methylcellulose sample showed significantly lower values than native egg white, nOVA, and rOVA. Both the nOVA and rOVA samples showed higher chewiness values than methylcellulose. The results are presented in Table 31.

[0289] Table 31: Results of texture profile analysis (TPA) for cooked cohesiveness regarding hardness, cohesiveness, elasticity, and chewiness. Data not sharing the same letter within a particular trait are significantly different from each other (p < 0.05). Results are averaged over n = 3. [Table 31] (Example 18) Egg white patty

[0290] In this example, the suitability of inclusion of native and recombinant protein OVA in the application of egg white patties as an example of cooked egg systems was evaluated. Parameters such as the nutritional value of fresh egg white when replaced by OVA, and the effect on texture (from a functionality perspective), and appearance were evaluated.

[0291] [Table 32]

[0292] Mixing: The dry ingredients from Table 32 were mixed together, excluding sodium alginate. Tapioca syrup, sodium alginate, and lemon yellow color were blended separately in water. All the ingredients were mixed with oil and vortexed until all the ingredients were dissolved. The mixture was equilibrated by leaving it for 10 minutes.

[0293] Cooking: The samples were cooked using a griddle. The griddle was set to 250°F, and a 1 / 2-inch diameter ring mold was used to cook the samples. The mold was sprayed with oil, and the mixture was placed into the mold. 1 / 2 cube ice was added to the mold to generate steam. The patty was cooked, and another cube ice was added. The patty was cooked for 5 minutes, and the lid was opened. The ring molds the cooked samples to fit the serving plate.

[0294] The textural properties of the egg white patties were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). The TPA compression test was used to compress and measure the hardness of the egg white patties. Four samples from each set were analyzed and compared. The following test parameters were used.

Table 33

Table 34

[0295] Findings: All samples of native egg white, nOVA, and rOVA were statistically similar with respect to hardness, adhesiveness, fracturability, cohesiveness, and gumminess. For chewiness, the native egg white patties were individually similar to nOVA and rOVA, however, nOVA and rOVA were statistically different from each other. nOVA had a higher chewiness value compared to the other samples. Overall, OVA proteins provide a good alternative to native egg white in both native and recombinant forms in non-animal patties (cooked egg applications). The rOVA liquid formulation was more viscous than the nOVA samples and the egg white samples. The results are shown in Figure 11. (Example 19) Meringue

[0296] In this example, the functionality of rOVA in a meringue food system was evaluated compared to fresh egg white.

[0297] Materials: - rOVA (Lyo 008; remaining at pH: 6.7) - Fresh egg white (remaining at pH: 9) - Sugar (C&H Sugar, Pure Cane, granular white) - Xanthan - pre - hydrated Ticaxan - Tic Gums - TEC (triethyl citrate) - SLS (sodium lauryl sulfate) - Kitchen Aid, Classic Plus - Breville BOV800XL Smart electric oven

[0298] Method: The egg white was carefully separated from the egg yolk at refrigerated temperature, and after bringing the egg white to room temperature, it was whipped. The rOVA powder, SLS, xanthan gum, and TEC were re - constituted in DI water at room temperature. The mixture was whipped at speed 5 for 30 seconds (to obtain a homogeneous solution), then mixed at speed 8 until soft peaks formed. While stirring constantly, sugar was gradually added, and after each addition, it was stirred at high speed until the sugar dissolved before the next addition. Mixing was continued until a shiny, stable peak formed. The oven (Breville BOV800XL Smart electric oven) was heated to 200°F, and the meringue was baked for 70 minutes (or until it was light and fluffy but did not turn brown). After cooling, the meringue was stored in an airtight container. The whipping time until stable foam formed was recorded for each protein solution.

