Protein compositions and consumable products thereof

Abstract

Provided herein are compositions with enhanced protein content, proteins with high solubility, protein combinations and methods for the preparation thereof.

Claims

1. An ingredient composition for producing a food item, the ingredient composition comprising: recombinant ovomucoid protein (rOVD); and one or more additional consumable ingredients; wherein: the rOVD comprises at least one glycosylated asparagine residue, the rOVD is substantially devoid of N-linked mannosylation, and wherein: the rOVD is capable of forming a clear liquid at a pH of from about 2.5 to about 6, and/or the clear liquid comprising the rOVD shows substantially higher liquid clarity as compared to a whey protein fluid at a pH of from about 2 to about 6.

2. The ingredient composition of claim 1, wherein the rOVD comprises a polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO. 1-44 or an amino acid sequence having at least 97% sequence identity with SEQ ID NO. 1-44.

3. The ingredient composition of claim 1, wherein the rOVD is expressed by a microbial organism selected from a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species, an Aspergillus species, and an E. coli species.

4. The ingredient composition of claim 1, wherein the concentration of rOVD is from about 0.1% w/v to about 30% w/v in an aqueous liquid, at a pH of from about 2.5 to about 6, and at room temperature.

5. The ingredient composition of claim 4, wherein the concentration of rOVD is about 10% w/v or less or about 20% w/v or less.

6. The ingredient composition of claim 4, wherein the composition is substantially optically clear.

7. The ingredient composition of claim 1, wherein the rOVD is substantially a full-length protein.

8. An ingredient composition for producing a food item, the ingredient composition comprising: a recombinant ovalbumin protein (rOVA); and one or more additional consumable ingredients; wherein: the pH of the ingredient composition, when solubilized in an aqueous solution, is above 3.5, and the ingredient composition provides to the food item at least one characteristic that is at least equivalent to a same characteristic in an otherwise similar food item that comprises native egg white and does not comprise rOVA.

9. The ingredient composition of claim 8, wherein the rOVA has a glycosylation, acetylation, or phosphorylation pattern different from wildtype OVA.

10. The ingredient composition of claim 8, wherein the rOVA may comprise one or more N-linked glycosylation sites having mannose linked to an N-acetyl glucosamine, and wherein the N-linked glycosylation sites lack galactose.

11. The ingredient composition of claim 8, wherein the amino acid sequence of the rOVA lacks an N-terminal methionine.

12. The ingredient composition of claim 8, wherein a glycosylation pattern of the rOVA is devoid of N-linked galactose units.

13. The ingredient composition of claim 8, wherein the ingredient composition does not comprise any natural egg white proteins or a natural egg white.

14. The ingredient composition of claim 8, wherein the rOVA comprises an amino acid sequence of a duck OVA, an ostrich OVA, or a chicken OVA.

15. The ingredient composition of claim 8, wherein the rOVA is expressed by a yeast host cell.

16. The ingredient composition of claim 15, wherein the host cell is selected from a Pichia species, and a Saccharomyces species.

17. The ingredient composition of claim 8, wherein the rOVA is expressed by a fungal host cell.

18. The ingredient composition of claim 17, wherein the host cell is selected from a Trichoderma species, and an Aspergillus species.

19. The ingredient composition of claim 8, wherein the rOVA comprises a polypeptide represented by an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with at least 97% identity with one of SEQ ID NO: 2.

20. The ingredient composition of claim 8, further comprising at least one plant protein selected from a group consisting of: a soy protein, a nut protein, a pea protein, a lentil protein, an almond protein, an oat protein, a flax seed protein, or a pulse protein.

21. The ingredient composition of claim 8, wherein the composition further comprises at least one starch selected from a group consisting of: corn, potato, sorghum, and arrowroot.

22. The ingredient composition of claim 8, wherein the composition further comprises at least one flour selected from a group consisting of: wheat flour, rice flour, corn flour, millet flour, spelt flour, and oat flour.

23. The ingredient composition of claim 8, wherein the rOVA provides to the food item a foam capacity higher than a foam capacity provided by native egg white in a similar food item.

24. A food item made with the ingredient composition of claim 8.

25. The food item of claim 24, further comprising at least one characteristic equivalent to or better than a similar food item made with a natural egg white or a natural whole egg, wherein the characteristic is selected from the group consisting of: hardness, cohesiveness, springiness, and chewiness foam capacity, foam stability, fluffing, clarification, resilience, hardness, chewiness, and gelling.

26. The food item of claim 24, wherein rOVA is present in the food item in an amount from about 2% to about 15% (weight rOVA/weight food item) before or after preparation of the food item.

27. The food item of claim 24, wherein rOVA is present in the food item in an amount less than 8% (weight rOVA/weight food item) before or after preparation of the food item.

28. The food item of claim 24, wherein the food item is a meat-based food item for which the ingredient composition binds together meat components, the meat-based food item further comprising: one or more fats or oils; one or more extruded proteins; at least one starch; and at least one gum.

29. The food item of claim 24, wherein the food item is a baked food item that further comprises: lecithin, a starch or a gum, and baking powder.

30. The food item of claim 29, wherein the food item comprises from about 1% to 5% (w/w) rOVA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also figure and FIG. herein), of which:

(2) FIG. 1A illustrates the vector constructs used for the expression of rOVD.

(3) FIG. 1B illustrates a comparison in the glycosylation pattern of native ovomucoid and a recombinant ovomucoid produced in P. pastoris and according to the present disclosure. Shown is a lack of the complex branched glycosylation (including a lack of mannose residues) on the recombinant ovomucoid when produced in a strain of P. pastoris comprising endoglycosidases.

(4) FIG. 1C illustrates the glycosylation patterns of the recombinant OVD produced by P. pastoris without an endoglycosidase treatment. rOVD thus produced have complex branched glycosylation patterns.

(5) FIG. 1D compares the molecular weight of native OVD, native OVD treated with an endoglycosidase, and recombinant OVD samples.

(6) FIG. 2 illustrates rOVD solution properties with 4.23% w/v rOVD.

(7) FIG. 3 illustrates rOVD solution clarity at about pH 4 and about pH 6 with 30% w/v rOVD after different heat treatments, measured using absorbance at 600 nm.

(8) FIG. 4 illustrates rOVD solution clarity after different heat treatments with 30% w/v rOVD in deionized water.

(9) FIG. 5A illustrates rOVD solution (9% w/v) appearance at pH 2.5, 4, and 6 after different heat treatment conditions.

(10) FIG. 5B are graphs showing absorbance of rOVD solution (9% w/v) at 600 nm after different heat treatment conditions at pH 2.5, 4 and 6. In each data pair, data in left columns relate to rOVD and data in right columns relates to Buffer.

(11) FIG. 6A illustrates rOVD solubility in different beverages.

(12) FIG. 6B is a graph showing absorbance of rOVD solution at 600 nm in different beverages. In each data pair, data in left columns relate to beverage and data in right columns relates to beverages with rOVD.

(13) FIG. 7 illustrates, left to right, a comparison of samples at room temperature: OVL+OVD with OVD control at pH 2.5, 4, 6.

(14) FIG. 8 illustrates, left to right, a comparison of Pasteurized (72? C.) samples: OVL+OVD with OVD control at pH 2.5, 4, 6.

(15) FIG. 9 illustrates, left to right, a comparison of Hot Fill (85? C.) samples of OVL+OVD with OVD control at pH 2.5, 4, 6.

(16) FIG. 10 illustrates, left to right, a comparison of retorted (121? C.) samples of OVL+OVD with OVD control at pH 2.5, 4, 6.

(17) FIG. 11 illustrates, left to right, a comparison of Pasteurized (72? C.) samples of OVL control with OVD control at pH 2.5, 4, 6.

(18) FIG. 12 illustrates, left to right, a comparison of Hot Fill (85? C.) samples of OVL control with OVD control at pH 2.5, 4, 6.

(19) FIG. 13 illustrates, left to right, a comparison of Retorted (121? C.) samples of OVL control with OVD control at pH 2.5, 4, 6.

(20) FIG. 14 illustrates, left to right, a comparison of rOVL+rOVD and rOVD samples at room temperature and after different heat treatments at pH 2.5, 4, 6.

(21) FIG. 15A and FIG. 15B illustrate comparisons of clarity for whey isolate (WPI1 and WPI3, 9% w/v) and rOVD solutions (9% w/v) at pH 2.5, 4 and 6.

(22) FIG. 16 illustrates protein water samples with 5% protein, from left to right, with whey protein isolate (neutral), whey protein isolate (acidic), nOVD, rOVD, 4%, pea protein (acidic), and soy protein.

(23) FIG. 17A and FIG. 17B illustrate samples of orange juice, from left to right, with 15% whey protein, 15% nOVD, 15% rOVD, 20% rOVD, 30% rOVD, or (no protein) control respectively. FIG. 17A: solution at time 0 hours and FIG. 17B: after 48 hours storage at 4? C.

(24) FIG. 18A illustrates jelly samples, from left to right, control (without protein supplementation), supplemented with 20% rOVD, supplemented with 20% nOVD and supplemented with 20% whey protein.

(25) FIG. 18B illustrates comparison of jelly samples with no protein (control), supplemented with 20% rOVD and supplemented with 20% whey protein.

(26) FIG. 18C to FIG. 18E illustrate jelly samples supplemented with 20% whey protein, supplemented with 16% gelatin and supplemented with 20% gelatin.

(27) FIG. 19A and FIG. 19B illustrate rOVD-H and rOVD-T samples solubilized in water at various concentrations.

(28) FIG. 20 illustrates the comparison in immunoreactivity for rOVD samples, native ovomucoid from chicken egg white (nOVD) and deglycosylated native ovomucoid (nOVD+PNGaseF).

(29) FIG. 21 indicates the color of an rOVD solution without (left) and with (right) hydrogen peroxide treatment.

(30) FIG. 22 illustrates a comparison of film formation using various protein samples.

(31) FIGS. 23A-23B illustrate glycosylation patterns of native OVA and rOVA produced in P. pastoris respectively.

(32) FIG. 24 illustrates pound cakes and their cross-sections made using rOVA compared to cakes made using eggs.

(33) FIG. 25 illustrates meringues made using rOVA compared to meringues made using eggs.

(34) FIG. 26 illustrates heat coagulation and foaming properties of whole egg, egg white and native OVA solutions.

(35) FIG. 27 illustrates heat coagulation and foaming properties of egg white and native OVA compared to rOVA.

(36) FIG. 28A illustrates gel electrophoresis migration of glycosylated native and recombinant OVA. Also shown are deglycosylated recombinant OVA treated with EndoH and PNGaseF enzymes.

(37) FIG. 28B illustrates a chromatogram depicting glycosylation patterns of rOVA produced in P. pastoris.

(38) FIG. 29 illustrates gelation results before and after foaming of various OVA samples compared to egg white.

(39) FIG. 30 illustrates film formation using nOVA, rOVA, whole egg wash and a commercial egg-white substitute.

(40) FIGS. 31A-B illustrates emulsification results of nOVA, rOVA and egg white protein at acidic and neutral pH.

(41) FIG. 32 illustrates foaming of rOVA and control samples in an alcohol-based drink.

(42) FIG. 33 illustrates egg patties made using nOVA, rOVA and egg white proteins.

(43) FIG. 34 illustrates meringues made using rOVA samples and egg white proteins.

(44) FIG. 35 illustrates protein bars made with egg white proteins (EWP), nOVA and rOVA at different protein inclusion levels.

DETAILED DESCRIPTION

(45) While various embodiments of the invention have been shown and described herein, it will be obvious 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

(46) Provided herein are compositions and methods of making compositions for non-animal-based sources of proteins as useful source of consumable protein for ingestion by an animal, including a human, such as for daily diet, dietary supplementation, consumer food and beverage, and nutrition.

(47) Provided herein are consumable compositions comprising ovomucoid (OVD). Such consumable compositions can be used in a food product, drink product, nutraceutical, pharmaceutical, cosmetic, or as an ingredient for a final product. In embodiments herein, the consumable composition is in a liquid form or a semi-solid form. In embodiments herein, the consumable composition is provided in a powdered form; this powder may be used to produce a liquid, solid, or semi-solid consumable composition. Preferably, the OVD in such consumable compositions is made recombinantly, and may be referred to herein as a recombinant OVD (rOVD).

(48) Unless indicated otherwise, the term OVD includes both native OVD (nOVD) and rOVD. The nOVD or rOVD in the consumable compositions herein is provided in concentrations that both increase the protein content of the consumable composition and also maintain one or more additional characteristics such as high clarity, high solubility, reduced turbidity, or substantial sensory neutrality.

(49) The use of rOVD in any of the consumable compositions herein allows for a non-animal-based source of protein, while providing additional features such as solubility, clarity, hardness, texture, mouthfeel, compatibility with heat treatment, compatibility with pH ranges and maintaining a consumer-favorable sensory profile. Various embodiments of such compositions, methods of making them, and methods of using them are provided herein.

(50) In some embodiments, the compositions and methods for making compositions herein increase the protein content of a consumable, and also provide additional features such as compatibility with other ingredients (such as, for example, compatibility with gluten, vitamins, minerals, and carbonation), coloration, smell, taste and compatibility with food and beverage preparation and/or storage conditions.

(51) Native ovomucoid (nOVD), such as isolated from a chicken or other avian egg, has a highly complex branched form of glycosylation. The glycosylation pattern comprises N-linked glycan structures such as N-acetylglucosamine units and N-linked mannose units. See, e.g., FIG. 1B (left-hand column). In some cases, the rOVD for use in a herein disclosed consumable composition and produced using the methods described herein has a glycosylation pattern which is different than the glycosylation pattern of nOVD. For example, when rOVD is produced in a Pichia sp., the protein may be highly glycosylated. FIG. 1C illustrates the glycosylation patterns of rOVD produced by P. pastoris, showing a complex branched glycosylation pattern. In some embodiments of the compositions and methods herein, rOVD is treated such that the glycosylation pattern is modified from that of nOVD and also modified as compared to rOVD produced by a Pichia sp. without such treatment. In some cases, the rOVD has no glycosylation. In other cases, the rOVD has reduced glycosylation. In some cases, the rOVD is modified by N-acetylglucosamine at one or more asparagine residues of the protein and lacks or is substantially devoid of N-linked mannosylation. See, e.g., FIG. 1B (right hand column). The changes in glycosylation described herein may lead to an increase in the solubility and clarity of rOVD as compared to other forms of protein such as whey proteins, soy proteins, pea proteins, and nOVD. The modifications in glycosylation of rOVD may lead to a change in the nitrogen to carbon ratio of the protein, such that reducing or removing substantially all of the mannose residues, the nitrogen to carbon ratio is increased (such as compared to nOVD or to rOVD produced without the modification to the glycosylation pattern).

(52) In some embodiments, the composition is a consumable food product. In some embodiments, the consumable food product is a finished product. In some embodiments, the composition is an ingredient of a finished product, e.g., a powder comprising rOVD or consisting essentially of rOVD.

(53) As used herein, the term consumable food composition refers to a composition, which comprises an isolated protein and may be consumed by an animal, including but not limited to humans and other mammals. Consumable food compositions include food products, beverage products, dietary supplements, food additives, and nutraceuticals, as non-limiting examples.

(54) Consumable food compositions also include compositions as an ingredient of a food or beverage or a product ingested as part of an animal diet.

(55) Since the rOVD of the present disclosure is not obtained from an animal source, a consumable composition comprising the rOVD is considered vegetarian and/or vegan.

(56) As used herein, a finished product refers to a consumable food composition directed to or suitable itself as a food or beverage for animal consumption. As used herein, an ingredient or component in reference to a consumable food composition refers to a composition that is used with other ingredient(s) or component(s) to create a finished product.

(57) Compositions with rOVD

(58) Provided herein are consumable food compositions and methods of making such compositions that increase the protein content of a consumable food composition through the addition of a recombinant ovomucoid protein (rOVD). In some embodiments, rOVD is added to a consumable food composition to increase the protein content, such as for added nutrition. In some embodiments, rOVD is present in the consumable food composition between about 1% and about 40% on a weight per total weight (w/w) and/or weight per total volume (w/v) of composition basis. For example, in a composition of 100 ml, rOVD is present at 30 g and the rOVD is thus at a 30% concentration. In some embodiments, the concentration of rOVD is or is about 1%, 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 of composition basis. In some embodiments, the rOVD is present at a concentration of or of about 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% or rOVD is present concentration greater than 5%, 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% w/w and/or w/v.

(59) A consumable product can include one or more other proteins, such as a non-OVD protein or a non-recombinant protein. The rOVD can increase amount of protein content in a consumable product, and/or it can also increase solubility of the one or more other proteins. For example, the consumable composition can include a whey protein, a pea protein, a soy protein, an almond protein, an oat protein, a flax seed protein, a vegetable protein, or an egg-white protein. In some cases, the one or more other proteins can comprise OVD having an amino acid sequence naturally found in an avian or a reptile.

(60) In some embodiments, the compositions and methods for making compositions increase the protein content, and provide solubility of the protein in the composition, as well as maintain or not substantially reduce the clarity of the composition. In some embodiments, the compositions and methods for making compositions increase the protein content, and provide solubility and maintain clarity, while not adversely affecting the stability, or one or more sensory qualities of the composition.

(61) In some embodiments, the consumable food compositions and methods for making consumable food compositions comprise rOVD and the rOVD increases the protein content of the consumable food composition and the rOVD is substantially soluble in the consumable food composition. The consumable food composition may be a finished product or an ingredient for making a finished product, e.g., a powdered rOVD composition.

(62) rOVD protein may be used on its own or in combination with other components to form a composition. In some embodiments, a composition may contain about or at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% protein, e.g., rOVD, by weight per total weight (w/w) and/or weight per total volume (w/v). In some cases, a composition described herein may contain up to about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% protein, e.g., rOVD, by w/w or w/v.

(63) In some embodiments, a composition described herein contains total protein at a concentration of about 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 total protein per 100 mL liquid (e.g., water). In some cases, a composition described herein contains total protein at a concentration of about 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 total protein per 100 g composition (e.g., powder).

(64) In some embodiments, a composition described herein contains total protein at a concentration of about 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 total protein per 100 mL liquid (e.g., water). In some cases, a composition described herein contains total protein at a concentration of about 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 total protein per 100 g composition (e.g., powder).

(65) In some embodiments, the rOVD consumable composition is a liquid composition. In such cases, the concentration of rOVD in the liquid composition may be between 0.1% to 40%. The concentration of rOVD in the liquid composition may be at least 0.1%. The concentration of rOVD in the liquid composition may be at most 40%. The concentration of rOVD in the liquid composition may be from 0.1% to 1%, 0.1% to 5%, 0.1% to 10%, 0.1% to 15%, 0.1% to 20%, 0.1% to 25%, 0.1% to 30%, 0.1% to 35%, 0.1% to 40%, 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%, 1% to 35%, 1% to 40%, 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%, 25% to 30%, 25% to 35%, 25% to 40%, 30% to 35%, 30% to 40%, or 35% to 40% in weight per total volume (w/v). The concentration of rOVD in the liquid composition may be about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% w/v. The concentration of rOVD in the liquid composition may be at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30% or 35% w/v. The concentration of rOVD in the liquid composition may be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% w/v.

(66) In some embodiments, the rOVD consumable composition is a solid composition. In such cases, the concentration of rOVD in the solid composition may be between 0.1% to 70%. The concentration of rOVD in the solid composition may be at least 0.1%. The concentration of rOVD in the solid composition may be at most 70%. The concentration of rOVD in the solid composition may be 0.1% to 1%, 0.1% to 10%, 0.1% to 20%, 0.1% to 30%, 0.1% to 40%, 0.1% to 50%, 0.1% to 60%, 0.1% to 70%, 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 40% to 50%, 40% to 60%, 40% to 70%, 50% to 60%, 50% to 70%, or 60% to 70% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of rOVD in the solid composition may be 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% w/w or w/v. The concentration of rOVD in the solid composition may be at least 0.1%, 1%, 10%, 20%, 30%, 40%, 50% or 60% w/w or w/v. The concentration of rOVD in the solid composition may be at most 1%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% w/w or w/v.

(67) In some embodiments, the rOVD consumable composition is a powdered composition. In such cases, the concentration of rOVD in the powder composition may be between 15% to 99% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of rOVD in the powder composition may be at least 15% w/w or w/v. In embodiments, the concentration of rOVD in the powder composition may be at most 99% w/w or w/v. The concentration of rOVD in the powder composition may be 15% to 30%, 15% to 45%, 15% to 60%, 15% to 75%, 15% to 80%, 15% to 85%, 15% to 90%, 15% to 95%, 15% to 99%, 30% to 45%, 30% to 60%, 30% to 75%, 30% to 80%, 30% to 85%, 30% to 90%, 30% to 95%, 30% to 99%, 45% to 60%, 45% to 75%, 45% to 80%, 45% to 85%, 45% to 90%, 45% to 95%, 45% to 99%, 60% to 75%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 99%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 99%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 99%, 85% to 90%, 85% to 95%, 85% to 99%, 90% to 95%, 90% to 99%, or 95% to 99% w/w or w/v. The concentration of rOVD 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 rOVD 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 rOVD in the powder composition may be at most 30%, 45%, 60%, 75%, 80%, 85%, 90%, 95%, or 99% w/w or w/v.