Table 35

Table 36

[0299] Finding: rOVA produced a meringue comparable to that of fresh egg white samples in terms of physical parameters. The appearance of the rOVA meringue was visually better than that of the fresh egg white control. The ridges were more distinct in the rOVA meringue, and the sample was whiter compared to the fresh egg white control. The results are shown in Figure 12. (Example 20) Effect of pH on Gelation Properties

[0300] In comparison with fresh egg white, the effect of different pH conditions on the gelation properties of the rOVA composition was evaluated in this example. [Table 37]

[0301] Method: A 1.7% protein solution was prepared for both rOVA and egg white protein. Based on the native pH, the pH of the solution was adjusted to pH 3, 4, 5, 6 using 1N HCl. The pH was also adjusted to the alkaline spectrum of pH 7, 8, 9, 10, 11, and 12 using microliter amounts of 1N and 3N sodium hydroxide. All solutions were gelled at 85°C for 5 minutes and then cooled to room temperature. All gels / solutions were taken and visually evaluated for gel properties. [Table 38]

[0302] Findings: Ovalbumin formed gels that were stable at pH 4 - 10 and showed gelling properties at all pH values. Solutions for both EWP and rOVA at pH 11 and pH 12 were clear liquids, however, only EWP gelled into a clear gel while rOVA remained as a solution at pH 11 and 12. The 7% rOVA solution gelled at pH 6, 7, 8, and 9. A dramatic increase in viscosity was observed for the rOVA solution at pH 5 and below. All EWP gels had a strong egg - like odor, while for rOVA, only the solutions / gels at pH 9 - 12 had an egg - like odor. For rOVA, pH 3.5 - 8 had no characteristic odor properties. Both EWP and rOVA gelled at pH 6 - 9, however, the EWP gels were stronger and more stable than the rOVA gels. Overall, EWP showed better gelling properties than rOVA over a wider pH spectrum, which was accompanied by the presence of a strong egg - like odor. rOVA provided gelling properties and functional neutrality (e.g., no odor) in the range of pH 6 - 8. At pH 8 and 9, rOVA provided clear and stable gels that could have unique value in embodiments requiring a clear visual appearance. (Example 21) Protein bar

[0303] rOVA was used as a protein source in the application of protein bars and compared with egg white proteins (effwhite proteins) and nOVA.

[0304] Cooking instructions:

[0305] In a small mixer, dates and nuts were chopped / blended. Dates, nuts, cocoa, and the target protein were added to a mixing bowl until a homogeneous mixture was formed. The mixture was divided into two equal parts, and one part was tested as an unbaked version. The other half was baked in an oven at 350°F for 10 minutes.

Table 39

[0306] For the formulation involving the inclusion of protein powder, the inclusion of dates and nuts was reduced, however, the ratio of dates:nuts was kept constant at a level of 4.5. [Table 40] [Table 41] [Table 42]

[0307] Texture analysis: The texture properties of protein bars (baked and unbaked) were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). A three-point bend test was used to measure the fold, bend, and hardness of the protein bars. One sample was analyzed for each protein inclusion level. The following test parameters were used. [Table 43] [Table 44] [Table 45]

[0308] For the un-baked samples, the protein-free control samples had the lowest hardness values. For all of the target proteins of EWP, nOVA, and rOVA, the hardness values increased with the increase in protein content. The egg white protein samples at 8, 12, 16, and 23% had higher hardness values than the nOVA and rOVA samples. The nOVA samples had a minimal increase in hardness from 12 - 23% protein inclusion. The hardness of the nOVA and rOVA samples was comparable at 4, 8, 12, and 16%. However, rOVA had a much higher hardness value for 23% protein inclusion.

[0309] Overall, the hardness of the baked samples was much higher than that of the un-baked samples. The control samples had the lowest hardness. All samples with protein inclusion were much harder even at lower protein inclusion ratios. The upper threshold value (load cell) for TA units is 1500 g. All baked protein samples reached the threshold, which made it difficult to identify subtle differences between the samples. The hardness of the nOVA and rOVA samples was comparable at 4, 8, 12, and 16% for both un-baked and baked protein bars. Photographs are shown in Figure 13.

[0310] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, variations, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby. Incorporation by reference

[0311] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0312] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be realized, the present disclosure is capable of other different embodiments and that several details thereof are capable of modifications in various obvious respects all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. The present invention provides, for example, the following items. (Item 1) A raw material composition for producing an egg-free food, wherein the composition contains recombinant ovalbumin (rOVA), when solubilized, the pH of the rOVA is from about 3.5 to about 7.0, when present in the egg-free food, the rOVA is in an amount of from about 2% to about 15% (w / w), the rOVA provides at least one egg white property selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturizing, water retention, clarification, and aggregating property to the egg-free food, A raw material composition. (Item 2) The raw material composition according to Item 1, wherein the composition is dried or is in powder form. (Item 3) The raw material composition according to Item 1 or Item 2, wherein the composition contains at least 75% of rOVA (w / w of total protein or w / w of total composition). (Item 4) The raw material composition according to Item 3, wherein the powdered composition is at least about 80%, at least about 85%, or at least about 90% rOVA (w / w). (Item 5) The raw material composition according to item 3 or item 4, wherein the powder is a concentrate. (Item 6) The raw material composition according to any one of items 2 to 5, wherein the powder composition is an isolate. (Item 7) The raw material composition according to item 1, wherein the composition is a liquid. (Item 8) The raw material composition according to item 7, wherein the liquid composition is a concentrate. (Item 9) The raw material composition according to item 7 or item 8, wherein the liquid composition contains at least 50% rOVA (w / w of total protein or w / w of the composition). (Item 10) The raw material composition according to item 7, wherein the liquid composition contains at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85% or at least about 90% rOVA (w / w). (Item 11) The raw material composition according to any one of items 1 to 3, wherein the rOVA provides equivalent properties or improved properties as compared to native egg white in a similar food. (Item 12) The raw material composition according to any one of items 1 to 11, wherein the rOVA provides a foam volume that is at least 20%, 30%, 40% or 50% higher than that of native egg white. (Item 13) The raw material composition according to any one of items 1 to 12, wherein the rOVA provides a time to foam that is at least 20%, 30%, 40% or 50% faster than that of native egg white. (Item 14) The raw material composition according to item 12 or item 13, wherein when solubilized, the pH of the rOVA is about 3.5 to about 4.5. (Item 15) The raw material composition according to any one of items 1 to 14, wherein the rOVA provides a hardness equal to or higher than that of native egg white in a food composition that does not contain eggs in a baked embodiment. (Item 16) The raw material composition according to any one of Items 1 to 15, wherein the rOVA provides a higher chewiness than native egg white to the food composition not containing the egg. (Item 17) The raw material composition according to Item 15 or Item 16, wherein the rOVA provides elasticity comparable to that of native egg white. (Item 18) The raw material composition according to any one of Items 1 to 17, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 19) The raw material composition according to any one of Items 1 to 18, wherein the rOVA comprises the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 20) The raw material composition according to Item 18 or Item 19, wherein the amino acid sequence of the rOVA lacks the N-terminal methionine. (Item 21) The raw material composition according to Item 20, wherein the rOVA further comprises the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. (Item 22) The raw material composition according to any one of Items 1 to 21, wherein the rOVA comprises the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, and the rOVA provides improved gelation. (Item 23) The raw material composition according to any one of Items 1 to 21, wherein the rOVA comprises the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, and the rOVA provides improved gelation. (Item 24) The raw material composition according to any one of Items 1 to 21, wherein when solubilized, the pH is about 6 to about 6.8. (Item 25) The raw material composition according to any one of items 1 to 21, wherein when solubilized, the pH of the rOVA is less than about 6.1. (Item 26) The raw material composition according to any one of items 1 to 25, wherein the rOVA is present in a food product that does not contain the egg in an amount of less