(68) In some embodiments, the rOVD consumable composition is a concentrated syrup composition. In such cases, the concentration of rOVD in the syrup composition may be between 10% to 60% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of rOVD in the syrup may be at least 10% w/w or w/v. The concentration of rOVD in the syrup may be at most 60% w/w or w/v. The concentration of rOVD in the syrup may be 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 30% to 40%, 30% to 50%, 30% to 60%, 40% to 50%, 40% to 60%, or 50% to 60% w/w or w/v. The concentration of rOVD in the syrup may be about 10%, 20%, 30%, 40%, 50%, or 60% w/w or w/v. The concentration of rOVD in the syrup may be at least 10%, 20%, 30%, 40% or 50% w/w or w/v. The concentration of rOVD in the syrup may be at most 20%, 30%, 40%, 50%, or 60% w/w or w/v. The syrup may include any solvent, e.g., water and juice.

(69) Solubility and Clarity

(70) Provided herein, in particular, are compositions of OVD where the OVD protein remains soluble in the composition. In some embodiments of any composition described herein, the proteins are fully soluble at a protein concentration between the lowest amounts of rOVD (e.g., 0.1 g or less) and in increasing amounts up to and including about 30 or 40 grams of rOVD protein per 100 mL of solution. In some embodiments of any composition described herein, the proteins are fully soluble at a concentration of about 1, 2, 5, 7, 10, 12 or 15 g, total OVD protein per 100 mL volume, for example when formulated in a liquid such as water. In some embodiments of any composition described herein, the proteins are fully soluble at a concentration of about 15, about 20, about 25, about 30, or about 40 g, total OVD protein per 100 mL volume, for example when formulated in a liquid such as water. In the compositions herein, the OVD may be native OVD or a recombinant OVD. In some embodiments, OVD is an isolated recombinant protein. In some embodiments, OVD is rOVD with modified glycosylation, such as having one or more asparagine residues modified by N-acetylglucosamine and substantially devoid of N-linked mannosylation.

(71) Solubility of rOVD may be measured by a variety of techniques including visual detection and measuring absorbance of the solution at a wavelength of 600 nm (OD600). In some embodiments, solubilized protein composition described herein have absorbance less than 1 (<1) as measured using 600 nm wavelength. In some embodiments, solubilized rOVD compositions described herein have an observed measured transmittance at 600 nm of greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the addition of rOVD to a composition does not change or only slightly changes the OD600 measurement as compared to the composition without rOVD.

(72) In some embodiments, the addition of rOVD to a composition may increase the OD600 measurement as compared to the composition without rOVD and the increase is less than what would be seen with the addition of another protein, such as whey protein or a native OVD added to the composition in the same amount.

(73) In some embodiments, the addition of rOVD to a composition has a solubility better than whey protein or native OVD, when compared at the same protein concentration and under equivalent conditions (such as pH and temperature treatment). In some embodiments, the addition of rOVD to a composition has a solubility better than whey protein or native OVD when compared at the same protein concentration and the composition is a consumable food composition such as an ingredient or a finished product.

(74) Clear or clarity as used herein refers to a lack of turbidity. Clarity may be assessed by visual observation, including by comparison to a solution that has no protein included. Such comparisons can be made by machine, by an individual or by a panel of testers, e.g., testers trained in the art of detecting clarity. Clarity of a solution can be tested by a panel of (at least 3, 5, 7, 10, or 12 individuals) or people skilled at such tests. Preferably, at least a majority of testers may be unable to visibly differentiate the rOVD composition from a solution comprising no protein, or a different protein at the same concentration.

(75) In some embodiments, the rOVD compositions exhibit improved clarity as compared to composition with other compositions having a different protein at an equivalent concentration, such as a composition containing pea protein, whey isolates or whey protein, native egg white proteins (e.g., nOVD), or whole egg white. In some embodiments, at least a majority or more of testers may be unable to visibly differentiate the rOVD added to a composition from a solution comprising no protein.

(76) A clear solution may be colored or may be colorless. In some embodiments, a solubilized rOVD protein in a composition may have a lack of color as measured by less than 0.15 absorbance at wavelengths between 350 nm and 850 nm. In some embodiments, a solubilized rOVD protein in a composition may provide a color such as yellow, green or brown or shades thereof to a consumable food composition. In some cases, rOVD and/or the solubilized rOVD protein may be treated with an oxidizing agent or oxygen generating agent to modify the color of the solution to a lighter or less intense color.

(77) In some embodiments, a composition of rOVD in solution, such as in a liquid consumable food composition, is essentially clear at a protein concentration between the lowest amounts of rOVD (e.g., 0.1 g) and in increasing amounts up to and including about 30 grams of rOVD protein per 100 mL of solution. In some embodiments, a composition of rOVD in solution, such as in a liquid consumable food composition, is essentially clear at a high protein concentration of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 grams of rOVD protein per 100 mL of solution. In some embodiments, an rOVD composition is essentially clear with at least about 9, 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 rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water).

(78) In some embodiments, an rOVD composition has a clarity better than whey protein, such as whey protein isolate or whey protein concentrate, when compared at the same protein concentration and under equivalent conditions (such as pH and temperature). In some embodiments, an rOVD composition has a clarity better than whey protein when compared at the same protein concentration and the rOVD composition is a component of a consumable food composition such as a finished product or as an ingredient in a finished product.

(79) In some embodiments, an rOVD composition has a clarity better than native OVD (nOVD) when compared at the same protein concentration and under equivalent conditions (such as pH and temperature). In some embodiments, an rOVD composition has a clarity better than an nOVD composition when compared at the same protein concentration and the rOVD composition is a component of a consumable food composition such as a finished product or as an ingredient in a finished product.

(80) In some embodiments herein, a composition of rOVD has both substantial solubility and is substantially clear at concentrations at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water).

(81) In some cases, rOVD remains soluble and clear in a consumable composition when the composition is heated to a temperature greater than 50? C., 60? C., or 70? C. or between about 70? C. and about 120? C., even when the rOVD is at a concentration of at least 2%, 4%, 10%, 20, 30%, 40%, or 50% on a w/v basis.

(82) In one instance, clarity of a consumable composition herein is determined using absorbance of visible light, such as by measuring absorbance of the solution at a wavelength of 600 nm (OD600). Preferably, a liquid or semi-liquid consumable composition herein has an absorbance that is less than 1.2, 1.1, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05 or 0.04 when determined using visible light at 600 nm. Other methods to measure solubility include examining solubility by centrifuge concentration followed by protein concentration assays such as Coomassie Plus (Bradford) Protein Assay (Thermo Scientific) and Bicinchoninic Acid (BCA) Protein Assay (Sigma-Aldrich).

(83) In some instances, clarity of a consumable composition is one that is not substantially different from the clarity of the solution before the addition of rOVD. For example, an addition of rOVD to a solution (consumable composition) does not change or does not substantially change (change of less than 0.03, 0.02, 0.01) the OD600 measurement as compared to the composition without rOVD.

(84) Thus, a consumable composition comprising rOVD may have a clarity less than 2 as measured at OD600 in room temperature, with a concentration of rOVD of at least or about 10%, 15%, 20%, 25%, or 30% rOVD weight per total weight (w/w) and/or weight per total volume (w/v). Alternatively, a solution comprising rOVD at a concentration greater than 10% w/w or w/v can have a clarity that is less than 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.08, 0.06, 0.04 or 0.02 as measured at OD 600 in room temperature. A substantially optically clear solution may refer to a solution where the OD600 measurement is less than or equal to about 0.1. In some cases, a substantially optically clear solution has an OD600 measurement of less than 0.08, 0.06, 0.05 or 0.02.

(85) In some embodiments, addition of rOVD increases the protein concentration by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% weight per total weight (w/w) and/or weight per total volume (w/v) without reducing clarity or increasing turbidity by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/w or w/v of the solution as compared to the solution before introduction of the rOVD.

(86) In some embodiments, rOVD protein may be added in an amount (such as a percentage by total weight or volume of the consumable food composition) that is greater than what could be added with other protein sources used in edible products such as whey proteins (such as whey protein isolate (WPI) and whey protein concentrate (WPC)), all embodiments of pea protein, soy protein, whole egg or egg white proteins (e.g., native OVD), while still maintaining the solubility, or solubility and clarity properties of the composition.

(87) Sensory Neutrality and Improved Sensory Appeal

(88) In some embodiments, in addition to the increased protein nutrition content, the addition of rOVD to a consumable food composition provides sensory neutrality or an improved sensory appeal as compared to other proteins in such compositions. As used herein sensory neutrality refers to the absence of a strong or distinctive taste, odor (smell) or combination of taste and smell, as well as texture, mouth-feel, aftertaste and color. A sensory panel such as one described in Kemp et al. 2009 may be used by a panel of trained analysts. Sensory neutrality may provide an improved sensory appeal to a taster, such as a tester of foods or a consumer, when a consumable food composition containing rOVD with another like composition that has a different protein such as whey protein, pea protein, soy protein, whole egg or egg white protein at the same concentration.

(89) In some embodiments, rOVD when added to a consumable food composition is substantially odorless, such as measured by a trained sensory panel, in comparison with different solutions with a different protein component present in an equal concentration to the rOVD containing solution, for example, in the comparison is whey, soy, collagen, pea, egg white solid isolates and/or native OVD. In some embodiments of the rOVD compositions described herein, such compositions are essentially odorless at a protein concentration between about 5-10%, 10-15%, 15-20%, 20-25%, 25-30% or greater than 30% rOVD weight per total weight (w/w) and/or weight per total volume (w/v) or at a protein concentration of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 g of total rOVD protein per 100 mL solution (e.g., per 100 mL water).

(90) In some embodiments, the addition of rOVD to a consumable food composition also provides a neutral taste in addition to the characteristics such as increased protein nutrition content, solubility, clarity, and/or odorless. A neutral taste can be measured for example, by a trained sensory panel in comparison with solutions containing a different protein present in an equal concentration to the rOVD, for example, whey, soy, collagen, pea, whole egg, and egg white solid isolates (including native OVD).

(91) In some embodiments, the addition of rOVD provides a reduction in a certain odor and/or taste that is associated with other proteins used for supplementation. For example, addition of rOVD has less of an egg-like odor or taste as compared to the addition of whole egg, fractionated egg or egg-white to a consumable food composition. In some embodiments, addition of rOVD has less of a metallic odor or taste as compared to other protein sources.

(92) In some embodiments, the addition of rOVD has an improved mouth-feel as compared to the addition of other protein sources. For example, the addition of rOVD is less grainy or has less precipitate or solids as compared to other protein sources.

(93) In some embodiments, the addition of rOVD has an improved texture, for example, as compared to other available supplemental protein sources.

(94) In some embodiments, the addition of rOVD has an improved or appealing color or visual appeal as compared to other available supplemental protein sources. For example, the addition of rOVD may maintain the clarity of a liquid (such as a carbonated drink, a protein water, sports drink) and provide visual appeal for the consumer.

(95) A consumable composition with rOVD may also have an improved sensory appeal as compared to the composition without rOVD or with a different protein present in an equal concentration to the rOVD. Such improved sensory appeal may relate to taste and/or smell. Taste and smell can be measured, for example, by a trained sensory panel. In some instances, a sensory panel compares a consumable composition with rOVD to one without it or with a different protein in an equivalent amount.

(96) As described herein, a consumable composition herein can be in a liquid form. A liquid form can be an intermediate product such as soluble rOVD solution. In some cases, a liquid form can be a final product, such as a beverage comprising rOVD. Example of different types of beverages contemplated herein include: a juice, a soda, a soft drink, a flavored water, a protein water, a fortified water, a carbonated water, a nutritional drink, an energy drink, a sports drink, a recovery drink, a heated drink, a coffee-based drink, a tea-based drink, a plant-based milk, a milk based drink, a non-dairy, plant based mild drink, infant formula drink, and a meal replacement drink.

(97) Non-limiting examples of juice drinks include Odwalla?, Naked?, and MinuteMaid?.

(98) Non-limiting examples of soda drinks include: Coca-Cola?, Pepsi?, Sprite? and 7Up?.

(99) Non-limiting examples of recovery drinks include Gatorade?, Pedialyte?, Poweradeo and Propel?.

(100) Non-limiting examples of an energy drink include Red Bull?, Monster?, Full Throttle?, AMP?, Rockstar?, Bang?, Reign?, NOS?, Venom?, and energy shots such as 5-Hour Energy?.

(101) Other examples of liquid form final products include broth, soup and liquid food.

(102) A liquid form can be a cold drink, a hot or warm drink, or a room-temperature drink

(103) Any of the liquid forms herein can be carbonated. Carbonation can be achieved using any safe gas such as carbon dioxide.

(104) In one embodiment, a consumable composition is sparkling water (such as San Pellegrino?) and has between 0.5 and 30% w/w or w/v rOVD. Such product has an OD 600 less than 0.2, preferably less than 0.15 while remaining essentially colorless, odorless and tasteless.

(105) In one embodiment, a consumable composition is a soda drink (such as Diet Coke? Pepsi?, Coke?) and has between 0.5 and 30% w/w or w/v rOVD. Such product retains a sensory profile (taste, odor, smell and clarity) comparable to the composition without the addition of rOVD.

(106) In some embodiments, a consumable composition is in a semi-sold form. Examples of semi-solid consumable compositions include: a jelly, a candy, a broth, a soup, a syrup, a gelatin-containing product, a gelled product, and a gummy product, or a combination thereof.

(107) Compatibility with Additional Ingredients

(108) Provided herein are compositions with rOVD wherein the rOVD is compatible with one or more additional ingredients that are used in the preparation of a consumable food composition, including a finished product. Such compatibility provides fortification of protein content to the consumable food composition, while maintaining one or more desired characteristics of the consumable food composition.

(109) In some embodiments, rOVD is compatible with gluten-containing ingredients. For example, rOVD can be added with a gluten-containing ingredient to achieve protein fortification and maintain gluten-structure necessary for the ingredient and/or finished product. For example, rOVD can be used as an ingredient for the production of protein fortified baked goods, a bread, a cookie, a cracker, a biscuit, a frozen dairy product, a frozen dairy-like product, a prepared meal, a meat product, a meatless product, a burger, a patty, a protein supplement, a snack bar, a protein bar, a nutrition bar, an energy bar, a dessert, a salad dressing, an egg-wash product, or an egg-like product, pastries, cakes and noodles. In the finished product, the rOVD does not substantially interfere with the gluten structure or has a substantially reduced interference with gluten structure as compared to other protein sources.

(110) In some embodiments, rOVD is compatible with gluten-free ingredients. For example, rOVD can be added with a gluten-free ingredient mix to achieve protein fortification and provide structure and/or texture to the finished product. Gluten-free ingredients and finished products include such grains and starches (rice, corn, sorghum, and other cereals), root tubers such as potato, and legumes and pulses such as chickpeas and lentils. For example, rOVD can be used as an ingredient for the production of protein fortified gluten-free products including baked goods, a bread, a cookie, a cracker, a biscuit, a frozen dairy product, a frozen dairy-like product, a prepared meal, a meat product, a meatless product, a burger, a patty, a protein supplement, a snack bar, a protein bar, a nutrition bar, an energy bar, a dessert, or an egg-like product, pastries, cakes and noodles.

(111) In some embodiments, rOVD is compatible with salts such that rOVD protein does not precipitate out from solution. For example, for use in foods and beverages such as protein smoothies, vegan milk and fruit juices fortified with rOVD, the protein remains substantially in solution. Addition of rOVD does not precipitate in vitamin/mineral fortified environment such as present with fruit juice and juice-like products, and rOVD provides increased protein content and nutrition.

(112) rOVD Combinations with a Second Source of Amino-Acids

(113) In some embodiments, rOVD is added to a consumable food composition and a second source of amino acids is added, such that the combination has an increased protein content and provides a desired amount or balance of amino acid content. In some embodiments, the second source of amino acids is a second protein (either a native protein or a recombinant protein). In some embodiments, the second source of amino acids is provided by adding one or more free amino acids.

(114) In some embodiments, rOVD is added to a consumable food composition and a second protein is added, such that the combination has an increased protein content and provides a desired amount or balance of amino acid content. In some embodiments, the second protein is a recombinant protein. In some embodiments, the second protein is a native protein, e.g., isolated from its native source.

(115) Protein content of compositions can be measured by various methods such as the protein digestibility-corrected amino acid score (PDCAAS) method. PDCAAS refers to a method for the measurement of the protein value in human nutrition. The method is based on comparison of the concentration of the first limiting essential amino acid in the test protein with the concentration of that amino acid in a reference (scoring) pattern. The method compares the amino acid profile of the specific food protein against a standard amino acid profile with the highest possible score being a 1.0, such 1.0 score meaning the specific food protein provides per unit of protein 100% or more of the indispensable amino acids required for human nutrition (see e.g., FAO/WHO/UNU Expert Consultation 1985).

(116) The formula for calculating the PDCAAS percentage is: (mg of limiting amino acid in 1 g of test protein/mg of same amino acid in 1 g of reference protein)?fecal true digestibility percentage. PDCAAS scores above 1.0 are truncated to 1.0. Amino acid score (not corrected or truncated) can exceed 1.0.

(117) In some embodiments, the combination of rOVD and a second protein increases the protein content and provides a PDCAAS of greater than about 0.75. In some embodiments, the combination provides a PDCAAS of or of about 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87. 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0. In some embodiments, the combination provides a PDCAAS of greater than or greater than about 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87. 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95. In some embodiments the combination provides a PDCAAS of or of about 1.0.

(118) In some embodiments, the ratio of rOVD and second protein is selected to provide a PDCAAS of at least about 0.75 and wherein the combination of rOVD and second protein remains soluble in the consumable food composition. In some embodiments of a herein-disclosed combination of rOVD and a second protein, rOVD is present in the combination at or at about 95%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% weight per total weight (w/w) and/or weight per total volume (w/v). In some embodiments of a herein-disclosed combination of rOVD and a second protein, rOVD is present in the combination at or at about 69%, 78%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% w/w or w/v. In some embodiments of the combination of rOVD and the second protein, rOVD is present in the combination in a percentage of total protein at least or at least about 60%, 65%, 70%, 75%, 80% or greater than 80% w/w or w/v. In some embodiments of a herein-disclosed combination of rOVD and a second protein, the second protein is present in the combination at an above percentage, such the rOVD is provided in a lesser amount than the second protein.

(119) In some embodiments, a second protein is selected based on its amino acid composition. In some embodiments, a second protein provides tryptophan to the composition. In some embodiments, a second protein provides tryptophan such that the combination with rOVD has a tryptophan content of at least about 1.7 g per 100 g total protein.

(120) In some embodiments, the second protein is lysozyme. In some embodiments, the second protein is egg white lysozyme. In some embodiments, the second protein is a recombinant protein. In some embodiments, the second protein is a recombinant egg white lysozyme (rOVL).

(121) The rOVD and rOVL can be processed or mixed together prior to mixing with any other food ingredients or consumable food products. Alternatively, either the rOVD or the rOVL can be processed or mixed individually, either at the same time or separately, with any other food ingredients or consumable food products. In embodiments, a single transformed cell expresses both rOVL and rOVD.

(122) In some embodiments, the second protein is rOVL and the combination of rOVD and rOVL provides protein fortification while remaining soluble in the composition and providing a PDCAAS of about 1.0. The ratio of rOVD to rOVL can be between about 60% rOVD:40% rOVL to about 82% rOVD:18% rOVL, or between about 75% rOVD:25% rOVL to about 82% rOVD:18% rOVL weight per total weight (w/w) and/or weight per total volume (w/v).

(123) Native OVD has a PDCAAS of approximately 0.02. Addition of rOVL to rOVD increases the amino acid score and PDCAAS of the combination. As an example, a 78.3% rOVD and 21.7% rOVL blend result in an amino acid score of 0.86 and a PDCAAS of 0.79. With a ratio of rOVD to rOVL from about 78.3% rOVD+21.7% rOVL to about 60% rOVD+40% rOVL provides a range of 0.86 to 1.06 amino acid score. In these exemplary ranges, the combination of rOVD and rOVL remains soluble.

(124) In some embodiments, a consumable composition comprises a protein mixture of rOVD and rOVL. In some cases, a composition comprising a mixture of rOVD and rOVL has about 20%-99% rOVD and 1-20% rOVL. In some examples, the concentration of rOVD in a protein mixture of rOVD and rOVL may be at least 20%. The concentration of rOVD in a protein mixture of rOVD and rOVL may be at most 99%. The concentration of rOVD in a protein mixture of rOVD and rOVL may be about 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 99%, 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 99%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80%, 40% to 90%, 40% to 99%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 99%, 60% to 70%, 60% to 80%, 60% to 90%, 60% to 99%, 70% to 80%, 70% to 90%, 70% to 99%, 80% to 90%, 80% to 99%, or 90% to 99% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of rOVD in a protein mixture of rOVD and rOVL may be about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% w/w or w/v. The concentration of rOVL in a protein mixture of rOVD and rOVL may be 1% to 20%. The concentration of rOVL in a protein mixture of rOVD and rOVL may be at least 1%. The concentration of rOVL in a protein mixture of rOVD and rOVL may be at most 20%. The concentration of rOVL in a protein mixture of rOVD and rOVL may be 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, 10% to 20%, or 15% to 20% w/w or w/v. The concentration of rOVL in a protein mixture of rOVD and rOVL may be about 1%, 5%, 10%, 15%, or 20% w/w or w/v.

(125) In some embodiments, the rOVD and second protein provide a PDCAAS similar to other protein sources such as whey protein and whey protein isolate, and the rOVD and second protein provide at least one feature improved as compared to the other protein source including solubility, clarity, sensory neutrality or improvement of taste and/or odor, improved mouthfeel, and compatibility with an additional ingredient. In some embodiments, the rOVD and second protein provide a PDCAAS similar to other protein sources and provided improved solubility and clarity in food preparation and processing conditions, such as pH, heating and carbonation.

(126) In some embodiments, the second source of amino acids added with rOVD is one or more free amino acids. In some embodiments, rOVD can be combined with free amino acids such as Tryptophan, Isoleucine, Leucine and Valine to selectively increase PDCAAS. In some embodiments, the addition of one or more free amino acids provides an amino acid balance similar to the addition of a second protein, such as similar to the PDCAAS achieved with the addition of rOVL. For example, one or more of the following can be added with rOVD: Tryptophan=1.7 g/100 g sample, Isoleucine=2.03 g/100 g sample, Leucine=4.55 g/100 g sample, Valine=4.94 g/100 g sample.

(127) Heating Conditions and pH of Compositions

(128) In some embodiments, the consumable food compositions and methods of making such compositions include a particular pH range, and in such range, the rOVD remains soluble in the composition. In some embodiments, the pH is between about 1.0 and about 8.0. In some embodiments, the pH is between about 2.0 and about 6.0, 6.5, or 7.0. In some embodiments, the pH is between about 2.0 to about 2.5, about 2.5 to about 3.0, about 2.5 to about 3.5, about 3.5 to about 4.0, about 2.5 to about 4.5, about 2.0 to about 4.0, about 4.0 to about 6.0, about 2.0 to about 6.0, about 4.0 to about 6.5, or about 2.0 to about 6.5. In some embodiments, the pH is less than 2.0, or equal to 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 or greater than 4.5. At such pH or pH range, rOVD remains soluble in the consumable food composition, when the rOVD is an ingredient of a finished product (e.g., as a powdered form for use in a finished product) or in a finished product itself. At such pH or pH range, rOVD remains soluble in the consumable food composition without affecting the texture or graininess of the composition. In semi-solid and solid foods, the solubility of rOVD enables protein fortification without jeopardizing functional and sensory properties of the food product. For instance, the addition of rOVD provides fortification and maintains sensory appeal such as a good mouth-feel and lack of graininess. In some embodiments, the addition of rOVD provides fortification, maintains solubility and as such provides the ability of the rOVD to blend with other ingredients.

(129) In some embodiments, the consumable food compositions and methods of making such compositions include a heating condition. For example, a consumable food composition may be a heated (e.g., fried, boiled, or baked) or may it may be a hot beverage, such as a warm or hot drink, a soup or a broth. In some cases, a consumable food composition may have a heating step as part of the preparation or sterilization process for producing an ingredient or a finished product. For example, a heating step may include pasteurization, hot fill, and/or retorting. In some embodiments, the heating step include heating to a temperature between about 72? C. and about 121? C. For example, a heating step may be a pasteurization, where the composition is heated to 72? C. for 1 minute and then cooled and stored, including storage at room temperature or refrigerated. For hot fill, a composition may be heated to 85? C. to 95? C., such as for 30 seconds and then placed at room temperature. Retorting may include heating to 121? C. under pressure, such as heating for 15 minutes at 19 psi, and then storing at room temperature.

(130) Preparation of a consumable composition can also include one or more heating steps. A heating step can comprise pasteurization, hot fill, and/or retorting. In some embodiments, the heating step includes heating to a temperature between about 70? C. and about 150? C.

(131) In one example, a pasteurization heating step is performed at temperatures ranging between 70? C. and 100? C.

(132) In one example, hot filling heating step is performed at about 90? C. to about 97? C.

(133) In one example, retorting is performed at about 100? C. to about 140? C. The retorting may be performed for about 10 or more minutes and at about or at least 12 psi.

(134) In some embodiments, the consumable food compositions and methods of making such compositions with rOVD provide a greater protein solubility or a greater protein solubility and improved clarity at pH ranges and/or with heating as compared to composition containing a different protein, such as whey protein, soy protein, pea protein, whole egg protein (e.g., native OVD), or whole egg white protein at the same concentration.

(135) In some cases, rOVD provides protein solubility in a consumable food composition at a pH between about 2 and about 6, at rOVD concentrations of concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis. In some cases, rOVD provides protein solubility and clarity in a consumable food composition at a pH between about 2 and about 6, at rOVD concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis.

(136) In some cases, rOVD provides protein solubility in a consumable food composition when the composition is heated to a temperature between about 72? C. and about 121? C. at rOVD concentrations of concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis. In some cases, rOVD provides protein solubility and clarity in a consumable food composition when the composition is heated to a temperature between about 72? C. and about 121? C. at rOVD concentrations of concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis.

(137) In some cases, rOVD provides protein solubility in a consumable food composition when the composition is heated to a temperature between about 72? C. and about 121? C. and where the composition has a pH between about 2 and about 4, or a pH about 2 to about 6, at rOVD concentrations of concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis. In some cases, rOVD provides protein solubility and clarity in a consumable food composition when the composition is heated to a temperature between about 72? C. and about 121? C., and where the composition has a pH between about 2 and about 4, or a pH about 2 to about 6, at rOVD concentrations of concentrations of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g or more than 30 g of total rOVD protein per 100 mL of solution (e.g., such as in 100 mL of water) or at a percentage of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 percent on a weight per total composition volume basis.

(138) Consumable Food Compositions

(139) Consumable food compositions described herein include food products, beverage products, dietary supplements, food additives, and nutraceuticals as non-limiting examples, and also include compositions as an ingredient of a food or beverage or a product ingested as part of an animal diet. In some embodiments, a consumable food composition is a finished product, such as a food or beverage for animal consumption or for human consumption, a dietary supplement, or a nutraceutical product.

(140) In some embodiments, a finished product is a beverage containing rOVD, and optionally a second protein, such as rOVL. The beverage can be a clear beverage, and can be selected from a juice, a soda, a soft drink, a flavored water, an unflavored water, a fortified water, a carbonated water, a nutritional drink, an energy drink, a sports drink, a recovery drink, a heated drink, a coffee-based drink, a tea-based drink, a cocoa based drink, a smoothie, a milk shake, coconut water, beer, wine, alcoholic beverage, nut milks, juice-based beverages, dairy-based beverages, and a plant-based milk. Many of these beverages have a pH that is between about 2 and about 7, and rOVD and/or rOVD and second protein combination remains soluble in such beverages. In some embodiments, the beverage is a heated beverage. In some embodiments, the beverage is a cold beverage or a beverage served or stored at room temperature. In some embodiments, the beverage contains alcohol from 3 to 40% weight per total weight (w/w) and/or weight per total volume (w/v).

(141) In some embodiments the beverage is carbonated. The carbonation may be created by, for example, carbon dioxide, carbonic acid, sodium bicarbonate, and potassium bicarbonate. A composition described herein may be carbonated. In some cases, a composition described herein has about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, or 4 volumes of carbon dioxide gas present per volume of beverage. In some cases, a composition described herein has up to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, or 4 volumes of carbon dioxide gas present per volume of beverage. In some cases, a composition described herein has about 0.1 volumes to about 4 volumes or about 1.5 volumes to about 3.5 volumes of carbon dioxide gas present per volume of beverage.

(142) In some embodiments, a protein composition may comprise carbon dioxide, and wherein the amount of carbon dioxide added to the soluble protein composition may be in a proportion between 0.01 g and 4.4 g in 355 mL or the gaseous carbon dioxide may be between 0.02 volumes and 5 volumes for every 1 volume of soluble protein composition, and wherein the beverage may have a pH range between about 2 and about 6 or about 2 and about 4. In some embodiments, a carbonated beverage has a pH between 1.0 and 6.0 or about 1.0 and about 4 or between about 1.6 and about 3.4.

(143) In some embodiments, the beverage preparation includes a heating step, such as hot fill, pasteurization or retorting and the rOVD in the beverage remains soluble during and subsequent to the heating step. In some embodiments, the addition of rOVD to the beverage does not substantially alter the visible appearance, smell, flavor or mouthfeel of the beverage as compared to a beverage that does not contain the composition. In some embodiments, the addition of rOVD to the beverage is sensory neutral and provides an improved sensory appeal as compared to other proteins when added to the beverage at the same concentration, such as whey protein, soy protein, pea protein, egg white proteins or whole egg proteins. In some embodiments, the beverage preparation also includes a second protein such as rOVL and the combination of rOVD and the second protein remains soluble during and subsequent to the heating step.

(144) In some embodiments, a finished product is a food product containing rOVD. The food product can be a jelly, a candy, a broth, a soup, a gelatin-containing product, a gelled product and a gummy product. Additional exemplary categories of food products in which rOVD can be added include sauces, dressings, condiments.

(145) rOVD can also be added to seasoning mixes and spices. rOVD can also be used in coating and breadings. rOVD may also be used to increase the protein content of snacks such as fruit and vegetable-based snacks.

(146) rOVD may be used as an egg wash to promote adhesion of seeds or grains to a baked good and/or to improve the visual appearance, such as browning, of the baked good.

(147) In some embodiments herein, a consumable food composition containing rOVD is a composition that is used as an ingredient with other ingredient(s) or component(s) to create a finished product. For example, rOVD can be mixed with water or other liquid, and then this mixture used as an ingredient to create a beverage, food product, dietary supplement or nutraceutical. In some cases, rOVD is mixed with other ingredients, such as other liquids (e.g., nut milks, fruit juices, vegetable extracts or carbonated solutions. This solution can be an ingredient that is then mixed with other ingredients to make a final product for an end-user; for example, the solution may be a syrup containing concentrated rOVD. A final or finished product is one that is ready for an end-user's consumption. The finished product can be a processed product, such as processed food or a processed drink. In some instances, the rOVD is provided in a separate container to be mixed into the final product by the end-user. In some cases, rOVD is mixed with other ingredients, such as gelling agents to make candies, gummy products, gelled products (such as a Jello?) or sports gels.

(148) During or after preparation of a consumable food product containing rOVD may be formulated as a liquid, solid, syrup, or powder. A composition may be refrigerated, frozen, stored warm, stored at room temperature or held at a heated temperature. Preparation of the food product can include a heating step or the food product is stored or served at a heated temperature, and the rOVD remains soluble in the food product during and subsequent to the heating step. In some cases, the food product can have a pH that is between about 2 and about 6, and rOVD remains soluble in the food product.

(149) Examples of liquid consumable compositions or beverages include: a soda, a vitamin drink, a protein shake, a meal replacement shake, a juice, a refreshment drink, a milk-based drink or a non-dairy based drink, flavored water, a carbonated drink, coffee, caffeinated drink, tea, flower-based drink, beer, liquor, and a sports drink.

(150) Any of the liquid or semi-solid consumable compositions herein can be created by mixing a powdered rOVD into a solution. The solution can be the final product or an intermediate solution which is then further modified to generate a final product.

(151) Examples of solvents that can be used to prepare an rOVD solution include still water, carbonated water, alcohol, juices, and any other commercially available drink including those described in more detail herein.

(152) A method of generating a consumable composition comprising rOVD may comprise mixing rOVD with a solvent and, optionally, one or more other components. The mixing may be performed by any conventionally used mixing method including mortar and pestle, mechanical grinder, blending, homogenization process or a sonication process.

(153) The amount of rOVD added to the solution can be one that generates an rOVD concentration as derived herein (either in the final product or an intermediate product).

(154) Preferably, addition of the rOVD to the solution results in most or nearly all of the rOVD solubilized into the solution at room temperature. In one instance, solubility is determined based on clarity or degree of lack of turbidity.

(155) The consumable compositions herein can also be subjected to a heating step. Such a step can modify or increase solubility of the rOVD. For example, it was found that performing a heating step in the process of making a product such as retorting, hot filling, or pasteurization can increase solubility and hence clarity of an rOVD solution herein.

(156) Preparation of a consumable food product containing rOVD may include processing steps, for example, freezing, chilling, heating, baking, roasting, broiling, boiling, blanching, packaging, canning, bleaching, enriching, drying, pressing, grinding, mixing, par cooking, cooking, proofing, marinating, cutting, slicing, dicing, crushing, shredding, chopping, shaking, coring, spiralizing, rolling, juicing, straining, filtering, kneading, whisking, beating, whipping, grating, stuffing, peeling, deseeding, smoking, curing, salting, preserving, pickling, fermenting, homogenizing, pasteurizing, sterilizing, irradiating, cold plasma processing, high pressure processing, pulse electric field processing, microwave assisted thermal sterilization, stabilizing, blending, pureeing, fortifying, refining, hydrogenating, aging, extending shelf life, or adding enzymes.

(157) Preparation of a consumable food product containing rOVD may include drying and/or concentrating. In some cases, drying forms a dry, dehydrated, concentrated, and/or solid protein or composition. Some non-limiting examples of drying methods include thermal drying, evaporation (e.g., by means of vacuum or air), distillation, boiling, heating in an oven, vacuum drying, spray drying, freeze drying, and lyophilization, or any combination thereof.

(158) Preparation of a consumable food product containing rOVD may include diluting and/or hydrating. In some cases, the diluting may comprise addition of a liquid, which may be water or another liquid form. For example, a composition can be diluted (e.g., from 20% water to 99.9% water). In another example, a dry composition can be hydrated (e.g., from a dry solid to 99.9% water).

(159) In some embodiments, the consumable food composition containing rOVD is in powder form and when the powdered composition is formulated into a solution, the rOVD is substantially fully soluble. In some embodiments, when the powdered composition is formulated into a solution, the rOVD is substantially fully soluble and the solution is substantially clear. In some embodiments, when the powdered composition is formulated into a solution, the rOVD is substantially fully soluble, the solution is substantially clear and the solution is essentially sensory neutral or has an improved sensory appeal as compared to solutions made with other powder zed proteins such whey protein, soy protein, pea protein, egg white protein or whole egg proteins. In some embodiments, the powdered composition is solubilized in water where the concentration of rOVD is or is about 1%, 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% weight per total weight (w/w) and/or weight per total volume (w/v) of composition.

(160) In some embodiments of the consumable food compositions described herein, the composition is essentially free of animal-derived component, whey protein, caseinate, fat, lactose, hydrolyzed lactose, soy protein, collagen, hydrolyzed collagen, or gelatin, or any combination thereof. A composition described herein may be essentially free of cholesterol, glucose, fat, saturated fat, trans fat, or any combination thereof. In some cases, a composition described herein comprises less than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% fat by dry weight. In some embodiments, the composition may be fat-containing (e.g., such as a mayonnaise) and such composition may include up to about 60% fat or a reduced-fat composition (e.g., reduced fat mayonnaise) and such composition may include lesser percentages of fat. A composition that free of an animal-derived component can be considered vegetarian and/or vegan.

(161) In some embodiments, an rOVD powder composition comprises less than 5% ash. The term ash is an art-known term and represents inorganics such as one or more ions, elements, minerals, and/or compounds In some cases, the rOVD powder composition comprises 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 weight per total weight (w/w) and/or weight per total volume (w/v).

(162) In some embodiments, the moisture content of an rOVD powder composition may be less than 15%. The rOVD powder composition may have less than 15%, 12%, 10%, 8%, 6%, 5%, 3%, 2% or 1% moisture weight per total weight (w/w) and/or weight per total volume (w/v). In some embodiments, the carbohydrate content of an rOVD powder composition may be less than 30%. The rOVD powder composition may have less than 30%, 27%, 25%, 22%, 20%, 17%, 15%, 12%, 10%, 8%, 5%, 3% or 1% carbohydrate content w/w or w/v.

(163) In some cases, the protein content of an rOVD powder composition may be 30% to 99% weight per total weight (w/w) and/or weight per total volume (w/v). In some cases, the protein content of an rOVD powder composition may be at least 30% w/w or w/v. In some cases, the protein content of an rOVD powder composition may be at most 99% w/w or w/v. In some cases, the protein content of an rOVD powder composition may be 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 75%, 30% to 80%, 30% to 85%, 30% to 90%, 30% to 95%, 30% to 99%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 75%, 40% to 80%, 40% to 85%, 40% to 90%, 40% to 95%, 40% to 99%, 50% to 60%, 50% to 70%, 50% to 75%, 50% to 80%, 50% to 85%, 50% to 90%, 50% to 95%, 50% to 99%, 60% to 70%, 60% to 75%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 99%, 70% to 75%, 70% to 80%, 70% to 85%, 70% to 90%, 70% to 95%, 70% to 99%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 99%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 99%, 85% to 90%, 85% to 95%, 85% to 99%, 90% to 95%, 90% to 99%, or 95% to 99% w/w or w/v. In some cases, the protein content of an rOVD powder composition may be about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w or w/v. In some cases, the protein content of an rOVD powder composition may be at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% w/w or w/v. In some cases, the protein content of an rOVD powder composition may be at most 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% w/w or w/v.

(164) Additional Components of Compositions

(165) The consumable food compositions containing rOVD disclosed herein and the methods of making such compositions may including adding or mixing the rOVD with one or more ingredients. For example, food additives may be added in or mixed with the compositions. Food additives can add volume and/or mass to a composition. A food additive may improve functional performance and/or physical characteristics. For example, a food additive may prevent gelation or increased viscosity due to the lipid portion of the lipoproteins in the freeze-thaw cycle. An anticaking agent may be added to make a free-flowing composition. Carbohydrates can be added to increase resistance to heat damage, e.g., less protein denaturation during drying and improve stability and flowability of dried compositions. Food additives include, but are not limited to, food coloring, pH adjuster, natural flavoring, artificial flavoring, flavor enhancer, batch marker, food acid, filler, anticaking agent (e.g., sodium silico aluminate), antigreening agent (e.g., citric acid), food stabilizer, foam stabilizer or binding agent, antioxidant, acidity regulatory, bulking agent, color retention agent, whipping agent (e.g., ester-type whipping agent, triethyl citrate, sodium lauryl sulfate), emulsifier (e.g., lecithin), humectant, thickener, excipient, solid diluent, salts, nutrient, sweetener, glazing agent, preservative, vitamin, dietary elements, carbohydrates, polyol, gums, starches, flour, oil, or bran.

(166) Food coloring includes, but is 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, ?-carotene), anthocyanins, annatto, betanin, butterfly pea, caramel coloring, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, Ponceau 4R, Patent Blue V, and Green S.

(167) Ingredients 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.

(168) 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 chlorides, zinc salts, and zinc chloride.

(169) Nutrient includes, but is not limited to, macronutrient, micronutrient, essential nutrient, non-essential nutrient, dietary fiber, amino acid, essential fatty acids, omega-3 fatty acids, and conjugated linoleic acid.

(170) Sweeteners include, but are not limited to, sugar substitute, artificial sweetener, acesulfame potassium, advantame, alitame, aspartame, sodium cyclamate, dulcin, glucin, neohesperidin dihydrochalcone, neotame, P-4000, saccharin, aspartame-acesulfame salt, sucralose, brazzein, curculin, glycyrrhizin, glycerol, inulin, mogroside, mabinlin, malto-oligosaccharide, mannitol, miraculin, monatin, monellin, osladin, pentadin, stevia, trilobatin, and thaumatin.

(171) Carbohydrates include, but are not limited to, sugar, sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allulose, tagatose, xylose, arabinose, high fructose corn syrup, high maltose corn syrup, corn syrup (e.g., glucose-free corn syrup), sialic acid, monosaccharides, disaccharides, and polysaccharides (e.g., polydextrose, maltodextrin).

(172) Polyols include, but are not limited to, xylitol, maltitol, erythritol, sorbitol, threitol, arabitol, hydrogenated starch hydrolysates, isomalt, lactitol, mannitol, and galactitol (dulcitol).

(173) Gums include, but are not limited to, gum arabic, gellan gum, guar gum, locust bean gum, acacia gum, cellulose gum, and xanthan gum.

(174) 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.

(175) Dietary 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.

(176) Packaging

(177) One of the benefits of the consumable compositions disclosed herein is that they allow for simpler packaging. In one instance, a consumable liquid composition disclosed herein may be packaged in a clear container as the lack of turbidity in the composition results in a more consumer-appealing product.