than about 8%. (Item 27) The raw material composition according to any one of items 1 to 25, wherein the rOVA is present in a food product that does not contain the egg in an amount of about 7% or less. (Item 28) The raw material composition according to any one of items 1 to 27, wherein the rOVA does not contaminate the composition with Salmonella. (Item 29) Recombinant ovalbumin (rOVA), wherein when solubilized, the pH of the rOVA is from about 3.5 to about 7.0, rOVA, at least one fat or oil, at least one cereal starch, and at least one sweetener A baked food product comprising wherein the rOVA provides at least one egg white property selected from binding, elasticity, air incorporation, browning, texturization, water retention, and cohesiveness to the baked food product, the baked food product does not contain any natural egg white protein or natural egg white, Baked food product. (Item 30) The baked food product according to item 29, wherein the rOVA is present in the product at about 2% to 15% (w / w of the total protein before baking or w / w of the total food product). (Item 31) The baked food product according to item 30, wherein the rOVA is present in the product at about 2% to about 5% (w / w). (Item 32) The baked food product according to item 29 or item 30, further comprising a milk component or a leavening agent, or a combination thereof. (Item 33) The baked food product according to any one of items 29 to 32, wherein the product is a cake, bread, roll, pastry, cracker, muffin, scone, bagel, biscuit or cookie. (Item 34) The baked product according to item 33, wherein the baked product has a crumb structure equivalent to or better than a similar baked product made using natural egg white or natural whole egg. (Item 35) The baked product according to any one of items 29 to 34, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 36) The baked product according to any one of items 29 to 35, wherein the rOVA comprises the amino acid sequence of duck OVA, goose OVA or chicken OVA. (Item 37) The baked product according to any one of items 29 to 36, wherein the percentage of weight loss is lower in the baked product made using rOVA compared to an equivalent baked product made using whole egg. (Item 38) Recombinant ovalbumin (rOVA), At least one fat or oil, Water A emulsified product comprising The emulsified product, wherein the rOVA is present in the product at about 2% to 15% (w / w). (Item 39) The emulsified product according to item 38, further comprising an acidifying agent. (Item 40) The emulsified product according to item 38 or item 39, wherein the product is a salad dressing, sauce, mayonnaise, commercially available mayonnaise substitute, sandwich spread or gravy. (Item 41) Recombinant ovalbumin (rOVA), wherein when solubilized, the pH of the rOVA is about 3.5 to about 7.0, rOVA At least one sweetener, and optionally, an ingestible liquid A food product comprising wherein the rOVA is present in the food product at about 2% to about 15% (w / w), and the rOVA provides the food product with foaming properties, whipability, fluffiness or air miscibility Food product (Item 42) The food product according to item 41, wherein the rOVA further provides gelation to the food product (Item 43) The food product according to item 42, wherein the rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, and the rOVA provides improved gelation (Item 44) The food product according to item 42, wherein the rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, and the rOVA provides improved gelation (Item 45) The food product according to items 41 to 44, wherein the food product is a meringue, a whipped dessert, a whipped topping or a soufflé (Item 46) The food product according to any one of items 41 to 45, wherein the rOVA provides the food product with a foam volume that is at least 20%, 30%, 40% or 50% higher than that of native egg white (Item 47) The food product according to any one of items 41 to 46, wherein the rOVA provides the food product with a time to foam that is at least 20%, 30%, 40% or 50% faster than that of native egg white (Item 48) The food product according to item 46 or item 47, wherein the pH of the rOVA is about 3.5 to about 4.5 when solubilized (Item 49) The food product according to any one of items 41 to 48, wherein the rOVA is present in the food product at about 5% to about 10% (w / w) (Item 50) The food product according to item 49, wherein the rOVA is present in the food product at about 7% to about 8% (w / w). (Item 51) The food product according to item 46 or item 47, wherein the rOVA is present in the food product at about 4%, about 7% or about 12% (w / w). (Item 52) The food product according to item 51, wherein the pH of the rOVA is about 6 when solubilized. (Item 53) The food product according to item 46 or item 47, wherein the rOVA is present in the food product at about 9% to about 10% (w / w). (Item 54) The food product according to item 53, wherein the pH of the rOVA is about 7 when solubilized. (Item 55) The food product according to item 41, wherein the product is a beverage. (Item 