(178) A consumable composition can be refrigerated, frozen, stored warm, stored at room temperature or held at a heated temperature.

(179) An rOVD composition may be packaged as a powder, a concentrated syrup, a consumable food product, a beverage, a ready-to-use foodstuff, an ingredient, or a finished product.

(180) Recombinant OVD and OVL

(181) In any composition described herein, the protein may be recombinantly expressed in a host cell. The recombinant protein may be OVD, a first non-recombinant protein (e.g., OVD) and a second recombinant protein such as lysozyme (e.g. rOVL), or OVD and at least one second protein may both be recombinantly produced (for example rOVD and rOVL).

(182) rOVD or rOVL can have an amino acid sequence from any species. For example, an rOVD can have an amino acid sequence of OVD native to a bird (avian) or a reptile or Platypus and a rOVL can have an amino acid sequence of OVL native to a bird or a reptile or Platypus. An rOVD and/or rOVL having an amino acid sequence from an avian OVD and/or OVL can be selected from the group consisting of: poultry, fowl, waterfowl, game bird, chicken, quail, turkey, turkey vulture, hummingbird, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. An rOVD and/or rOVL can have an amino acid sequence native to a single species, such as Gallus gallus domesticus. Alternatively, an rOVD and/or rOVL can have an amino acid sequence native to two or more species, and as such be a hybrid.

(183) Exemplary OVD and OVL amino acid sequences contemplated herein are provided in Table 1 below as SEQ ID NOs: 1-44 and 45-51, respectively.

(184) TABLE-US-00001 TABLE1 Sequences SEQ Sequence ID Description NOs SEQUENCES Ovomucoid SEQID AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGT (canonical) NO:1 NISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYD maturechicken NECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLC OVD GSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQID AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSVEFGT variantofSEQID NO:2 NISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYD 1 NECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLC GSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC G162MF167A SEQID AEVDCSRFPNATDMEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSVEFGT Ovomucoid NO:3 NISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYD VariantofChicken NECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLC OVDinGenbank GSDNKTYMNKCNACNAVVESNGTLTLSHFGKC Ovomucoid SEQID MAMAGVFVLFSFVLCGFLPDAAFGAEVDCSRFPNATDKEGKDVLVCNKDLR isoform1 NO:4 PICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSED precursorfull GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKE length LAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSH FGKC Ovomucoid SEQID MAMAGVFVLFSFVLCGFLPDAVFGAEVDCSRFPNATDMEGKDVLVCNKDLR [Gallusgallus] NO:5 PICGTDGVTYTNDCLLCAYSVEFGTNISKEHDGECKETVPMNCSSYANTTSED GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKE LAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSH FGKC Ovomucoid SEQID MAMAGVFVLFSFVLCGFLPDAAFGAEVDCSRFPNATDKEGKDVLVCNKDLR isoform2 NO:6 PICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSED precursor GKVMVLCNRAFNPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKE [Gallusgallus] LAAVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFG KC Ovomucoid SEQID AEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYNNECLLCAYSIEFGT [Gallusgallus] NO:7 NISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYD NECLLCAHKVEQGASVDKRHDGECRKELAAVSVDCSEYPKPDCTAEDRPLC GSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQID MAMAGVFVLFSFALCGFLPDAAFGVEVDCSRFPNATNEEGKDVLVCTEDLRP [Numidameleagris] NO:8 ICGTDGVTYSNDCLLCAYNIEYGTNISKEHDGECREAVPVDCSRYPNMTSEEG KVLILCNKAFNPVCGTDGVTYDNECLLCAHNVEQGTSVGKKHDGECRKELA AVDCSEYPKPACTMEYRPLCGSDNKTYDNKCNFCNAVVESNGTLTLSHFGKC PREDICTED: SEQID MQTITWRQPQGDHLRSRAPAATCRAGQYLTMAMAGIFVLFSFALCGFLPDAA Ovomucoid NO:9 FGVEVDCSRFPNTTNEEGKDVLVCTEDLRPICGTDGVTHSECLLCAYNIEYGT isoformX1 NISKEHDGECREAVPMDCSRYPNTTNEEGKVMILCNKALNPVCGTDGVTYD [Meleagrisgallopavo] NECVLCAHNLEQGTSVGKKHDGGCRKELAAVSVDCSEYPKPACTLEYRPLC GSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQID VEVDCSRFPNTTNEEGKDVLVCTEDLRPICGTDGVTHSECLLCAYNIEYGTN [Meleagrisgallopavo] NO:10 ISKEHDGECREAVPMDCSRYPNTTSEEGKVMILCNKALNPVCGTDGVTYDNE CVLCAHNLEQGTSVGKKHDGECRKELAAVSVDCSEYPKPACTLEYRPLCGSD NKTYGNKCNFCNAVVESNGTLTLSHFGKC PREDICTED: SEQID MQTITWRQPQGDHLRSRAPAATCRAGQYLTMAMAGIFVLFSFALCGFLPDAA Ovomucoid NO:11 FGVEVDCSRFPNTTNEEGKDVLVCTEDLRPICGTDGVTHSECLLCAYNIEYGT isoformX2 NISKEHDGECREAVPMDCSRYPNTTNEEGKVMILCNKALNPVCGTDGVTYD [Meleagrisgallopavo] NECVLCAHNLEQGTSVGKKHDGGCRKELAAVDCSEYPKPACTLEYRPLCGS DNKTYGNKCNFCNAVVESNGTLTLSHFGKC Ovomucoid SEQID EYGTNISIKHNGECKETVPMDCSRYANMTNEEGKVMMPCDRTYNPVCGTDG [Bambusicola NO:12 VTYDNECQLCAHNVEQGTSVDKKHDGVCGKELAAVSVDCSEYPKPECTAEE thoracicus] RPICGSDNKTYGNKCNFCNAVVYVQP Ovomucoid SEQID VDCSRFPNTTNEEGKDVLACTKELHPICGTDGVTYSNECLLCYYNIEYGTNIS [Callipeplasquamata] NO:13 KEHDGECTEAVPVDCSRYPNTTSEEGKVLIPCNRDFNPVCGSDGVTYENECLL CAHNVEQGTSVGKKHDGGCRKEFAAVSVDCSEYPKPDCTLEYRPLCGSDNK TYASKCNFCNAVVIWEQEKNTRHHASHSVFFISARLVC Ovomucoid SEQID MLPLGLREYGTNTSKEHDGECTEAVPVDCSRYPNTTSEEGKVRILCKKDINPV [Colinusvirginianus] NO:14 CGTDGVTYDNECLLCSHSVGQGASIDKKHDGGCRKEFAAVSVDCSEYPKPAC MSEYRPLCGSDNKTYVNKCNFCNAVVYVQPWLHSRCRLPPTGTSFLGSEGRE TSLLTSRATDLQVAGCTAISAMEATRAAALLGLVLLSSFCELSHLCFSQASCD VYRLSGSRNLACPRIFQPVCGTDNVTYPNECSLCRQMLRSRAVYKKHDGRCV KVDCTGYMRATGGLGTACSQQYSPLYATNGVIYSNKCTFCSAVANGEDIDLL AVKYPEEESWISVSPTPWRMLSAGA Ovomucoid-like SEQID MSWWGIKPALERPSQEQSTSGQPVDSGSTSTTTMAGIFVLLSLVLCCFPDAAF isoformX2 NO:15 GVEVDCSRFPNTTNEEGKEVLLCTKDLSPICGTDGVTYSNECLLCAYNIEYGT [Ansercygnoides NISKDHDGECKEAVPVDCSTYPNMTNEEGKVMLVCNKMFSPVCGTDGVTYD domesticus] NECMLCAHNVEQGTSVGKKYDGKCKKEVATVDCSDYPKPACTVEYMPLCG SDNKTYDNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQID MSSQNQLHRRRRPLPGGQDLNKYYWPHCTSDRFSWLLHVTAEQFRHCVCIY isoformX1 NO:16 LQPALERPSQEQSTSGQPVDSGSTSTTTMAGIFVLLSLVLCCFPDAAFGVE [Ansercygnoides VDCSRFPNTTNEEGKEVLLCTKDLSPICGTDGVTYSNECLLCAYNIEYGTN domesticus] ISKDHDGECKEAVPVDCSTYPNMTNEEGKVMLVCNKMFSPVCGTDGVTYDN ECMLCAHNVEQGTSVGKKYDGKCKKEVATVDCSDYPKPACTVEYMPLCGSD NKTYDNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid SEQID VEVDCSRFPNTTNEEGKDEVVCPDELRLICGTDGVTYNHECMLCFYNKEYGT [Coturnixjaponica] NO:17 NISKEQDGECGETVPMDCSRYPNTTSEDGKVTILCTKDFSFVCGTDGVTYDNE CMLCAHNVVQGTSVGKKHDGECRKELAAVSVDCSEYPKPACPKDYRPVCGS DNKTYSNKCNFCNAVVESNGTLTLNHFGKC Ovomucoid SEQID MAMAGVFLLFSFALCGFLPDAAFGVEVDCSRFPNTTNEEGKDEVVCPDELRLI [Coturnixjaponica] NO:18 CGTDGVTYNHECMLCFYNKEYGTNISKEQDGECGETVPMDCSRYPNTTSED GKVTILCTKDFSFVCGTDGVTYDNECMLCAHNIVQGTSVGKKHDGECRKEL AAVSVDCSEYPKPACPKDYRPVCGSDNKTYSNKCNFCNAVVESNGTLTLNHF GKC Ovomucoid SEQID MAGVFVLLSLVLCCFPDAAFGVEVDCSRFPNTTNEEGKDVLLCTKELSPVCG [Anasplatyrhynchos] NO:19 TDGVTYSNECLLCAYNIEYGTNISKDHDGECKEAVPADCSMYPNMTNEEGK MTLLCNKMFSPVCGTDGVTYDNECMLCAHNVEQGTSVGKKYDGKCKKEVA TVDCSGYPKPACTMEYMPLCGSDNKTYGNKCNFCNAVVDSNGTLTLSHFGEC Ovomucoid, SEQID QVDCSRFPNTTNEEGKEVLLCTKELSPVCGTDGVTYSNECLLCAYNIEYGTNI partial NO:20 SKDHDGECKEAVPADCSMYPNMTNEEGKMTLLCNKMFSPVCGTDGVTYDN [Anasplatyrhynchos] ECMLCAHNVEQGTSVGKKYDGKCKKEVATVSVDCSGYPKPACTMEYMPLC GSDNKTYGNKCNFCNAVV Ovomucoid-like SEQID MTMPGAFVVLSFVLCCFPDATFGVEVDCSTYPNTTNEEGKEVLVCSKILSPIC [Tytoalba] NO:21 GTDGVTYSNECLLCANNIEYGTNISKYHDGECKEFVPVNCSRYPNTTNEEGK VMLICNKDLSPVCGTDGVTYDNECLLCAHNLEPGTSVGKKYDGECKKEIATV DCSDYPKPVCSLESMPLCGSDNKTYSNKCNFCNAVVDSNETLTLSHFGKC Ovomucoid SEQID MTMAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC [Balearica NO:22 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVVPVDCSRYPNSTNEEGK regulorum VVMLCSKDLNPVCGTDGVTYDNECVLCAHNVESGTSVGKKYDGECKKETA gibbericeps] TVDCSDYPKPACTLEYMPFCGSDSKTYSNKCNFCNAVVDSNGTLTLSHFGKC Turkeyvulture SEQID MTTAGVFVLLSFALCSFPDAAFGVEVDCSTYPNTTNEEGKEVLVCTKILSPI [Cathartesaura] NO:23 CGTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEFVPVDCSRYPNTTNEDG OVD(native KVVLLCNKDLSPICGTDGVTYDNECLLCARNLEPGTSVGKKYDGECKKEIAT sequence) VDCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLTLSHFGKC boldedisnative signalsequence Ovomucoid-like SEQID MTTAGVFVLLSFTLCSFPDAAFGVEVDCSPYPNTTNEEGKEVLVCNKILSPI [Cuculuscanorus] NO:24 CGTDGVTYSNECLLCAYNLEYGTNISKDYDGECKEVAPVDCSRHPNTTNEEG KVELLCNKDLNPICGTNGVTYDNECLLCARNLESGTSIGKKYDGECKKEIAT VDCSDYPKPVCTLEEMPLCGSDNKTYGNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid SEQID MTTAVVFVLLSFALCCFPDAAFGVEVDCSTYPNSTNEEGKDVLVCPKILGPIC [Antrostomus NO:25 GTDGVTYSNECLLCAYNIQYGTNVSKDHDGECKEIVPVDCSRYPNTTNEEGK carolinensis] VVFLCNKNFDPVCGTDGDTYDNECMLCARSLEPGTTVGKKHDGECKREIAT VDCSDYPKPTCSAEDMPLCGSDSKTYSNKCNFCNAVVDSNGTLTLSRFGKC Ovomucoid SEQID MTMTGVFVLLSFAICCFPDAAFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC [Cariamacristata] NO:26 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVVPVDCSKYPNTTNEEGKV VLLCSKDLSPVCGTDGVTYDNECLLCARNLEPGSSVGKKYDGECKKEIATIDC SDYPKPVCSLEYMPLCGSDSKTYDNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQID MTTAGVFVLLSFVLCCFPDAVFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC isoformX2 NO:27 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVVPVNCSRYPNTTNEEGK [Pygoscelisadeliae] VVLRCSKDLSPVCGTDGVTYDNECLMCARNLEPGAVVGKNYDGECKKEIAT VDCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQID MTTAGVFVLLSIALCCFPDAAFGVEVDCSAYSNTTSEEGKEVLSCTKILSPIC [Nipponianippon] NO:28 GTDGVTYSNECLLCAYNIEYGTNISKDHDGECKEVVSVDCSRYPNTTNEEGKA VLLCNKDLSPVCGTDGVTYDNECLLCAHNLEPGTSVGKKYDGACKKEIATV DCSDYPKPVCTLEYLPLCGSDSKTYSNKCDFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQID MTTAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC [Phaethonlepturus] NO:29 GTDGTTYSNECLLCAYNIEYGTNVSKDHDGECKVVPVDCSKYPNTTNEDGK VVLLCNKALSPICGTDRVTYDNECLMCAHNLEPGTSVGKKHDGECQKEVAT VDCSDYPKPVCSLEYMPLCGSDGKTYSNKCNFCNAVVNSNGTLTLSHFEKC Ovomucoid-like SEQID MTTAGVFVLLSFVLCCFFPDAAFGVEVDCSTYPNTTNEEGKEVLVCAKILSPV isoformX1 NO:30 CGTDGVTYSNECLLCAHNIENGTNVGKDHDGKCKEAVPVDCSRYPNTTDEE [Melopsittacus GKVVLLCNKDVSPVCGTDGVTYDNECLLCAHNLEAGTSVDKKNDSECKTED undulatus] TTLAAVSVDCSDYPKPVCTLEYLPLCGSDNKTYSNKCRFCNAVVDSNGTLTL SRFGKC Ovomucoid SEQID MTTAGVFVLLSFALCCSPDAAFGVEVDCSTYPNTTNEEGKEVLACTKILSPIC [Podicepscristatus] NO:31 GTDGVTYSNECLLCAYNMEYGTNVSKDHDGKCKEVVPVDCSRYPNTTNEEG KVVLLCNKDLSPVCGTDGVTYDNECLLCARNLEPGASVGKKYDGECKKEIA TVDCSDYPKPVCSLEHMPLCGSDSKTYSNKCTFCNAVVDSNGTLTLSHFGKC Ovomucoid-like SEQID MTTAGVFVLLSFALCCFPDAAFGVEVDCSTYPNTTNEEGREVLVCTKILSPIC [Fulmarus NO:32 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVAPVGCSRYPNTTNEEGK glacialis] VVLLCNKDLSPVCGTDGVTYDNECLLCARHLEPGTSVGKKYDGECKKEIATV DCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVLDSNGTLTLSHFGKC Ovomucoid SEQID MTTAGVFVLLSFALCCFPDAVFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC [Aptenodytes NO:33 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVVPVDCSRYPNTTNEEGK forsteri] VVLRCNKDLSPVCGTDGVTYDNECLMCARNLEPGAIVGKKYDGECKKEIAT VDCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLILSHFGKC Ovomucoid-like SEQID MTTAGVFVLLSFVLCCFPDAVFGVEVDCSTYPNTTNEEGKEVLVCTKILSPIC isoformX1 NO:34 GTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEVVPVDCSRYPNTTNEEGK [Pygoscelis VVLRCSKDLSPVCGTDGVTYDNECLMCARNLEPGAVVGKNYDGECKKEIAT adeliae] VDCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVVDSNGTLTLSHFGKC Ovomucoid SEQID MSSQNQLPSRCRPLPGSQDLNKYYQPHCTGDRFCWLFYVTVEQFRHCICIYLQ isoformX1 NO:35 LALERPSHEQSGQPADSRNTSTMTTAGVFVLLSFALCCFPDAVFGVEVDCSTY [Aptenodytesforsteri] PNTTNEEGKEVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKDHDGE CKEVVPVDCSRYPNTTNEEGKVVLRCNKDLSPVCGTDGVTYDNECLMCARN LEPGAIVGKKYDGECKKEIATVDCSDYPKPVCSLEYMPLCGSDSKTYSNKCN FCNAVVDSNGTLILSHFGKC Ovomucoid, SEQID MTTAVVFVLLSFALCCFPDAAFGVEVDCSTYPNSTNEEGKDVLVCPKILGPIC partial NO:36 GTDGVTYSNECLLCAYNIQYGTNVSKDHDGECKEIVPVDCSRYPNTTNEEGK [Antrostomus VVFLCNKNFDPVCGTDGDTYDNECMLCARSLEPGTTVGKKHDGECKREIAT carolinensis] VDCSDYPKPTCSAEDMPLCGSDSKTYSNKCNFCNAVV rOVDas SEQID EAEAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSI expressedinpichia NO:37 EFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGV secretedform1 TYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDR PLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC rOVDas SEQID EEGVSLEKREAEAAEVDCSRFPNATDKEGKDVLVCNKDLRPICGTDGVTYTN expressedinpichia NO:38 DCLLCAYSIEFGTNISKEHDGECKETVPMNCSSYANTTSEDGKVMVLCNRAF secretedform2 NPVCGTDGVTYDNECLLCAHKVEQGASVDKRHDGGCRKELAAVSVDCSEYP KPDCTAEDRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC rOVD[gallus] SEQID MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA codingsequence NO:39 VLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAAEVDCSRFPNATDK containingan EGKDVLVCNKDLRPICGTDGVTYTNDCLLCAYSIEFGTNISKEHDGECKETVP alphamating MNCSSYANTTSEDGKVMVLCNRAFNPVCGTDGVTYDNECLLCARKVEQGA factorsignal SVDKRHDGGCRKELAAVSVDCSEYPKPDCTAEDRPLCGSDNKTYGNKCNFC sequence(bolded) NAVVESNGTLTLSHFGKC asexpressedin pichia Turkeyvulture SEQID MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA OVDcoding NO:40 VLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAVEVDCSTYPNTTNE sequence EGKEVLVCTKILSPICGTDGVTYSNECLLCAYNIEYGTNVSKDHDGECKEFVP containing VDCSRYPNTTNEDGKVVLLCNKDLSPICGTDGVTYDNECLLCARNLEPGTSV secretionsignals GKKYDGECKKEIATVDCSDYPKPVCSLEYMPLCGSDSKTYSNKCNFCNAVV asexpressedin DSNGTLTLSHFGKC pichia boldedisanalpha matingfactor signalsequence Turkeyvulture SEQID EAEAVEVDCSTYPNTTNEEGKEVLVCTKILSPICGTDGVTYSNECLLCAYNIE OVDinsecreted NO:41 YGTNVSKDHDGECKEFVPVDCSRYPNTTNEDGKVVLLCNKDLSPICGTDGVT formexpressedin YDNECLLCARNLEPGTSVGKKYDGECKKEIATVDCSDYPKPVCSLEYMPLCG Pichia SDSKTYSNKCNFCNAVVDSNGTLTLSHFGKC Hummingbird SEQID MTMAGVFVLLSFILCCFPDTAFGVEVDCSIYPNTTSEEGKEVLVCIETLSPIC OVD(native NO:42 GSDGVTYNNECQLCAYNVEYGTNVSKDHDGECKEIVPVDCSRYPNTTEEGR sequence) VVMLCNKALSPVCGTDGVTYDNECLLCARNLESGTSVGKKFDGECKKEIAT boldedisthe VDCTDYPKPVCSLDYMPLCGSDSKTYSNKCNFCNAVMDSNGTLTLNHFGKC nativesignal sequence Hummingbird SEQID MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA OVDcoding NO:43 VLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAVEVDCSIYPNTTSEE sequenceas GKEVLVCIETLSPICGSDGVTYNNECQLCAYNVEYGTNVSKDHDGECKEIVP expressedin VDCSRYPNTIEEGRVVMLCNKALSPVCGTDGVTYDNECLLCARNLESGTSV Pichia GKKFDGECKKEIATVDCTDYPKPVCSLDYMPLCGSDSKTYSNKCNFCNAVM boldedisanalpha DSNGTLTLNHFGKC matingfactor signalsequence Hummingbird SEQID EAEAVEVDCSIYPNTTSEEGKEVLVCIETLSPICGSDGVTYNNECQLCAYNVE OVDinsecreted NO:44 YGTNVSKDHDGECKEIVPVDCSRYPNTTEEGRVVMLCNKALSPVCGTDGVT formfromPichia YDNECLLCARNLESGTSVGKKFDGECKKEIATVDCTDYPKPVCSLDYMPLCG SDSKTYSNKCNFCNAVMDSNGTLTLNHFGKC rOVLasexpressed SEQID MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVA inpichia NO:45 VLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAKVFGRCELAAANIK boldedisanalpha RHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWC matingfactor NDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCK signalsequence GTDVQAWIRGCRL rOVLasfound SEQID EAEAKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRN aftersecretion NO:46 TDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAK fromPichia KIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL Lysozyme(OVL) SEQID KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGS fromGallusgallus NO:47 TDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGN (withoutsignal GMNAWVAWRNRCKGTDVQAWIRGCRL sequence) Lysozyme SEQID KVFGRCELAAAMKRHGLDNYRGYSLGNWVCVAKFESNFNTQATNRNTDGS NO:48 TDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGN GMSAWVAWRNRCKGTDVQAWIRGCRL LysozymeC SEQID KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDR (Human) NO:49 STDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRD PQGIRAWVAWRNRCQNRDVRQYVQGCGV LysozymeC(Bos SEQID KVFERCELARTLKKLGLDGYKGVSLANWLCLTKWESSYNTKATNYNPSSEST taurus) NO:50 DYGIFQINSKWWCNDGKTPNAVDGCHVSCRELMENDIAKAVACAKHIVSEQ GITAWVAWKSHCRDHDVSSYVEGCTL Lysozyme(OVL) SEQID MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA fromGallusgallus NO:51 KFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLS Nativesecretion SDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL signalisbolded OCH1:EndoH SEQID MAKADGSLLYYNPHNPPRRYYFYMAIFAVSVICVLYGPSQQLSSPKIDASAPA fusionprotein NO:52 PVKQGPTSVAYVEVNNNSMLNVGKYTLADGGGNAFDVAVIFAANINYDTGT KTAYLHFNENVQRVLDNAVTQIRPLQQQGIKVLLSVLGNHQGAGFANFPSQQ AASAFAKQLSDAVAKYGLDGVDFDDEYAEYGNNGTAQPNDSSFVHLVTALR ANMPDKIISLYNIGPAASRLSYGGVDVSDKFDYAWNPYYGTWQVPGIALPKA QLSPAAVEIGRTSRSTVADLARRTVDEGYGVYLTYNLDGGDRTADVSAFTRE LYGSEAVRTP

(185) An rOVD or rOVL can include additional sequences. Expression of rOVD and rOVL in a host cell, for instance a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species may lead to an addition of peptides to the OVD or OVL sequence as part of post-transcriptional or post-translational modifications. Such peptides may not be part of the native OVD or OVL sequences. For instance, expressing an OVD sequence in a Pichia species, such as Komagataella phaffii and Komagataella pastoris may lead to addition of a peptide at the N-terminus or C-terminus. In some cases, a tetrapeptide EAEA (SEQ ID NO: 53) is added to the N-terminus of the OVD sequence upon expression in a host cell. In some embodiments, rOVD or rOVL or both include the amino acids EAEA at the N-terminus. An OVD or OVL protein sequence can include a signal sequence, such as for directing secretion from a host cell. In some cases, the signal sequence may be a native signal sequence. In some cases, a signal sequence may be a heterologous signal sequence. For instance, an alpha mating factor signal sequence can be fused to an OVD or OVL sequence for expression and secretion in a yeast cell such as a Pichia sp. In some cases, the signal sequence is removed in whole or in part when the protein, such as an rOVD or rOVL, is secreted from the host cell.

(186) An rOVD and/or rOVL can be a non-naturally occurring variant of an OVD and/or OVL. Such variant can comprise one or more amino acid insertions, deletions, or substitutions relative to a native OVD or native OVL sequence.

(187) Such an rOVD variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1-44. A rOVL variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 45-51. The term sequence identity as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

(188) In some embodiments, a variant is one that confers additional features, such as reduced allergenicity. For example, an rOVD can include G162M and/or F167A (such as in SEQ ID NO: 3) relative to a wild type OVD sequence SEQ ID NO: 2 and have reduced allergenicity as compared to the wild type OVD sequence.

(189) Depending on the host organism used to express the rOVD and/or rOVL, the rOVD and/or rOVL can have a glycosylation, acetylation, or phosphorylation pattern different from wildtype OVD (e.g., native OVD) or wildtype OVL (e.g., native OVL). For example, the rOVD and/or rOVL herein may or may not be glycosylated, acetylated, or phosphorylated. An rOVD and/or rOVL may have an avian, non-avian, microbial, non-microbial, mammalian, or non-mammalian glycosylation, acetylation, or phosphorylation pattern.

(190) An rOVD and/or rOVL is recombinantly expressed in a host cell. As used herein, a host or host cell denotes here any protein production host selected or genetically modified to produce a desired product. Exemplary hosts include fungi, such as filamentous fungi, as well as bacteria, yeast, plant, insect, and mammalian cells. A host cell may be 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., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicilliumfuniculo sum, 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. A host cell can be an organism that is approved as generally regarded as safe by the U.S. Food and Drug Administration.

(191) A recombinant protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, recombinant protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pastoris), a Saccharomyces species, a Trichoderma species, a Trichoderma species, a Pseudomonas species or an E. coli species.

(192) A host cell may be transformed to include one or more expression cassettes. As examples, a host cell may be transformed to express one expression cassette, two expression cassettes, three expression cassettes or more expression cassettes.

(193) In some cases, rOVD and/or rOVL may be deglycosylated or modified in its glycosylation (e.g., chemically, enzymatically through endoglucanases (such as EndoH), endoglycosidases, mannosidases (such as alpha-1,2 mannosidase), PNGase F, O-Glycosidase, OCH1, Neuraminidase, ?,1-4 Galactosidase, ?-N-acetylglucosaminidases, etc.), 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 may produce a protein that is more uniform or is capable of producing a composition with less variation.

(194) The present disclosure contemplates modifying glycosylation of the recombinant OVD to alter or enhance one or more functional characteristics of the protein and/or its production. A host cell may comprise heterologous enzymes that modify the glycosylation pattern of ovomucoid. In some cases, one or more enzymes may be used for modifying the glycosylation of rOVD protein. The enzymes used modifying glycosylation of rOVD may be an enzyme or a fusion protein comprising an enzyme or active fragment of an enzyme, for example EndoH or a fusion of OCH1 to EndoH (such as to provide for Golgi retention of the EndoH enzyme) may be provided in a host cell.

(195) Native ovomucoid (nOVD), such as isolated from a chicken or other avian egg, has a highly complex branched form of glycosylation. The glycosylation pattern comprises N-linked glycan structures such as N-acetylglucosamine units and N-linked mannose units. See, e.g., FIG. 1B (left-hand column). In some cases, the rOVD for use in a herein disclosed consumable composition and produced using the methods described herein has a glycosylation pattern which is different than the glycosylation pattern of nOVD. For example, when rOVD is produced in a Pichia sp., the protein may be highly glycosylated. FIG. 1C illustrates the glycosylation patterns of rOVD produced by P. pastoris, showing a complex branched glycosylation pattern. In some embodiments of the compositions and methods herein, rOVD is treated such that the glycosylation pattern is modified from that of nOVD and also modified as compared to rOVD produced by a Pichia sp. without such treatment. In some cases, the rOVD has no glycosylation. In other cases, the rOVD has reduced glycosylation. In some cases, the rOVD is modified by N-acetylglucosamine at one or more asparagine residues of the protein and lacks or is substantially devoid of N-linked mannosylation. See, e.g., FIG. 1B (right hand column). The changes in glycosylation described herein may lead to an increase in the solubility and clarity of rOVD as compared to other forms of protein such as whey proteins, soy proteins, pea proteins, and nOVD.

(196) In some cases, an enzyme used for modifying glycosylation may be transformed into a host cell. In some cases, the enzyme used for modifying glycosylation may be transformed into the same host cell that produces rOVD. In some cases, the enzyme may be provided transiently to the host cell, such as by an inducible expression system. In some cases, when a host cell expresses an enzyme used for modifying glycosylation, the recombinant protein (e.g., rOVD and rOVL) is secreted from the host cell in the modified state.

(197) In one example, a host cell producing OVD comprises a fusion of EndoH and OCH1 enzymes. An exemplary OCH1-EndoH protein sequence is provided as SEQ ID No: 52. In such cases, an rOVD produced from the host cell comprises a glycosylation pattern substantially different from an rOVD which is produced in a cell without such enzymes. The rOVD produced in such cases is also substantially different as compared to a native OVD (e.g., produced by a chicken or other avian egg). FIG. 1B shows a comparison of nOVD (with mannose residues) and rOVD glycosylation patterns wherein the rOVD was treated with EndoH and comprises an N-acetylglucosamine residue at the asparagine but no mannose residues. FIG. 1C shows the glycosylation pattern of rOVD produced in a host cell such as P. pastoris and where rOVD was not treated with EndoH and has both N-acetylglucosamine resides as well as the chains of N-linked mannose residues. Modification of the glycosylation of rOVD may provide nutritional benefits to rOVD, such as a higher nitrogen to carbon ratio, and may improve the clarity and solubility of the protein. In some cases, the modification of the glycosylation of rOVD is performed within the host cell that produces rOVD before the rOVD is secreted from the host cell and/or before isolating the rOVD. In some cases, modification of the glycosylation of rOVD is performed after its secretion and/or after isolating rOVD from the host cell.

(198) The molecular weight or rOVD may be different as compared to nOVD. The molecular weight of the protein may be less than the molecular weight of nOVD or less than rOVD produced by the host cell where the glycosylation of rOVD is not modified. In embodiments, the molecular weight of an rOVD may be between 20 kDa and 40 kDa. In some cases, an rOVD with modified glycosylation has a different molecular weight, such as compared to a native OVD (as produced by an avian host species) or as compared to a host cell that glycosylates the rOVD, such as where the rOVD includes N-linked mannosylation. In some cases, the molecular weight of rOVD is greater than the molecular weight of the rOVD that is completely devoid of post-translational modifications. or an rOVD that lacks all forms of N-linked glycosylation.

(199) Expression of an rOVD or rOVL can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, a vector for expression can include: (a) a promoter element, (b) a signal peptide, (c) a heterologous OVD or OVL sequence, and (d) a terminator element.

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

(201) To aide in the amplification of the vector prior to transformation into the host microorganism, a replication origin (e) may be contained in the vector (such as PUC_ORIC and PUC (DNA2.0)). To aide in the selection of microorganism stably transformed with the expression vector, the vector may also include a selection marker (f) such as URA3 gene and Zeocin resistance gene (ZeoR). The expression vector may also contain a restriction enzyme site (g) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vectors stable integration into the host genome. In some embodiments the expression vector may contain any subset of the elements (b), (e), (f), and (g), including none of elements (b), (e), (f), and (g). Other expression elements and vector element known to one of skill in the art can be used in combination or substituted for the elements described herein.

(202) Exemplary promoter elements (a) may include, but are not limited to, a constitutive promoter, inducible promoter, and hybrid promoter. Promoters include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, ?-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular 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, ?-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUTi), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, ?-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PH089, 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, a sequence or subsequence chosen from SEQ ID Nos: 121 to 132, and any combination thereof. Illustrative inducible promoters include methanol-induced promoters, e.g., DAS1 and pPEX11.

(203) A signal peptide (b), also known as a signal sequence, targeting signal, localization signal, localization sequence, signal peptide, transit peptide, leader sequence, or leader peptide, may support secretion of a protein or polynucleotide. Extracellular secretion of a recombinant or heterologously expressed protein from a host cell may facilitate protein purification. A signal peptide may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. Signal peptides can be derived from a precursor of a protein other than the signal peptides in native OVD and/or OVL.

(204) Any nucleic acid sequence that encodes OVD and/or OVL can be used as (c). Preferably such sequence is codon optimized for the host cell.

(205) Exemplary transcriptional terminator elements include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, ?-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular 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, ?-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUTi), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, ?-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PH089, 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.

(206) Exemplary selectable markers (f) may include but are not limited to: an antibiotic resistance gene (e.g. zeocin, ampicillin, blasticidin, kanamycin, nurseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), an auxotrophic marker (e.g. ade1, arg4, his4, ura3, met2, and any combination thereof).

(207) In one example, a vector for expression in Pichia sp. can include an AOX1 promoter operably linked to a signal peptide (alpha mating factor) that is fused in frame with a nucleic acid sequence encoding OVD and/or OVL, and a terminator element (AOX1 terminator) immediately downstream of the nucleic acid sequence encoding OVD and/or OVL.

(208) In another example, a vector comprising a DAS1 promoter is operably linked to a signal peptide (alpha mating factor) that is fused in frame with a nucleic acid sequence encoding OVD and/or OVL and a terminator element (AOX1 terminator) immediately downstream of OVD and/or OVL.

(209) A recombinant protein described herein may be secreted from the one or more host cells. In some embodiments, rOVD and/or rOVL protein is secreted from the host cell. The secreted rOVD and/or rOVL may be isolated and purified by methods such as centrifugation, fractionation, filtration, affinity purification and other methods for separating protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, rOVD and/or rOVL is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rOVD and/or rOVL is then separated from other media components for further use.

(210) In some cases, multiple vectors comprising OVD may be transfected into one or more host cells. A host cell may comprise more than one copy of OVD. A single host cell may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 copies of OVD. A single host cell may comprise one or more vectors for the expression of OVD. A single host cell may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 vectors for OVD expression. Each vector in the host cell may drive the expression of OVD using the same promoter. Alternatively, different promoters may be used in different vectors for OVD expression.

(211) The consumable products and rOVD and/or rOVL compositions herein can be essentially free of any microbial cells or microbial cell contaminants. For instance, rOVD and/or rOVL may be isolated from a culture comprising microbial growth.

(212) rOVD may be treated chemically or enzymatically before it is purified for use in a consumable composition. Such treatments may be performed to reduce impurities in an rOVD protein composition. Such treatments may be performed to improve the sensory attributes of the rOVD protein composition. Treatments may include but are not limited to purification steps, filtration, chemical treatments, and enzymatic treatments.

(213) In some cases, rOVD protein and compositions containing rOVD protein, including forms of rOVD with modified glycosylation (e.g., such forms with N-acetylglucosamine but lacking N-linked mannose residues) may be treated with oxidizing agent or an oxygen-generating agent to modify components of the rOVD composition, such as impurities. The oxidizing agent or oxygen-generating agent may comprise hydrogen peroxide, sodium percarbonate, activated chlorine dioxide, bubbled oxygen or ozone. The treatment may improve the solubility and clarity of an rOVD composition. The treatment may reduce the odor of an rOVD composition. The treatment may neutralize the color of an rOVD composition; for instance, the rOVD composition may lose color after a treatment, e.g., to a less intense/lighter coloration. In embodiments, the color may change form greenish to yellowish and/or from yellowish to essentially colorless.

(214) In some examples, rOVD may be treated with an oxidizing agent or an oxygen-generating agent, e.g., hydrogen peroxide or sodium percarbonate, before it is purified for use in a consumable composition. A culture medium comprising secreted or isolated rOVD may be treated with an oxygen-generating agent, e.g., hydrogen peroxide or sodium percarbonate. Using hydrogen peroxide as an example, a hydrogen peroxide treatment may be followed by one or more wash steps and/or filtration steps to remove hydrogen peroxide from the resulting rOVD compositions. Such steps may be performed following treatments with other oxygen-generating agents, e.g., sodium percarbonate.

(215) In some cases, the concentration of hydrogen peroxide used for treating rOVD may be from 1% to 20%. The concentration of hydrogen peroxide used for treating rOVD may be at least 1%. The concentration of hydrogen peroxide used for treating rOVD may be at most 20%. The concentration of hydrogen peroxide used for treating rOVD may be 1% to 2%, 1% to 5%, 1% to 7%, 1% to 10%, 1% to 12%, 1% to 15%, 1% to 17%, 1% to 20%, 2% to 5%, 2% to 7%, 2% to 10%, 2% to 12%, 2% to 15%, 2% to 17%, 2% to 20%, 5% to 7%, 5% to 10%, 5% to 12%, 5% to 15%, 5% to 17%, 5% to 20%, 7% to 10%, 7% to 12%, 7% to 15%, 7% to 17%, 7% to 20%, 10% to 12%, 10% to 15%, 10% to 17%, 10% to 20%, 12% to 15%, 12% to 17%, 12% to 20%, 15% to 17%, 15% to 20%, or 17% to 20% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of hydrogen peroxide used for treating rOVD may be about 1%, 2%, 5%, 7%, 10%, 12%, 15%, 17%, or 20% w/w or w/v. The concentration of hydrogen peroxide used for treating rOVD may be at least 1%, 2%, 5%, 7%, 10%, 12%, 15% or 17% w/w or w/v. The concentration of hydrogen peroxide used for treating rOVD may be at most 2%, 5%, 7%, 10%, 12%, 15%, 17%, or 20% w/w or w/v.

(216) rOVD may be treated with hydrogen peroxide for a limited duration of time. For instance, rOVD may be exposed to hydrogen peroxide for at least 1 hour, 2 hours, 3 hours, 5 hours, 7 hours, 10 hours, 12 hours, 15 hours, 17 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 34 hours, 36 hours, 40 hours, 44 hours or 48 hours. Hydrogen peroxide may be added to the rOVD culture media throughout the culturing process.

(217) rOVD may be treated with hydrogen peroxide at a pH of about 3 to 6. rOVD may be treated with hydrogen peroxide at a pH of about 3, 3.2, 3.4, 3.6, 3.8, 4, 4.1, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8 or 6. rOVD may treated with hydrogen peroxide at a pH of at least 3, 3.2, 3.4, 3.6, 3.8, 4, 4.1, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6 or 5.8. rOVD may treated with hydrogen peroxide at a pH of at most 3.2, 3.4, 3.6, 3.8, 4, 4.1, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8 or 6.

(218) rOVD may be filtered before treatment with an oxygen-generating agent. In some cases, rOVD may be filtered before and after treatment with an oxygen-generating agent.

(219) rOVA

(220) Proteins are important dietary nutrients and food ingredients. They can serve as a fuel source or as sources of amino acids, including the essential amino acids that cannot be synthesized by the body. The daily recommended intake of protein for healthy adults is 10% to 35% of a person's total calorie needs, and currently the majority of protein intake for most humans 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 sourced from animals. With the world population growth and the coinciding growth in global food demand, there is a need to provide alternative sustainable, non-animal-based sources of proteins as useful source of protein for daily diet, food ingredients and food products.

(221) While various embodiments of the invention have been shown and described herein, it will be obvious 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

(222) Provided herein are compositions and methods of making compositions for non-animal-based sources of proteins which provide nutritional as well as functional properties to food ingredients and consumable products for ingestion by an animal, including a human, such as for daily diet, ingredients for human food and treats and for human and animal nutrition.

(223) The compositions and methods provided herein contain fermentation-derived ovalbumin, produced through recombinant technology, i.e., a recombinant ovalbumin (rOVA). The compositions and methods for making compositions comprising rOVA can increase the protein content of a consumable or food ingredient, and also provide functional features for use in the preparation of food ingredients and consumable food products for animal and human ingestion.

(224) In some embodiments, the rOVA provides one or more functional characteristics such as of gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, humectant, clarification, and cohesiveness. The rOVA with such feature(s) can be a food ingredient that provides for production of an egg-less or animal-free food ingredient or food product.

(225) As used herein native in the context of native egg white, native egg protein, native ovalbumin and native egg, refers to the egg white, egg protein, ovalbumin or whole egg, respectively, produced by an animal or collected from an animal, in particular an egg-laying animal such as a bird. The 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 egg protein, native ovalbumin or native egg. In some cases, the ingredients or food products made using rOVA do not include any egg-white proteins other than rOVA. The 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 products.

(226) In some embodiments, the rOVA can (alone or with other ingredients) substitute for the use of whole egg or egg white in the production of a food product. In some embodiments, the feature(s) provided by the rOVA is substantially the same or better than the same characteristic provided by a native egg white or native egg. For example, the rOVA and compositions containing rOVA can have gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, preserving moisture (humectant), clarification, and cohesiveness, improved color, such as a whiter color, as compared to native egg white or native whole egg and compositions made with native egg white.