56) The food product according to item 55, wherein the beverage is an ingestible alcohol. (Item 57) The food product according to item 56, wherein the rOVA provides foaming property, whipping property, fluffiness or air-mixability to the ingestible alcohol beverage. (Item 58) The food product according to item 55, wherein the beverage is a coffee drink. (Item 59) The food product according to item 58, wherein the rOVA provides foaming property, whipping property, fluffiness or air-mixability to the coffee drink. (Item 60) The food product according to item 58 or item 59, wherein the coffee drink lacks a milk component or contains a milk component. (Item 61) The food product according to any one of items 41 to 60, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 62) The food product according to any one of items 41 to 61, wherein the rOVA contains the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 63) The food product according to any one of items 41 to 62, wherein the rOVA does not contaminate the food product with Salmonella. (Item 64) The food product according to any one of items 41 to 63, wherein the food product is a protein bar, an energy bar, a nutrition bar or a granola bar. (Item 65) The food product according to item 64, wherein the food product contains about 4% to about 8% (w / w) of rOVA. (Item 66) The food product according to item 64 or item 65, wherein the bar is baked or unbaked. (Item 67) Recombinant ovalbumin (rOVA), at least one fat or oil, and plant-derived protein A meat analogue food product comprising wherein the rOVA is present in the food product at about 2% to about 15% (w / w), The rOVA acts as a binder or gelling agent, or a combination thereof, in the meat analogue food product. (Item 68) The meat analogue food product according to item 67, wherein the plant protein is an extruded plant protein. (Item 69) The meat analogue food product according to item 67, wherein the plant protein is a non-extruded plant protein. (Item 70) The meat analogue food product according to any one of items 67 to 69, wherein the meat analogue food product is selected from hamburgers, patties, sausages, hot dogs, sliced deli meats, jerky, bacon, nuggets, ground meat-like compositions and formed meat-like compositions. (Item 71) The meat analogue food product according to any one of items 67 to 70, wherein the rOVA provides the food product with a higher hardness than native egg white. (Item 72) The meat analogue food product according to any one of items 67 to 71, wherein the rOVA provides the food product with a higher chewiness than native egg white. (Item 73) The meat analogue food product according to item 71 or item 72, wherein the rOVA provides elasticity comparable to that of native egg white. (Item 74) The meat analogue food product according to any one of items 67 to 73, wherein the rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, and the rOVA provides improved gelation. (Item 75) The meat analogue food product according to any one of items 67 to 73, wherein the rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, and the rOVA provides improved gelation. (Item 76) The meat analogue food product according to any one of items 67 to 73, wherein the rOVA is present in the food product at about 4%, about 5% or about 6% (w / w). (Item 77) The meat analogue food product according to any one of items 67 to 73, wherein the rOVA contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 78) The meat analogue food product according to any one of items 67 to 76, wherein the rOVA contains the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 79) Recombinant ovalbumin (rOVA), at least one fat or oil, and a polysaccharide or a polysaccharide-containing raw material comprising an egg white substitute, The rOVA is present in the composition at about 2% to 15% (w / w), the composition has one or more properties selected from hardness, adhesiveness, crushability, cohesiveness, tackiness, and chewiness, and when the egg white substitute is cooked, the one or more properties are equivalent to or improved compared to natural egg white. Egg white substitute. (Item 80) The egg white substitute according to item 79, further comprising a flavoring agent or a coloring agent, or a combination thereof. (Item 81) The egg white substitute according to item 79 or item 80, wherein the polysaccharide or polysaccharide-containing raw material is starch. (Item 82) The egg white substitute according to any one of items 79 to 81, wherein the polysaccharide or polysaccharide-containing raw material is selected from gellan gum, sodium alginate, and psyllium, or any combination thereof. (Item 83) The egg white substitute according to any one of items 79 to 82, wherein the rOVA provides a higher hardness to the food product than native egg white. (Item 84) The egg white substitute according to any one of items 79 to 83, wherein the rOVA provides a higher chewiness to the food product than native egg white. (Item 85) The egg white substitute according to item 83 or item 84, wherein the rOVA provides the same level of tackiness and / or elasticity as native egg white. (Item 86) The egg white substitute according to any one of items 79 to 85, wherein the rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, and the rOVA provides improved gelation. (Item 87) The egg white substitute according to any one of items 79 to 85, wherein the rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, and the rOVA provides improved gelation. (Item 88) The ovalbumin (rOVA) according to any one of Items 79 to 85, wherein the rOVA is present in a food product at about 10% to about 12% (w / w). (Item 89) The ovalbumin (rOVA) according to any one of Items 79 to 88, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 90) The ovalbumin (rOVA) according to any one of Items 79 to 88, wherein the rOVA comprises the amino acid sequence of duck OVA, goose OVA or chicken OVA. (Item 91) A powdered raw material composition comprising recombinant ovalbumin (rOVA), wherein when solubilized, the pH of the rOVA is from about 3.5 to about 7.0, wherein the rOVA is at least 75% w / w of the composition, wherein the rOVA comprises one or more N-linked glycosylation sites having mannose linked to N-acetylglucosamine, and the N-linked glycosylation sites lack galactose, Powdered raw material composition. (Item 92) The powdered raw material composition according to Item 91, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 93) The powdered raw material composition according to Item 91 or Item 92, wherein the rOVA comprises the amino acid sequence of duck OVA, goose OVA or chicken OVA. (Item 94) The powdered raw material composition according to Item 92 or 93, wherein the amino acid sequence of the rOVA lacks an N-terminal methionine. (Item 95) The powdered raw material composition according to any one of Items 91 to 94, wherein the rOVA further comprises an EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. (Item 96) The powdered composition according to any one of items 91 to 95, wherein the composition contains at least about 80%, at least about 85% or at least about 90% rOVA (w / w). (Item 97) A liquid composition containing recombinant ovalbumin (rOVA), wherein the composition contains at least 50% rOVA (w / w of total protein or w / w of total composition), liquid composition. (Item 98) The liquid composition according to item 97, wherein the composition contains at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85% or at least about 90% rOVA (w / w). (Item 99) The liquid composition according to item 97 or item 98, wherein the rOVA contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 100) The liquid composition according to any one of items 97 to 99, wherein the rOVA contains the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 101) The liquid composition according to item 99 or item 100, wherein the amino acid sequence of the rOVA lacks the N-terminal methionine. (Item 102) The liquid composition according to item 101, wherein the rOVA further contains the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. (Item 103) The liquid composition according to any one of items 97 to 102, wherein the pH of the solubilized rOVA is about 3.5 to about 7.0. (Item 104) The liquid composition according to any one of items 97 to 103, wherein the pH of the solubilized rOVA is about 6 to about 6.8. (Item 105) The liquid composition according to any one of items 97 to 104, wherein when solubilized, the pH of the rOVA is less than about 6.1. (Item 106) The liquid composition according to any one of Items 97 to 105, wherein the rOVA provides at least one property of egg white selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturizing, water retention, clarification and aggregating property to a food not containing eggs. (Item 107) The liquid composition according to Item 106, wherein the rOVA provides equivalent properties or improved properties as compared with native egg white in a food not containing similar eggs. (Item 108) The liquid composition according to any one of Items 97 to 107, wherein the rOVA provides a foam volume at least 20%, 30%, 40% or 50% higher than that of native egg white to a food not containing eggs. (Item 109) The liquid composition according to any one of Items 97 to 108, wherein the rOVA provides a time to foam at least 20%, 30%, 40% or 50% faster than that of native egg white to a food not containing eggs. (Item 110) The liquid composition according to any one of Items 106 to 109, wherein the pH of the rOVA is about 3.5 to about 4.5 when solubilized. (Item 111) The liquid composition according to any one of Items 106 to 110, wherein the rOVA is present in a food not containing eggs at about 5% to about 10% (w / w). (Item 112) The liquid composition according to Item 111, wherein the rOVA is present in a food not containing eggs at about 7% to about 8% (w / w). (Item 113) The liquid composition according to any one of Items 106 to 109, wherein the rOVA is present in a food not containing eggs at about 4%, about 7% or about 12% (w / w). (Item 114) The liquid composition according to Item 113, wherein the pH of the rOVA is about 6 when solubilized. (Item 