(227) Food Ingredients and Food Products with rOVA

(228) Food ingredients and food products disclosed herein include compositions that comprise, consists essentially of, or consist of rOVA, where rOVA provides at least one functional feature to the composition, food ingredient, or food product. In some cases, at least one functional feature provided by the rOVA is comparable or substantially similar to a native egg or egg white or native OVA (nOVA). For instance, it may provide any one of gelling, foaming, whipping, fluffing, binding, springiness, aeration, coating, film forming, emulsification, browning, thickening, texturizing, preserving moisture (humectant), clarification, and cohesiveness comparable to a whole egg, egg-white or nOVA composition. In some embodiments, the at least one functional feature is provided by or provided substantially by the inclusion of rOVA in the food ingredient or food product, for example, in the absence of any other whole egg proteins or egg white proteins.

(229) Such compositions can include rOVA in an amount between 0.1% and 25% on a weight/weight (w/w) or weight/volume (w/v) basis. rOVA may be present at or at least 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. These concentrations can be based on the dry weight of the composition. Additionally, or alternatively, the concentration of rOVA in such compositions 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 the food ingredient or food product can be at a concentration range of 0.1%-20%, 1%-20%, 0.1%-10%, 1%-10%, 0.1%-5%, 1%-5%, 2-10%, 4-8%, 4-10%, 4-12%, 0.1%-2%, 1%-2% or 0.1-1%.

(230) Provided herein are consumable food compositions and methods of making such compositions where rOVA provides at least one feature of whole egg or egg-whites to a consumable food composition. In some embodiments, rOVA is added to a consumable food composition to increase the protein content, such as for added nutrition. In some embodiments, rOVA is present in the consumable food composition between about 1% and about 40% on a weight per total weight (w/w) and/or weight per total volume (w/v) of composition basis. For example, in a composition of 100 ml, rOVA is present at 30 g and the rOVA is thus at a 30% concentration (w/v) or for example, in a composition of 100 g, rOVA is present at 30 g and the rOVA is thus at a 30% concentration (w/w). In some embodiments, the concentration of rOVA is or is 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% on a w/w and/or w/v of composition basis. In some embodiments, the rOVA is present at a concentration of or of 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 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% w/w and/or w/v.

(231) A consumable product can include one or more other proteins, such as a non-OVA protein or a non-recombinant protein. The rOVA can increase amount of protein content in a consumable product, and/or provide one or more egg-white like features. For example, the consumable composition can include a whey protein, a pea protein, a soy protein, an almond protein, an oat protein, a flax seed protein, a vegetable protein, or an egg-white protein. The consumable protein may include an extruded plant protein or a non-extruded plant protein. In some cases, the one or more other proteins can comprise OVA having an amino acid sequence naturally found in a bird or a reptile.

(232) In some embodiments, the compositions and methods for making compositions have an egg-white like property and increase the protein content in the composition. In some embodiments, the compositions and methods for making compositions with an egg-white like property increase the protein content, while not adversely affecting the stability, or one or more sensory qualities of the composition.

(233) In some embodiments, the consumable food compositions and methods for making consumable food compositions comprise rOVA and the addition of rOVA generates an egg-white like composition. The consumable food composition may be a finished product or an ingredient for making a finished product, e.g., a liquid or a powdered rOVA composition.

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

(235) In some embodiments, a composition described herein contains total protein at a concentration of about 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 total protein per 100 mL liquid (e.g., water). In some cases, a composition described herein contains total protein at a concentration of about 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 total protein per 100 g composition (e.g., powder).

(236) In some embodiments, a composition described herein contains rOVA at a concentration of about 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 liquid (e.g., water). In some cases, a composition described herein contains rOVA at a concentration of about 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 total protein per 100 g composition (e.g., powder)

(237) In some embodiments, a composition described herein contains total protein at a concentration of about 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 total protein per 100 mL liquid (e.g., water). In some cases, a composition described herein contains total protein at a concentration of about 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 total protein per 100 g composition (e.g., powder).

(238) In some embodiments, a composition described herein contains rOVA at a concentration of about 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 liquid (e.g., water). In some cases, a composition described herein contains rOVA at a concentration of about 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 composition (e.g., powder).

(239) In some embodiments, the rOVA consumable composition is a liquid composition. In such cases, the concentration of rOVA in the liquid composition may be between 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 at most 90%. The concentration of rOVA in the liquid composition may be from 0.1% to 1%, 0.1% to 5%, 0.1% to 10%, 0.1% to 15%, 0.1% to 20%, 0.1% to 25%, 0.1% to 30%, 0.1% to 35%, 0.1% to 40%, 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%, 1% to 35%, 1% to 40%, 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 5% to 35%, 5% to 40%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%, 20% to 25%, 20% to 30%, 20% to 35%, 20% to 40%, 25% to 30%, 25% to 35%, 25% to 40%, 30% to 35%, 30% to 40%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, or 90% to 95% in weight per total volume (w/v). The concentration of rOVA in the liquid composition may be about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% w/v. The concentration of rOVA in the liquid composition may be 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% w/v. The concentration of rOVA in the liquid composition may be at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% w/v. In some embodiments, rOVA is the sole protein in the liquid composition. In other embodiments, a liquid composition comprises proteins other than rOVA.

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

(241) In some embodiments, the rOVA consumable composition is a powdered composition. In such cases, the concentration of rOVA in the powder composition may be between 15% to 99% weight per total weight (w/w) and/or weight per total volume (w/v). The concentration of rOVA in the powder composition may be at least 15% w/w or w/v. In embodiments, 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% to 30%, 15% to 45%, 15% to 60%, 15% to 75%, 15% to 80%, 15% to 85%, 15% to 90%, 15% to 95%, 15% to 99%, 30% to 45%, 30% to 60%, 30% to 75%, 30% to 80%, 30% to 85%, 30% to 90%, 30% to 95%, 30% to 99%, 45% to 60%, 45% to 75%, 45% to 80%, 45% to 85%, 45% to 90%, 45% to 95%, 45% to 99%, 60% to 75%, 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 99%, 75% to 80%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 99%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 99%, 85% to 90%, 85% to 95%, 85% to 99%, 90% to 95%, 90% to 99%, or 95% to 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 sole protein in the powder composition. In other embodiments, a powder composition comprises proteins other than rOVA.

(242) In some cases, a powder composition may be a concentrate which comprises at least 70% rOVA w/w. In some cases, a powder composition may be a concentrate which comprises at least 80% rOVA w/w. In some cases, a powder composition may be an isolate which comprises at least 90% rOVA w/w. In some cases, a powder composition may be an isolate which comprises at least 95% rOVA w/w.

(243) In some embodiments, the rOVA consumable composition is a concentrated liquid composition. In such cases, the concentration of rOVA in the concentrated liquid composition may be between 10% to 60% 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% w/w or w/v. The concentration of rOVA in the concentrated liquid may be at most 60% w/w or w/v. The concentration of rOVA in the concentrated liquid may be 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 30% to 40%, 30% to 50%, 30% to 60%, 40% to 50%, 40% to 60%, or 50% to 60% w/w or w/v. The concentration of rOVA in the concentrated liquid may be about 10%, 20%, 30%, 40%, 50%, or 60% w/w or w/v. The concentration of rOVA in the concentrated liquid may be at least 10%, 20%, 30%, 40% or 50% w/w or w/v. The concentration of rOVA in the concentrated liquid may be at most 20%, 30%, 40%, 50%, or 60% w/w or w/v. The liquid may include any consumable solvent, e.g., water, dairy, oil, or other cooking base.

(244) In some embodiments, the rOVA consumable composition is a prepared food for example, as a baked good, a salad dressing, an egg-like dish (such as an egg-patty or scramble), a dessert or dairy-like product or a meat-analog (such as a vegan meat patty, sausage or hot dog). Such compositions can include rOVA in an amount between 0.1% and 20% on a weight/weight (w/w) or weight/volume (w/v) basis. rOVA may be present at or at least 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% on a weight/weight (w/w) or weight/volume (w/v) basis. Additionally, or alternatively, the concentration of rOVA in such compositions 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 the food ingredient or food product can be at a concentration range of 0.1%-20%, 1%-20%, 0.1%-10%, 1%-10%, 0.1%-5%, 1%-5%, 0.1%-2%, 1%-2% or 0.1-1%.

(245) Features and Characteristics of rOVA Compositions and Food Ingredients and Food Products Containing rOVA

(246) The rOVA containing compositions herein can provide one or more functional features to food ingredients and food products. In some embodiments, the rOVA provides a nutritional feature such as protein content, protein fortification and amino acid content to a food ingredient or food product. The nutritional feature provided by rOVA in the composition may be comparable or substantially similar to an egg, egg white or native OVA (nOVA). The nutritional feature provided by rOVA in the composition may be better than that provided by a native whole egg or native egg white. In some cases, rOVA provides the one or more functional features of egg-white in absence of any other egg-white proteins.

(247) rOVA compositions disclosed herein can provide foaming and foam capacity to a composition. For example, rOVA can be used for forming a foam to use in baked products, such as cakes, for meringues and other foods where rOVA can replace egg white to provide foam capacity. In some cases, rOVA provides foaming and foam capacity of egg-white in absence of any other egg-white proteins.

(248) A composition comprising rOVA may have a foam height greater than a foam height of an egg white or a composition comprising nOVA. In some cases, a composition comprising rOVA may have a foam height of about 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% relative to an egg white, nOVA compositions or a substitute egg white. In some cases, a composition comprising rOVA may have a foam height of 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% relative to an egg white, nOVA compositions or a substitute egg white. Substitute egg whites may include products such as aquafaba, chia seeds, flax seeds, starches; apple sauce, banana puree; condensed milk, etc. which are commonly used as egg white substitutes.

(249) A composition comprising rOVA may have a foam stability greater than a foam stability of an egg white, nOVA compositions or a substitute egg white. In some cases, a composition comprising rOVA may have a foam stability of about 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% relative to an egg white or a substitute egg white. In some cases, a composition comprising rOVA may have a foam stability of 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% relative to an egg white. Foam stability may be calculated by measuring drainage of a foamed solution. The drainage may be measured in 10-minute increments for 30 minutes to gather data for foam stability. The drained volume after 30 minutes may be compared to the initial liquid volume (5 mL) for instance, foam Stability (%): (Initial volume?drained volume)/initial volume*100.

(250) A composition comprising rOVA may have a foam capacity greater than a foam capacity of an egg white, nOVA compositions or a substitute egg white. In some cases, a composition comprising rOVA may have a foam capacity of about 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% relative to an egg white, nOVA or a substitute egg white. In some cases, a composition comprising rOVA may have a foam capacity of 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% relative to an egg white, nOVA compositions or a substitute egg white. Foam capacity may be determined by measuring the initial volume of foam following the whipping and compare against the initial volume of 5 mL. Foam Capacity (%)=(volume of foam/initial volume)*100.

(251) A liquid composition may foam faster than a composition comprising egg whites, nOVA or a substitute egg white. In some cases, an 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 an egg white, nOVA or substitute egg-white composition. In some cases, an 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 an egg white, nOVA or substitute egg-white composition.

(252) A composition comprising rOVA may have a gel strength greater than a gel strength of an egg white, nOVA composition or a egg white substitutes. In some cases, the rOVA composition may have a gel strength within the range from 100 g to 1500 g, from 500 g to 1500 g, or from 700 g to 1500 g. In some cases, an rOVA composition has a gel strength of about 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, an 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, an rOVA composition has a gel strength of about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to an egg white, nOVA or egg white substitutes. In some cases, an rOVA composition has a gel strength of up to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% relative to an egg white, nOVA or egg white substitutes.

(253) rOVA compositions disclosed herein can provide structure, texture or a combination of structure and texture. In some embodiments, rOVA is added to a food ingredient or food product for baking and the rOVA provides structure, texture or a combination of structure and texture to the baked product. rOVA can be used in such baked products in place of native egg white, native egg or native egg protein. The addition of rOVA to baked products can also provide protein fortification to improve the nutritional content. In some embodiments, rOVA is used in a baked product in an amount between 0.1% and 25% on a weight/weight or weight/volume basis. In some embodiments, rOVA is used in a baked product in an amount between 0.1% and 5%. In some cases, rOVA provides the structure and/or texture of egg-white in absence of any other egg-white proteins.

(254) rOVA compositions disclosed herein can be compatible with gluten formation, such that the rOVA can be used where gluten formation provides structure, texture and/or form to a food ingredient or food product.

(255) Exemplary baked products in which rOVA can be used as an ingredient include, but are not limited to cake, cookie, bread, bagel, biscuits, muffin, cupcake, scone, pancake, macaroon, choux pastry, meringue, and souffl?. For example, rOVA can be used as an ingredient to make cakes such as pound cake, sponge cake, yellow cake, or angel food cake, where such cakes do not contain any native egg white, native whole egg or native egg protein. Along with rOVA, baked products may contain additional ingredients such as flour, sweetening agents, gum, hydrocolloids, starches, fibers, flavorings (such as flavoring extracts) and other protein sources. In some embodiments, a baked product may include rOVA and at least one fat or oil, at least one grain starch, and optionally at least one sweetener. Grain starch for use in such compositions include flours such as wheat flour, rice flour, corn flour, millet flour, spelt flour, and oat flour, and starches such as from corn, potato, sorghum, and arrowroot. Oil and fat for use in such compositions include plant-derived oils and fats, such as olive oil, corn oil, avocado oil, nut oils (e.g., almond, walnut and peanut) and safflower oil. rOVA may provide such baked goods with at least one characteristic of an egg white such as binding, springiness, aeration, browning, texturizing, humectant, and cohesiveness of the baked product. In some cases, the baked product does not comprise any natural egg white or natural egg, and/or does not include any other egg white derived proteins except rOVA. In some cases, rOVA is provided to the baked composition as an ingredient, such as starting with a concentrate, isolate or powder form of rOVA. In some cases, the rOVA provided as an ingredient for baked products is at a pH range between about 3.5 and 7.0. In some cases, a sweetener is included in the baked product such as a sugar, syrup, honey or sugar-substitute.

(256) rOVA compositions disclosed herein can also be used to prepare egg-less food products, such as food products made where native whole egg or native egg white is a primary or featured ingredient such as scramble, omelet, patty, souffl?, quiche and frittata. In some embodiments, rOVA provides one or more functional features to the preparation including foaming, coagulation, binding, structure, texture, film-formation, nutritional profile, absence of cholesterol (i.e., cholesterol free) and protein fortification. Such egg-less preparations can be vegan, vegetarian, halal, or kosher, or a combination thereof. An egg-less preparation (also referred to as an egg-white substitute) may include rOVA and at least one fat or oil, a polysaccharide or polysaccharide-containing ingredient, and a starch. In some cases, the egg-less preparation may also include a flavoring agent (such as to provide a salty, sulfur-like or umami flavor), and/or a coloring agent (for example to provide yellow-like or off-white color to the baked product). In some cases, the inclusion or rOVA in the egg-less preparation provides a characteristic of natural (native) egg white such as hardness, adhesiveness, fracturability, cohesiveness, gumminess and chewiness when the composition is heated or cooked. Exemplary polysaccharide or polysaccharide-containing ingredients for such compositions include gellan gum, sodium alginate, and psyllium. Oil and fat for use in such compositions include plant-derived oils and fats, such as olive oil, corn oil, avocado oil, and safflower oil.

(257) rOVA compositions disclosed herein can be used for a processed meat product or meat-like product, or for fish-like or shell-fish-like products. In such products, rOVA can provide one or more functional characteristics such as protein content and protein supplementations as well as binding, texturizing properties. Exemplary meat and meat-like products include burger, patty, sausage, hot dog, sliced deli meat, jerky, bacon, nugget and ground meat-like mixtures. Meat-like products can resemble beef, pork, chicken, lamb and other edible and consumed meats for humans and for other animals. Fish-like and shell-fish like products can resemble, for example, fish cakes, crab cakes, shrimp, shrimp balls, fish sticks, seafood meat, crab meat, fish fillets and clam strips. In some embodiments, rOVA is present in an amount between about 0.1% and 30% w/w/or w/v in the meat or meat-like product. In some embodiments, rOVA is used for a meat-like product (also referred to as a meat-analog and includes at least one fat or oil; and a plant-derived protein. Oil and fat 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 soy protein, nut proteins, pea protein, lentil and other pulse proteins and whey protein. In some cases, such plant protein is extruded, in other cases, such plant protein is non-extruded protein. In some cases, a meat analog include rOVA at about 2% to 15% (w/w). In some cases for meat analog compositions, rOVA acts as a binding agent, a gelling agent or a combination of a binding and gelling agent for such compositions.

(258) rOVA compositions disclosed herein can be employed in coatings for food products. For example, rOVA can provide binding or adhesion characteristics to adhere batter or breading to another food ingredient. rOVA can be used as an egg-less egg wash where the rOVA protein provides appearance, color and texture when coated onto other food ingredients or food products, such as baked products. In one example, the egg-less egg wash may be used to coat a baked good such that the baked good adheres to a coating (e.g., seed, salt, spice, and herb). The addition of rOVA as a coating to a food product can provide a crunchy texture or increase the hardness, for example, of the exterior of a food product such as when the product is cooked, baked or fried.

(259) rOVA compositions disclosed herein include sauces and dressings, such as an eggless mayonnaise, commercial mayonnaise substitutes, gravy, sandwich spread, salad dressing or food sauce. Inclusion of rOVA in a sauce or dressing, and the like, can provide one or more characteristics such as binding, emulsifying, odor neutrality, and mouthfeel. In some embodiments rOVA is present in such sauces and dressing in an amount between 0.1% and 3% or between about 3% and about 5% w/w/ or w/v. In some cases, the amount of rOVA in a sauce or dressing may be substantially similar to the amount of whole egg, egg-white or nOVA used in a commercially available or commonly used recipe. Exemplary sauces and dressing include mayonnaise, commercial mayonnaise substitutes, alfredo sauce, and hollandaise sauce. In some embodiments, the rOVA-containing sauce or dressing does not contain whole egg, egg white, or any other protein extracted from egg. In some cases, the sauce, dressing or other emulsified product made with rOVA includes 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.

(260) rOVA compositions can be used to prepare confectionaries such as eggless, animal-free, vegetarian and vegan confectionaries. rOVA can provide one or more functional features to the confectionary including odor neutrality, flavor, mouthfeel, texture, gelling, cohesiveness, foaming, frothiness, nutritional value and protein fortification. In some embodiments, the prepared confectionary containing rOVA does not contain any native egg protein or native egg white. rOVA in such confectionaries can provide a firm or chewy texture. In some embodiments, rOVA is present between about 0.1% and 15% in a confectionary. Exemplary confectionaries include a gummy, a taffy, a divinity candy, meringue, marshmallow, and a nougat. In some embodiments, a confectionary includes rOVA, at least one sweetener and optionally a consumable liquid. Exemplary sweetners include sugar, honey, sugar-substitutes and plant-derived syrups. In some cases, the rOVA is provided as an ingredient for making confectionaries at a pH between about 3.5 and about 7. In some cases, the rOVA is present in the confectionary composition at about 2% to about 15% (w/v). In some embodiments, the confectionary is a food product such as a meringue, a whipped dessert, or a whipped topping. In some embodiments, rOVA in the confectionary provides foaming, whipping, fluffing or aeration to the food product, and/or provides gelation. In some cases, the confectionary is a liquid, such as a foamed drink. In some cases, the liquid may include a consumable alcohol (such as in a sweetened cocktail or after-dinner drink).

(261) rOVA compositions herein can be used in dairy products, dairy-like products or dairy containing products. For example, rOVA can be used in preparations of beverages such as a smoothie, milkshake, egg-nog, and coffee beverage. In some embodiments, rOVA is added to additional ingredients where at least one ingredient is a dairy ingredient or 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 or native egg. In some embodiments, rOVA is an ingredient in a beverage that does not contain any animal-derived ingredients, such as one that does not contain any native egg-derived or any dairy-derived ingredients. Examples of such non-dairy derived drinks include nut milks, such as soy milk or almond milk. rOVA can also be used to create beverage additions, such as creamer or milk to provide protein, flavor, texture and mouthfeel to a beverage such as a coffee, tea, alcohol-based beverages or cocoa. In some embodiments, rOVA is present in a beverage ingredient or beverage addition in an amount between about 0.1% and 20% w/w or w/v.

(262) In some embodiments herein, rOVA can be used to prepare a dairy-like product such as yogurt, cheese or butter. Dairy products with rOVA can include other animal-based dairy components or proteins. In some embodiments, dairy products prepared with rOVA do not include any animal-based ingredients.

(263) Preparations of dessert products can be prepared using rOVA. In dessert products rOVA can provide one or more characteristics such as creamy texture, low fat content, odor neutrality, flavor, mouthfeel, texture, binding, and nutritional value. rOVA may be present in an ingredient or set of ingredients that is used to prepare a dessert product. Exemplary dessert products suitable for preparation with rOVA include a mousse, a cheesecake, a custard, a pudding, a popsicle and an ice cream. In some embodiments, dessert products prepared to include rOVA are vegan, vegetarian or dairy-free. Dessert products that include rOVA can have an amount of rOVA that is between about 0.1% and about 10% rOVA w/w or w/v.

(264) rOVA can be used to prepare a snack food, such as a protein bar, an energy bar, a nutrition bar or a granola bar. The rOVA can provide characteristics to the snack food including one or more of binding, protein supplementation, flavor neutrality, odor neutrality, coating and mouth feel. In some embodiments, rOVA is added to a preparation of a snack food in an amount between about 0.1% and 30% w/w or w/v.

(265) rOVA can be used for nutritional supplements such as in parenteral nutrition, protein drink supplements, protein shakes where rOVA provides a high protein supplement. In some embodiments, rOVA can be added to such compositions in an amount between about 10% and 30% w/w or w/v.

(266) In some embodiments, rOVA compositions can be used as an egg-replacer and an egg white-replacer. rOVA can be mixed or combined with at least one additional component to form the egg white replacer. rOVA can provide one or more characteristics to the egg-replacer or egg white-replacer, such as gelling, foaming, whipping, fluffing, binding, springiness, aeration, creaminess and cohesiveness. In some embodiments, characteristic is the same or better than a native egg or native egg white provided in the same amount or concentration (w/w or w/v). In some embodiments, the egg-replacer or egg white-replacer, does not contain any egg, egg white, protein extracted or isolated from egg.

(267) The rOVA-containing food ingredient and food products, such as described herein, can contain additional ingredients or components. For example, rOVA compositions can be prepared with an additional component such as one or more of a sweetener, a gum, a flavoring, a thickener, an acidulant and an emulsifier. Other ingredients such as flour, grains, oils and fats, fiber, fruit and vegetables can be combined with rOVA. Such rOVA compositions can be vegan, vegetarian, halal, kosher and animal-free, or a combination thereof. In some embodiments, rOVA can be a food ingredient or prepared for a food product that is normally animal based or normally contains animal-derived components, such as meat, dairy or eggs.

(268) Compositions including rOVA including food ingredients and food products can be compatible with one or more steps of consumables preparation such as heated, baked, grilled, roasted, braised, microwaved, broiled, boiled, steamed, extruded, deep fried, or pan-fried, or processed using ohmic heating, Sue Vide, freezing, chilling, blanching, packaging, canning, bleaching, enriching, drying, pressing, grinding, mixing, par cooking, cooking, proofing, marinating, cutting, slicing, dicing, crushing, shredding, chopping, shaking, coring, spiralizing, rolling, juicing, straining, filtering, kneading, whisking, beating, whipping, grating, stuffing, peeling, smoking, curing, salting, preserving, pickling, fermenting, homogenizing, pasteurizing, sterilizing, irradiating, cold plasma processing, high pressure processing, pulse electric field processing, microwave assisted thermal sterilization, stabilizing, blending, pureeing, fortifying, refining, hydrogenating, aging, extending shelf life, or adding enzymes.

(269) Food ingredients and food products prepared with rOVA can be essentially free of any microbial cells or microbial cell debris. For instance, rOVA may be secreted from a microbial host cell and isolated from microbial cells, culture media and/or microbial cell debris.

(270) In some embodiments, rOVA may be prepared as a whole cell extract or fractionated extract such that an rOVA composition contains microbial cells and/or microbial cell components.

(271) In one embodiment, an rOVA composition is prepared for animal consumption where the rOVA is present in a whole cell extract or fractionated extract such that an rOVA composition contains microbial cells and/or microbial cell components. In some embodiments, an rOVA composition is prepared for animal consumption where rOVA is isolated from microbial cells, culture media and microbial cell debris. Exemplary compositions for animal consumption can include a pet food, an animal feed, a chewy treat, bone broth, smoothie or other liquid for animal nutrition and a solid nutritional supplement suitable for animal consumption. In these cases, the microbial cell extract or microbial cell debris may provide additional nutritional value.

(272) Animals which may consume rOVA compositions can include companion animals (e.g., dog, cat, horse), farm animals, exotic animals (lion, tiger, zebra) as well as livestock (such as cow, pig, sheep, goat). rOVA compositions as described herein can also be used for aquaculture (such as for fish and shell fish) and for avian nutrition (such as for bird pets, zoo birds, wild birds, fowl and birds raised for human and animal food).

(273) In some embodiments of the consumable food compositions described herein, the composition is essentially free of animal-derived components, whey protein, caseinate, fat, lactose, hydrolyzed lactose, soy protein, collagen, hydrolyzed collagen, or gelatin, or any combination thereof. A composition described herein may be essentially free of cholesterol, glucose, fat, saturated fat, trans fat, or any combination thereof. In some cases, a composition described herein comprises less than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% fat by dry weight. In some embodiments, the composition may be fat-containing (e.g., such as a mayonnaise and commercial mayonnaise substitutes) and such composition may include up to about 60% fat or a reduced-fat composition (e.g., reduced fat mayonnaise and commercial mayonnaise substitutes) and such composition may include lesser percentages of fat. A composition that free of an animal-derived component can be considered vegetarian and/or vegan.

(274) In some embodiments, an rOVA powder composition comprises less than 5% ash. The term ash is an art-known term and represents inorganics such as one or more ions, elements, minerals, and/or compounds In some cases, the rOVA powder composition comprises 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 weight per total weight (w/w) and/or weight per total volume (w/v).

(275) In some embodiments, the moisture content of an 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% moisture weight per total weight (w/w) and/or weight per total volume (w/v). In some embodiments, the carbohydrate content of an 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 content w/w or w/v.

4. Sensory Neutrality and Improved Sensory Appeal

(276) In some embodiments, in addition to the egg-white like properties, the addition of rOVA to a consumable food composition provides increased protein nutritional content, sensory neutrality or an improved sensory appeal as compared to other proteins in such compositions. As used herein sensory neutrality refers to the absence of a strong or distinctive taste, odor (smell) or combination of taste and smell, as well as texture, mouth-feel, aftertaste and color. A sensory panel such as one described in Kemp et al. 2009 may be used by a trained sensory analyst. Sensory neutrality may provide an improved sensory appeal to a taster, such as a tester of foods or a consumer, when a consumable food composition containing rOVA is compared with another like composition that has a different protein such as nOVA, whey protein, pea protein, soy protein, whole egg or egg white protein at the same concentration.

(277) In some embodiments, rOVA when added to a consumable food composition is substantially odorless, such as measured by a trained sensory analyst, in comparison with different solutions/products with a different protein component present in an equal concentration to the rOVA containing solution/product, for example, in the comparison is whey, soy, collagen, pea, egg white solid isolates and/or nOVA. In some embodiments of the rOVA compositions described herein, such compositions are essentially odorless at a protein concentration between about 0.5-1%, 1%-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% rOVA weight per total weight (w/w) and/or weight per total volume (w/v) or at a protein concentration of 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 per 100 mL solution (e.g., per 100 mL water).

(278) In some embodiments, the addition of rOVA to a consumable food composition also provides a neutral taste in addition to the characteristics such as egg-white like properties and increased protein nutrition content. A neutral taste can be measured for example, by a trained sensory analyst in comparison with solutions containing a different protein present in an equal concentration to the rOVA, for example, whey, soy, collagen, pea, whole egg, and egg white solid isolates (including native OVA).

(279) In some embodiments, the addition of rOVA provides a reduction in a certain odor and/or taste that is associated with other proteins or egg-whites. For example, addition of rOVA has less of an egg-like odor or taste as compared to the addition of whole egg, fractionated egg or egg-white to a consumable food composition. In some embodiments, addition of rOVA has less of a metallic odor or taste as compared to other protein sources.

(280) In some embodiments, the addition of rOVA has an improved mouth-feel as compared to the addition of other protein sources used to produce egg-white like properties. For example, the addition of rOVA is less grainy or has less precipitates or solids as compared to other protein sources.

(281) In some embodiments, the addition of rOVA has an improved texture, for example, as compared to other available supplemental protein sources.

(282) A consumable composition with rOVA may also have an improved sensory appeal as compared to the composition without rOVA or with a different protein present in an equal concentration to the rOVA. Such improved sensory appeal may relate to taste and/or smell. Taste and smell can be measured, for example, by a trained sensory analyst. In some instances, a sensory analyst compares a consumable composition with rOVA to one without it or with a different protein or protein source in an equivalent amount.

(283) As described herein, a consumable composition herein can be in a liquid form. A liquid form can be an intermediate product such as soluble rOVA solution. In some cases, a liquid form can be a final product, such as a beverage comprising rOVA. Example of different types of beverages contemplated herein include: a juice, a soda, a soft drink, a flavored water, a protein water, a fortified water, a carbonated water, a nutritional drink, an energy drink, a sports drink, a recovery drink, an alcohol-based drink, a heated drink, a coffee-based drink, a tea-based drink, a plant-based milk, a nut milk, a milk based drink, a non-dairy, plant based mild drink, infant formula drink, and a meal replacement drink.

(284) pH of Compositions

(285) The pH of an rOVA composition may be 3.5 to 8. The pH of an rOVA composition may be at least 3.5. The pH of an rOVA composition may be at most 8. The pH of an 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 an rOVA composition may be 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8. An rOVA composition with a pH between 3.5 to 7 may have one or more improved functionalities as compared to nOVA, egg white or egg-white substitute compositions.

(286) The pH of an rOVA composition may be 2 to 3.5. The pH of an rOVA composition may be at least 2. The pH of an rOVA composition may be at most 3.5. The pH of an 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 an rOVA composition may be 2, 2.5, 3, or 3.5.

(287) The pH of an rOVA composition may be 7 to 12. The pH of an rOVA composition may be at least 7. The pH of an rOVA composition may be at most 12. The pH of an rOVA composition may be 7 to 7.5, 7 to 8, 7 to 8.5, 7 to 9, 7 to 9.5, 7 to 10, 7 to 10.5, 7 to 11, 7 to 11.5, 7 to 12, 7.5 to 8, 7.5 to 8.5, 7.5 to 9, 7.5 to 9.5, 7.5 to 10, 7.5 to 10.5, 7.5 to 11, 7.5 to 11.5, 7.5 to 12, 8 to 8.5, 8 to 9, 8 to 9.5, 8 to 10, 8 to 10.5, 8 to 11, 8 to 11.5, 8 to 12, 8.5 to 9, 8.5 to 9.5, 8.5 to 10, 8.5 to 10.5, 8.5 to 11, 8.5 to 11.5, 8.5 to 12, 9 to 9.5, 9 to 10, 9 to 10.5, 9 to 11, 9 to 11.5, 9 to 12, 9.5 to 10, 9.5 to 10.5, 9.5 to 11, 9.5 to 11.5, 9.5 to 12, 10 to 10.5, 10 to 11, 10 to 11.5, 10 to 12, 10.5 to 11, 10.5 to 11.5, 10.5 to 12, 11 to 11.5, 11 to 12, or 11.5 to 12. The pH of an rOVA composition may be 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12.

(288) In some embodiments, the pH of rOVA may be adjusted prior to its inclusion in a composition or its use as an ingredient. In some embodiments, the pH of rOVA is adjusted during the purification and/or isolation processes. In some embodiments, the pH of the rOVA for use in an ingredient or in production of a food product composition is adjusted to between about 3.5 to about 7.0. In some cases, the pH of rOVA may be adjusted to more than one pH during the production process. For example rOVA may be expressed in a host cell such as a a microbial cell, and in some cases the rOVA is secreted by the host cell into the growth media (e.g., liquid media). rOVA is separated from the host cells and such separation step may be performed at a selected pH, for example at a pH of about 3.5. In some cases, the rOVA at such separation pH may not be soluble or may not be fully soluble and the pH is adjusted to a higher pH, such as about pH 12. The rOVA may then be adjusted to a final pH between about 3.5 and about 7.0. Separation of rOVA from other components of the host cells or other components of the liquid media can include one or more of ion exchange chromatography, such as cation exchange chromatography and/or anion exchange chromatography, filtration and ammonium sulfate precipitation.

(289) Additional Components of Compositions

(290) The consumable food compositions containing rOVA disclosed herein and the methods of making such compositions may including adding or mixing the rOVA with one or more ingredients. For example, food additives may be added in or mixed with the compositions. Food additives can add volume and/or mass to a composition. A food additive may improve functional performance and/or physical characteristics. For example, a food additive may prevent gelation or increased viscosity due to the lipid portion of the lipoproteins in the freeze-thaw cycle. An anticaking agent may be added to make a free-flowing composition. Carbohydrates can be added to increase resistance to heat damage, e.g., less protein denaturation during drying and improve stability and flowability of dried compositions. Food additives include, but are not limited to, food coloring, pH adjuster, natural flavoring, artificial flavoring, flavor enhancer, batch marker, food acid, filler, anticaking agent (e.g., sodium silico aluminate), antigreening agent (e.g., citric acid), food stabilizer, foam stabilizer or binding agent, antioxidant, acidity regulatory, bulking agent, color retention agent, whipping agent (e.g., ester-type whipping agent, triethyl citrate, sodium lauryl sulfate), emulsifier (e.g., lecithin), humectant, thickener, excipient, solid diluent, salts, nutrient, sweetener, glazing agent, preservative, vitamin, dietary elements, carbohydrates, polyol, gums, starches, flour, oil, or bran.

(291) Food coloring includes, but is 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, ?-carotene), anthocyanins, annatto, betanin, butterfly pea, caramel coloring, chlorophyllin, elderberry juice, lycopene, carmine, pandan, paprika, turmeric, curcuminoids, quinoline yellow, carmoisine, Ponceau 4R, Patent Blue V, and Green S.

(292) Ingredients 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.

(293) 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 chlorides, zinc salts, and zinc chloride.

(294) Nutrient includes, but is not limited to, macronutrient, micronutrient, essential nutrient, non-essential nutrient, dietary fiber, amino acid, essential fatty acids, omega-3 fatty acids, and conjugated linoleic acid.

(295) Sweeteners include, but are not limited to, sugar substitute, artificial sweetener, acesulfame potassium, advantame, alitame, aspartame, sodium cyclamate, dulcin, glucin, neohesperidin dihydrochalcone, neotame, P-4000, saccharin, aspartame-acesulfame salt, sucralose, brazzein, curculin, glycyrrhizin, glycerol, inulin, mogroside, mabinlin, malto-oligosaccharide, mannitol, miraculin, monatin, monellin, osladin, pentadin, stevia, trilobatin, and thaumatin.

(296) Carbohydrates include, but are not limited to, sugar, sucrose, glucose, fructose, galactose, lactose, maltose, mannose, allulose, 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.

(297) Polyols include, but are not limited to, xylitol, maltitol, erythritol, sorbitol, threitol, arabitol, hydrogenated starch hydrolysates, isomalt, lactitol, mannitol, and galactitol (dulcitol).

(298) Gums include, but are not limited to, gum arabic, gellan gum, guar gum, locust bean gum, acacia gum, cellulose gum, and xanthan gum.

(299) 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.

(300) Dietary 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.

(301) rOVA Protein and Production of rOVA Protein

(302) rOVA can have an amino acid sequence from any species. For example, an rOVA can have an amino acid sequence of OVA from a bird or a reptile or other egg-laying species. An rOVA having an amino acid sequence from an avian can be selected from the group consisting of: poultry, fowl, waterfowl, game bird, chicken, quail, turkey, duck, ostrich, goose, gull, guineafowl, pheasant, emu, and any combination thereof. An rOVA can have an amino acid sequence derived from a single species, such as Gallus gallus domesticus. Alternatively, an rOVA can have an amino acid sequence derived from two or more species, and as such be a hybrid.

(303) Exemplary OVA amino acid sequences contemplated herein are provided in Table 43 below as SEQ ID NOs: 60-133.