115) The liquid composition according to any one of items 97 to 114, wherein the rOVA provides a higher hardness to a food not containing the egg than native egg white. (Item 116) The liquid composition according to any one of items 97 to 115, wherein the rOVA provides a higher chewiness to a food not containing the egg than native egg white. (Item 117) The liquid composition according to item 115 or item 116, wherein the rOVA provides an elasticity comparable to that of native egg white to a food not containing the egg. (Item 118) The liquid composition according to any one of items 97 to 117, wherein the rOVA contains the amino acid sequence of chicken OVA and has a pH of about 6.5 to 7.0 when solubilized, and the rOVA provides improved gelation. (Item 119) The liquid composition according to any one of items 97 to 117, wherein the rOVA contains the amino acid sequence of ostrich OVA and has a pH of less than about 6.0 and greater than about 3.7 when solubilized, and the rOVA provides improved gelation. (Item 120) The liquid composition according to any one of items 97 to 119, wherein the rOVA does not contaminate a food not containing the egg with Salmonella. (Item 121) A dry or powdered composition containing recombinant ovalbumin (rOVA), wherein the composition contains at least 50% rOVA (w / w of total protein or w / w of total composition), the dry or powdered composition. (Item 122) The dry or powdered composition according to item 121, wherein the composition contains at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least 95% rOVA (w / w). (Item 123) The dried or powdered composition according to item 121 or item 122, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 124) The dried or powdered composition according to any one of items 121 to 123, wherein the rOVA comprises the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 125) The dried or powdered composition according to item 123 or item 124, wherein the amino acid sequence of the rOVA lacks the N-terminal methionine. (Item 126) The dried or powdered composition according to item 125, wherein the rOVA further comprises the EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. (Item 127) The dried or powdered composition according to any one of items 121 to 126, wherein the rOVA provides at least one property of egg white selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturization, water retention, clarification and aggregability to a food not containing eggs. (Item 128) The dried or powdered composition according to item 127, wherein the rOVA provides equivalent properties or improved properties as compared with native egg white in a similar food not containing eggs. (Item 129) The dried or powdered composition according to any one of items 121 to 128, wherein the rOVA provides a foam volume that is at least 20%, 30%, 40% or 50% higher than that of native egg white to a food not containing eggs. (Item 130) The dried or powdered composition according to any one of items 121 to 129, wherein the rOVA provides a time to foam that is at least 20%, 30%, 40% or 50% faster than that of native egg white to a food not containing eggs. (Item 131) The dried or powdered composition according to any one of items 121 to 129, wherein when solubilized, the pH of the rOVA is from about 3.5 to about 4.5. (Item 132) The dried or powdered composition according to any one of items 121 to 129, wherein the rOVA is present in the egg-free food at about 4%, about 7% or about 12% (w / w). (Item 133) The food product according to item 132, wherein when solubilized, the pH of the rOVA is about 6. (Item 134) The dried or powdered composition according to any one of items 121 to 133, wherein the rOVA provides a higher hardness to the egg-free food than native egg white. (Item 135) The dried or powdered composition according to any one of items 121 to 134, wherein the rOVA provides a higher chewiness to the egg-free food than native egg white. (Item 136) The dried or powdered composition according to item 134 or item 135, wherein the rOVA provides an elasticity comparable to that of native egg white to the egg-free food. (Item 137) The dried or powdered composition according to any one of items 121 to 136, wherein the rOVA contains the amino acid sequence of chicken OVA and, when solubilized, has a pH of about 6.5 to 7.0, and the rOVA provides improved gelation. (Item 138) The dried or powdered composition according to any one of items 121 to 136, wherein the rOVA contains the amino acid sequence of ostrich OVA and, when solubilized, has a pH of less than about 6.0 and greater than about 3.7, and the rOVA provides improved gelation. (Item 139) A method for producing a food product, comprising: providing a recombinant ovalbumin (rOVA) that, when solubilized, has a pH of from about 3.5 to about 7.0; Combining the rOVA in an amount of 2% to 15% (w / w) with one or more ingestible raw materials to form a food product comprising wherein the rOVA provides at least one egg white property selected from gelling property, foaming property, whipping property, fluffiness, binding property, elasticity, air miscibility, coating, film-forming property, emulsifying action, browning, thickening property, texturization , water retention, clarification and aggregability, to the food product. (Item 140) A method for producing a raw material composition, the method comprising expressing recombinant ovalbumin (rOVA) in microbial cells, wherein the rOVA is secreted by the microbial cells into a liquid medium, recovering the liquid medium containing the secreted rOVA, performing a separation step at a pH of about 3.5, solubilizing the rOVA at a pH of about 12, adjusting the final pH of the rOVA to about 3.5 to about 7.0 to prepare the raw material composition comprising. (Item 141) The method according to item 140, wherein the separation step comprises ion exchange chromatography or ammonium sulfate precipitation. (Item 142) The method according to item 141, wherein the ion exchange chromatography is cation exchange chromatography or anion exchange chromatography, or a combination thereof. (Item 143) The method according to any one of items 140 to 142, wherein the method further comprises a filtration step after the solubilization step. (Item 144) The method according to any one of items 140 to 143, wherein the microbial cells are fungal cells. (Item 145) The method according to item 144, wherein the fungal cells are Pichia sp. (Item 146) The method according to item 144 or item 145, wherein the microbial cell expresses a recombinant helper factor, and the helper factor enhances the level of expression or accumulation of rOVA. (Item 147) The method according to any one of items 140 to 146, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 148) The method according to any one of items 140 to 147, wherein the rOVA comprises the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 149) The method according to item 147 or item 148, wherein the amino acid sequence of the secreted rOVA lacks an N-terminal methionine. (Item 150) The method according to any one of items 140 to 149, wherein the secreted rOVA further comprises an EAEA amino acid sequence (SEQ ID NO: 75) at its N-terminus. (Item 151) An egg-free food product comprising recombinant ovalbumin (rOVA) in an amount of about 15% to about 25% (w / w of total protein or w / w of food product). (Item 152) The egg-free food product according to item 151, comprising the rOVA in an amount of up to about 23% (w / w). (Item 153) Use of recombinant ovalbumin (rOVA) as a raw material in the production of baked goods. (Item 154) Use of recombinant ovalbumin (rOVA) as a raw material in the production of egg-free food products. (Item 155) Use of recombinant ovalbumin (rOVA) as a raw material in the production of meat analogue food products. (Item 156) Use of recombinant ovalbumin (rOVA) as a raw material in the production of egg white substitutes. (Item 157) Use of recombinant ovalbumin (rOVA) as an alternative egg liquid for baked goods products, wherein said alternative egg liquid provides membrane formation equivalent to or better than an egg liquid containing natural egg white or natural whole egg. (Item 158) The use according to any one of Items 153 to 157, wherein the rOVA comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1, or an amino acid sequence having at least 70% identity with SEQ ID NO: 2 or SEQ ID NO: 1. (Item 159) The use according to any one of Items 153 to 157, wherein the rOVA comprises the amino acid sequence of duck OVA, ostrich OVA or chicken OVA. (Item 160) The use according to any one of Items 153 to 158, wherein the rOVA is present in an amount of 8% to 9% (w / w) in the egg liquid. (Item 161) Large-scale production of recombinant ovalbumin (rOVA), wherein said large-scale production comprises at least 1 liter of liquid culture of microbial cells expressing the rOVA. (Item 162) The large-scale production of rOVA according to Item 161, wherein said large-scale production comprises at least 10 liters of liquid culture of microbial cells expressing the rOVA. (Item 163) The large-scale production of rOVA according to Item 162, wherein said large-scale production comprises at least 100 liters of liquid culture of microbial cells expressing the rOVA. (Item 164) The large-scale production of rOVA according to Item 162, wherein said large-scale production comprises at least 1,000 liters of liquid culture of microbial cells expressing the rOVA. (Item 165) The large-scale production of rOVA according to Item 162, wherein said large-scale production comprises at least 10,000 liters of liquid culture of microbial cells expressing the rOVA. (Item 166) The large-scale production of rOVA according to item 162, wherein the large-scale production comprises a liquid culture of at least 100,000 liters of microbial cells expressing the rOVA. (Item 167) The large-scale production of rOVA according to item 162, wherein the large-scale production comprises a liquid culture of approximately 200,000 liters of microbial cells expressing the rOVA.

Claims

【Claim 1】 The invention described in the specification.

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