(304) TABLE-US-00002 TABLE43 OVASequences SEQ Name ID Sequence Chicken 60 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYS Ovalbumin DLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDK withbolded REAEAGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSAL signal AMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVH sequence SSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKEL YRGGLEPINFQTAADQARELINSWVESQINGIIRNVLQPSSVD SQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPV QMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVS GLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYN LTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINE AGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFF GRCVSP Chicken 61 EAEAGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALA OVA MVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHS sequenceas SLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELY secreted RGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDS frompichia QTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQ MMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSG LEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLT SVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEA GREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFG RCVSP Predicted 62 MRVPAQLLGLLLLWLPGARCGSIGAASMEFCFDVFKELKVH Ovalbumin HANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLP [Achromobacter GFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLY denitrificans] AEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWV ESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDE DTQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILEL PFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEE RKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISS AESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFR ADHPFLFCIKHIATNAVLFFGRCVSPLEIKRAAAHHHHHH OLLAS 63 MTSGFANELGPRLMGKLTMGSIGAASMEFCFDVFKELKVHH epitope- ANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGF tagged GDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAE ovalbumin ERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVES QTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDEDT QAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFA SGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKI KVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAES LKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADH PFLFCIKHIATNAVLFFGRCVSPSR Serpin 64 MGGRRVRWEVYISRAGYVNRQIAWRRHHRSLTMRVPAQLL family GLLLLWLPGARCGSIGAASMEFCFDVFKELKVHHANENIFYC protein PIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQC [Achromobacter GTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEY denitrificans] LQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNV LQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVT EQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSML VLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRM KMEEKYNLTSVLMAMGITDVESSSANLSGISSAESLKISQAVH AAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHI ATNAVLFFGRCVSPLEIKRAAAHHHHHH PREDICTED: 65 MGSIGAVSMEFCFDVFKELKVHHANENIFYSPFTIISALAMVY ovalbumin LGAKDSTRTQINKVVRFDKLPGFGDSVEAQCGTSVNVHSSLR isoformX1 DILNQITKPNDVYSFSLASRLYAEETYPILPEYLQCVKELYRG [Meleagris GLESINFQTAADQARGLINSWVESQTNGMIKNVLQPSSVDSQ gallopavo] TAMVLVNAIVFKGLWEKAFKDEDTQAIPFRVTEQESKPVQM MYQIGLFKVASMASEKMKILELPFASGTMSMWVLLPDEVSG LEQLETTISFEKMTEWISSNIMEERRIKVYLPRMKMEEKYNLT SVLMAMGITDLFSSSANLSGISSAGSLKISQAVHAAYAEIYEA GREVIGSAEAGADATSVSEEFRVDHPFLYCIKHNLTNSILFFGR CISP Ovalbumin 66 MGSIGAVSMEFCFDVFKELKVHHANENIFYSPFTIISALAMVY precursor LGAKDSTRTQINKVVRFDKLPGFGDSVEAQCGTSVNVHSSLR [Meleagris DILNQITKPNDVYSFSLASRLYAEETYPILPEYLQCVKELYRG gallopavo] GLESINFQTAADQARGLINSWVESQTNGMIKNVLQPSSVDSQ TAMVLVNAIVFKGLWEKAFKDEDTQAIPFRVTEQESKPVQM MYQIGLFKVASMASEKMKILELPFASGTMSMWVLLPDEVSG LEQLETTISFEKMTEWISSNIMEERRIKVYLPRMKMEEKYNLT SVLMAMGITDLESSSANLSGISSAGSLKISQAAHAAYAEIYEA GREVIGSAEAGADATSVSEEFRVDHPFLYCIKHNLTNSILFFGR CISP Hypothetical 67 YYRVPCMVLCTAFHPYIFIVLLFALDNSEFTMGSIGAVSMEFC protein FDVFKELRVHHPNENIFFCPFAIMSAMAMVYLGAKDSTRTQI [Bambusicola NKVIRFDKLPGFGDSTEAQCGKSANVHSSLKDILNQITKPNDV thoracicus] YSFSLASRLYADETYSIQSEYLQCVNELYRGGLESINFQTAAD QARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFRG LWEKAFKDEDTQTMPFRVTEQESKPVQMMYQIGSFKVASMA SEKMKILELPLASGTMSMLVLLPDEVSGLEQLETTISFEKLTE WTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDLFR SSANLSGISLAGNLKISQAVHAAHAEINEAGRKAVSSAEAGV DATSVSEEFRADRPFLFCIKHIATKVVFFFGRYTSP Egg 68 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLAMV albumin FLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSVNVHSSLR DILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQCVKELYR GGLESVNFQTAADQARGLINAWVESQTNGIIRNILQPSSVDSQ TAMVLVNAIAFKGLWEKAFKAEDTQTIPFRVTEQESKPVQM MYQIGSFKVASMASEKMKILELPFASGTMSMLVLLPDDVSGL EQLESIISFEKLTEWTSSSIMEERKVKVYLPRMKMEEKYNLTS LLMAMGITDLFSSSANLSGISSVGSLKISQAVHAAHAEINEAG RDVVGSAEAGVDATEEFRADHPFLFCVKHIETNAILLFGRCVS P Ovalbumin 69 MASIGAVSTEFCVDVYKELRVHHANENIFYSPFTIISTLAMVY isoformX2 LGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLR [Numida DILNQITKPNDVYSFSLASRLYAEETYPILPEYLQCVKELYRG meleagris] GLESINFQTAADQARELINSWVESQTSGIIKNVLQPSSVNSQTA MVLVNAIYFKGLWERAFKDEDTQAIPFRVTEQESKPVQMMS QIGSFKVASVASEKVKILELPFVSGTMSMLVLLPDEVSGLEQL ESTISTEKLTEWTSSSIMEERKIKVFLPRMRMEEKYNLTSVLM AMGMTDLFSSSANLSGISSAESLKISQAVHAAYAEIYEAGREV VSSAEAGVDATSVSEEFRVDHPFLLCIKHNPTNSILFFGRCISP Ovalbumin 70 MALCKAFHPYIFIVLLFDVDNSAFTMASIGAVSTEFCVDVYKE isoformX1 LRVHHANENIFYSPFTIISTLAMVYLGAKDSTRTQINKVVRFD [Numida KLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASR meleagris] LYAEETYPILPEYLQCVKELYRGGLESINFQTAADQARELINS WVESQTSGIIKNVLQPSSVNSQTAMVLVNAIYFKGLWERAFK DEDTQAIPFRVTEQESKPVQMMSQIGSFKVASVASEKVKILEL PFVSGTMSMLVLLPDEVSGLEQLESTISTEKLTEWTSSSIMEER KIKVFLPRMRMEEKYNLTSVLMAMGMTDLFSSSANLSGISSA ESLKISQAVHAAYAEIYEAGREVVSSAEAGVDATSVSEEFRV DHPFLLCIKHNPTNSILFFGRCISP PREDICTED: 71 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLAMV Ovalbumin FLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSANVHSSLR isoformX2 DILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQCVKELYR [Coturnix GGLESVNFQTAADQARGLINAWVESQTNGIIRNILQPSSVDSQ japonica] TAMVLVNAIAFKGLWEKAFKAEDTQTIPFRVTEQESKPVQM MHQIGSFKVASMASEKMKILELPFASGTMSMLVLLPDDVSGL EQLESTISFEKLTEWTSSSIMEERKVKVYLPRMKMEEKYNLTS LLMAMGITDLFSSSANLSGISSVGSLKISQAVHAAYAEINEAG RDVVGSAEAGVDATEEFRADHPFLFCVKHIETNAILLFGRCVS P PREDICTED: 72 MGLCTAFHPYIFIVLLFALDNSEFTMGSIGAASMEFCFDVFKE ovalbumin LKVHHANDNMLYSPFAILSTLAMVFLGAKDSTRTQINKVVHF isoformX1 DKLPGFGDSIEAQCGTSANVHSSLRDILNQITKQNDAYSFSLA [Coturnix SRLYAQETYTVVPEYLQCVKELYRGGLESVNFQTAADQARG japonica] LINAWVESQTNGIIRNILQPSSVDSQTAMVLVNAIAFKGLWEK AFKAEDTQTIPFRVTEQESKPVQMMHQIGSFKVASMASEKMK ILELPFASGTMSMLVLLPDDVSGLEQLESTISFEKLTEWTSSSI MEERKVKVYLPRMKMEEKYNLTSLLMAMGITDLFSSSANLS GISSVGSLKISQAVHAAYAEINEAGRDVVGSAEAGVDATEEF RADHPFLFCVKHIETNAILLFGRCVSP Egg 73 MGSIGAASMEFCFDVFKELKVHHANDNMLYSPFAILSTLAMV albumin FLGAKDSTRTQINKVVHFDKLPGFGDSIEAQCGTSANVHSSLR DILNQITKQNDAYSFSLASRLYAQETYTVVPEYLQCVKELYR GGLESVNFQTAADQARGLINAWVESQINGIIRNILQPSSVDSQ TAMVLVNAIAFKGLWEKAFKAEDTQTIPFRVTEQESKPVQM MHQIGSFKVASMASEKMKILELPFASGTMSMLVLLPDDVSGL EQLESTISFEKLTEWTSSSIMEERKVKVYLPRMKMEEKYNLTS LLMAMGITDLFSSSANLSGISSVGSLKIPQAVHAAYAEINEAG RDVVGSAEAGVDATEEFRADHPFLFCVKHIETNAILLFGRCVS P ovalbumin 74 MGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALAMVYL [Anas GARDNTRTQIDKVVHFDKLPGFGESMEAQCGTSVSVHSSLRD platyrhynchos] ILTQITKPSDNFSLSFASRLYAEETYAILPEYLQCVKELYKGGL ESISFQTAADQARELINSWVESQTNGIIKNILQPSSVDSQTTMV LVNAIYFKGMWEKAFKDEDTQAMPFRMTEQESKPVQMMYQ VGSFKVAMVTSEKMKILELPFASGMMSMFVLLPDEVSGLEQL ESTISFEKLTEWTSSTMMEERRMKVYLPRMKMEEKYNLTSVF MALGMTDLFSSSANMSGISSTVSLKMSEAVHAACVEIFEAGR DVVGSAEAGMDVTSVSEEFRADHPFLFFIKHNPTNSILFFGRW MSP PREDICTED: 75 MGSIGAASTEFCFDVFRELKVQHVNENIFYSPLSIISALAMVYL ovalbumin- GARDNTRTQIDQVVHFDKIPGFGESMEAQCGTSVSVHSSLRDI like[Anser LTEITKPSDNFSLSFASRLYAEETYTILPEYLQCVKELYKGGLE cygnoides SISFQTAADQARELINSWVESQTNGIIKNILQPSSVDSQTTMVL domesticus] VNAIYFKGMWEKAFKDEDTQTMPFRMTEQESKPVQMMYQV GSFKLATVTSEKVKILELPFASGMMSMCVLLPDEVSGLEQLE TTISFEKLTEWTSSTMMEERRMKVYLPRMKMEEKYNLTSVF MALGMTDLFSSSANMSGISSTVSLKMSEAVHAACVEIFEAGR DVVGSAEAGMDVTSVSEEFRADHPFLFFIKHNPSNSILFFGRW ISP PREDICTED: 76 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALSMVYL Ovalbumin- GARENTRAQIDKVLHFDKMPGFGDTIESQCGTSVSIHTSLKD like[Aquila MFTQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKGG chrysaetos LETISFQTAAEQARELINSWVESQTNGMIKNILQPSSVDPQTK canadensis] MVLVNAIYFKGVWEKAFKDEDTQEVPFRVTEQESKPVQMM YQIGSFKVAVMASEKMKILELPYASGQLSMLVLLPDDVSGLE QLESAITFEKLMAWTSSTTMEERKMKVYLPRMKIEEKYNLTS VLMALGVTDLFSSSANLSGISSAESLKISKAVHEAFVEIYEAGS EVVGSTEAGMEVTSVSEEFRADHPFLFLIKHNPTNSILFFGRCF SP PREDICTED: 77 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALSMVYL Ovalbumin- GARENTRTQIDKVLHFDKMTGFGDTVESQCGTSVSIHTSLKDI like FTQITKPSDNYSLSLASRLYAEETYPILPEYLQCVKELYKGGLE [Haliaeetus TVSFQTAAEQARELINSWVESQTNGMIKNILQPSSVDPQTKM albicilla] VLVNAIYFKGVWEKAFKDEDTQEVPFRVTEQESKPVQMMYQ IGSFKVAVMASEKMKILELPYASGQLSMLVLLPDDVSGLEQL ESAITSEKLMEWTSSTTMEERKMKVYLPRMKIEEKYNLTSVL MALGVTDLFSSSADLSGISSAESLKISKAVHEAFVEIYEAGSEV VGSTEGGMEVTSVSEEFRADHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: 78 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALSMVYL Ovalbumin- GARENTRTQIDKVLHFDKMTGFGDTVESQCGTSVSIHTSLKDI like FTQITKPSDNYSLSLASRLYAEETYPILPEYLQCVKELYKGGLE [Haliaeetus TVSFQTAAEQARELINSWVESQTNGMIKNILQPSSVDPQTKM leucocephalus] VLVNAIYFKGVWEKAFKDEDTQEVPFRVTEQESKPVQMMYQ IGSFKVAVMASEKMKILELPYASGQLSMLVLLPDDVSGLEQL ESAITSEKLMEWTSSTTMEERKMKVYLPRMKIEEKYNLTSVL MALGVTDLFSSSADLSGISSAESLKISKAVHEAFVEIYEAGSEV VGSTEGGMEVTSFSEEFRADHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: 79 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin GARENTRAQIDKVVHFDKITGFGETIESQCGTSVSVHTSLKDM [Fulmarus FTQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKGGLE glacialis] TTSFQTAADQARELINSWVESQTNGMIKNILQPGSVDPQTEM VLVNAIYFKGMWEKAFKDEDTQAVPFRMTEQESKTVQMMY QIGSFKVAVMASEKMKILELPYASGELSMLVMLPDDVSGLEQ LETAITFEKLMEWTSSNMMEERKMKVYLPRMKMEEKYNLTS VLMALGVTDLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAG SEVVGSTGAGMEVTSVSEEFRADHPFLFLIKHNPTNSILFFGRC FSP PREDICTED: 80 MGSIGAASTEFCFDVFKELRVQHVNENVCYSPLIIISALSLVYL Ovalbumin- GARENTRAQIDKVVHFDKITGFGESIESQCGTSVSVHTSLKDM like FNQITKPSDNYSLSVASRLYAEERYPILPEYLQCVKELYKGGL [Chlamydotis ESISFQTAADQAREAINSWVESQTNGMIKNILQPSSVDPQTEM macqueenii] VLVNAIYFKGMWQKAFKDEDTQAVPFRISEQESKPVQMMYQ IGSFKVAVMAAEKMKILELPYASGELSMLVLLPDEVSGLEQL ENAITVEKLMEWTSSSPMEERIMKVYLPRMKIEEKYNLTSVL MALGITDLFSSSANLSGISAEESLKMSEAVHQAFAEISEAGSEV VGSSEAGIDATSVSEEFRADHPFLFLIKHNATNSILFFGRCFSP PREDICTED: 81 MGSISAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin GARENTRAQIEKVVHFDKITGFGESIESQCSTSVSVHTSLKDM like FTQITKPSDNYSLSFASRFYAEETYPILPEYLQCVKELYKGGLE [Nipponia TINFRTAADQARELINSWVESQTNGMIKNILQPGSVDPQTDM nippon] VLVNAIYFKGMWEKAFKDEDTQALPFRVTEQESKPVQMMY QIGSFKVAVLASEKVKILELPYASGQLSMLVLLPDDVSGLEQL ETAITVEKLMEWTSSNNMEERKIKVYLPRIKIEEKYNLTSVLM ALGITDLFSSSANLSGISSAESLKVSEAIHEAFVEIYEAGSEVAG STEAGIEVTSVSEEFRADHPFLFLIKHNATNSILFFGRCFSP PREDICTED: 82 MVSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKITGFEETIESQCSTSVSVHTSLKDM likeisoform FTQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKGGLE X2[Gavia TISFQTAADQARELINSWVESQTDGMIKNILQPGSVDPQTEMV stellata] LVNAIYFKGMWEKAFKDEDTQAVPFRMTEQESKPVQMMYQI GSFKVAVMASEKMKILELPYASGGMSMLVMLPDDVSGLEQL ETAITFEKLMEWTSSNMMEERKMKVYLPRMKMEEKYNLTS VLMALGMTDLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAG SEAVGSTGAGMEVTSVSEEFRADHPFLFLIKHNPTNSILFFGRC FSP PREDICTED: 83 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin GARENTRAQIDKVVHFDKITGFGEPIESQCGISVSVHTSLKDMI [Pelecanus TQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKGGLET crispus] ISFQTAADQARELINSWVENQTNGMIKNILQPGSVDPQTEMV LVNAVYFKGMWEKAFKDEDTQAVPFRMTEQESKPVQMMY QIGSFKVAVMASEKIKILELPYASGELSMLVLLPDDVSGLEQL ETAITLDKLTEWTSSNAMEERKMKVYLPRMKIEKKYNLTSVL IALGMTDLFSSSANLSGISSAESLKMSEAIHEAFLEIYEAGSEV VGSTEAGMEVTSVSEEFRADHPFLFLIKHNPTNSILFFGRCLSP PREDICTED: 84 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKIPGFGDTTESQCGTSVSVHTSLKD like MFTQITKPSDNYSVSFASRLYAEETYPILPEFLECVKELYKGG [Charadrius LESISFQTAADQARELINSWVESQTNGMIKNILQPGSVDSQTE vociferus] MVLVNAIYFKGMWEKAFKDEDTQTVPFRMTEQETKPVQMM YQIGTFKVAVMPSEKMKILELPYASGELCMLVMLPDDVSGLE ELESSITVEKLMEWTSSNMMEERKMKVFLPRMKIEEKYNLTS VLMALGMTDLFSSSANLSGISSAEPLKMSEAVHEAFIEIYEAG SEVVGSTGAGMEITSVSEEFRADHPFLFLIKHNPTNSILFFGRC VSP PREDICTED: 85 MGSIGAVSTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKITGSGETIEAQCGTSVSVHTSLKD like MFTQITKPSENYSVGFASRLYADETYPIIPEYLQCVKELYKGG [Eurypyga LEMISFQTAADQARELINSWVESQTNGMIKNILQPGSVDPQTE helias] MILVNAIYFKGVWEKAFKDEDTQAVPFRMTEQESKPVQMMY QFGSFKVAAMAAEKMKILELPYASGALSMLVLLPDDVSGLE QLESAITFEKLMEWTSSNMMEEKKIKVYLPRMKMEEKYNFT SVLMALGMTDLFSSSANLSGISSADSLKMSEVVHEAFVEIYEA GSEVVGSTGSGMEAASVSEEFRADHPFLFLIKHNPTNSILFFGR CFSP PREDICTED: 86 MVSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKITGFEETIESQVQKKQCSTSVSVHT likeisoform SLKDMFTQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKEL X1[Gavia YKGGLETISFQTAADQARELINSWVESQTDGMIKNILQPGSVD stellata] PQTEMVLVNAIYFKGMWEKAFKDEDTQAVPFRMTEQESKPV QMMYQIGSFKVAVMASEKMKILELPYASGGMSMLVMLPDD VSGLEQLETAITFEKLMEWTSSNMMEERKMKVYLPRMKMEE KYNLTSVLMALGMTDLFSSSANLSGISSAESLKMSEAVHEAF VEIYEAGSEAVGSTGAGMEVTSVSEEFRADHPFLFLIKHNPTN SILFFGRCFSP PREDICTED: 87 MGSIGAASGEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKIIGFGESIESQCGTSVSVHTSLKDM like[Egretta FAQITKPSDNYSLSFASRLYAEETFPILPEYLQCVKELYKGGLE garzetta] TLSFQTAADQARELINSWVESQTNGMIKDILQPGSVDPQTEM VLVNAIYFKGVWEKAFKDEDTQTVPFRMTEQESKPVQMMY QIGSFKVAVVAAEKIKILELPYASGALSMLVLLPDDVSSLEQL ETAITFEKLTEWTSSNIMEERKIKVYLPRMKIEEKYNLTSVLM DLGITDLFSSSANLSGISSAESLKVSEAIHEAIVDIYEAGSEVVG SSGAGLEGTSVSEEFRADHPFLFLIKHNPTSSILFFGRCFSP PREDICTED: 88 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKITGSGEAIESQCGTSVSVHISLKDM like FTQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKEGLA [Balearica TISFQTAADQAREFINSWVESQTNGMIKNILQPGSVDPQTQMV regulorum LVNAIYFKGVWEKAFKDEDTQAVPFRMTKQESKPVQMMYQI gibbericeps] GSFKVAVMASEKMKILELPYASGQLSMLVMLPDDVSGLEQIE NAITFEKLMEWTNPNMMEERKMKVYLPRMKMEEKYNLTSV LMALGMTDLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAGS EVVGSTGAGIEVTSVSEEFRADHPFLFLIKHNPTNSILFFGRCFS P PREDICTED: 89 MGSIGEASTEFCIDVFRELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDQVVHFDKITGFGDTVESQCGSSLSVHSSLKDI like[Nestor FAQITQPKDNYSLNFASRLYAEETYPILPEYLQCVKELYKGGL notabilis] ETISFQTAADQARELINSWVESQTNGMIKNILQPSSVDPQTEM VLVNAIYFKGVWEKAFKDEETQAVPFRITEQENRPVQIMYQF GSFKVAVVASEKIKILELPYASGQLSMLVLLPDEVSGLEQLEN AITFEKLTEWTSSDIMEEKKIKVFLPRMKIEEKYNLTSVLVAL GIADLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAGSEVVGS SGAGIEAASDSEEFRADHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: 90 MGSIGAASTEFCFDIFNELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTKAQIDKVVHFDKITGFGESIESQCSTSASVHTSFKDM like FTQITKPSDNYSLSFASRLYAEETYPILPEYSQCVKELYKGGLE [Pygoscelis SISFQTAADQARELINSWVESQTNGMIKNILQPGSVDPQTELV adeliae] LVNAIYFKGTWEKAFKDKDTQAVPFRVTEQESKPVQMMYQI GSYKVAVIASEKMKILELPYASGELSMLVLLPDDVSGLEQLET AITFEKLMEWTSSNMMEERKVKVYLPRMKIEEKYNLTSVLM ALGMTDLFSPSANLSGISSAESLKMSEAIHEAFVEIYEAGSEVV GSTEAGMEVTSVSEEFRADHPFLFLIKCNLTNSILFFGRCFSP Ovalbumin- 91 MGSISTASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL like[Athene GARENTRAQIEKVVHFDKITGFGESIESQCGTSVSVHTSLKDM cunicularia] LIQISKPSDNYSLSFASKLYAEETYPILPEYLQCVKELYKGGLE SINFQTAADQARQLINSWVESQTNGMIKDILQPSSVDPQTEMV LVNAIYFKGIWEKAFKDEDTQEVPFRITEQESKPVQMMYQIG SFKVAVIASEKIKILELPYASGELSMLIVLPDDVSGLEQLETAIT FEKLIEWTSPSIMEERKTKVYLPRMKIEEKYNLTSVLMALGM TDLFSPSANLSGISSAESLKMSEAIHEAFVEIYEAGSEVVGSAE AGMEATSVSEFRVDHPFLFLIKHNPANIILFFGRCVSP PREDICTED: 92 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLTIISALSLVYL Ovalbumin- GARENTRAQIDKVFHFDKISGFGETTESQCGTSVSVHTSLKEM like FTQITKPSDNYSVSFASRLYAEDTYPILPEYLQCVKELYKGGL [Calidris ETISFQTAADQAREVINSWVESQTNGMIKNILQPGSVDSQTEM pugnax] VLVNAIYFKGMWEKAFKDEDTQTMPFRITEQERKPVQMMYQ AGSFKVAVMASEKMKILELPYASGEFCMLIMLPDDVSGLEQL ENSFSFEKLMEWTTSNMMEERKMKVYIPRMKMEEKYNLTSV LMALGMTDLFSSSANLSGISSAETLKMSEAVHEAFMEIYEAG SEVVGSTGSGAEVTGVYEEFRADHPFLFLVKHKPTNSILFFGR CVSP PREDICTED: 93 MGSIGAASTEFCFDIFNELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin GARENTKAQIDKVVHFDKITGFGETIESQCSTSVSVHTSLKDT [Aptenodytes FTQITKPSDNYSLSFASRLYAEETYPILPEYSQCVKELYKGGLE forsteri] TISFQTAADQARELINSWVESQTNGMIKNILQPGSVDPQTELV LVNAIYFKGTWEKAFKDKDTQAVPFRVTEQESKPVQMMYQI GSYKVAVIASEKMKILELPYASRELSMLVLLPDDVSGLEQLET AITFEKLMEWTSSNMMEERKVKVYLPRMKIEEKYNLTSVLM ALGMTDLFSPSANLSGISSAESLKMSEAVHEAFVEIYEAGSEV VGSTGAGMEVTSVSEEFRADHPFLFLIKCNPTNSILFFGRCFSP PREDICTED: 94 MGSISAASAEFCLDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKITGSGETIEFQCGTSANIHPSLKDM like FTQITRLSDNYSLSFASRLYAEERYPILPEYLQCVKELYKGGLE [Pterocles TISFQTAADQARELINSWVESQTNGMIKNILQPGSVNPQTEMV gutturalis] LVNAIYFKGLWEKAFKDEDTQTVPFRMTEQESKPVQMMYQV GSFKVAVMASDKIKILELPYASGELSMLVLLPDDVTGLEQLET SITFEKLMEWTSSNVMEERTMKVYLPHMRMEEKYNLTSVLM ALGVTDLFSSSANLSGISSAESLKMSEAVHEAFVEIYESGSQV VGSTGAGTEVTSVSEEFRVDHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin- 95 MGSIGAASVEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL like[Falco GARENTKAQIDKVVHFDKIAGFGEAIESQCVTSASIHSLKDMF peregrinus] TQITKPSDNYSLSFASRLYAEEAYSILPEYLQCVKELYKGGLE TISFQTAADQARDLINSWVESQTNGMIKNILQPGAVDLETEM VLVNAIYFKGMWEKAFKDEDTQTVPFRMTEQESKPVQMMY QVGSFKVAVMASDKIKILELPYASGQLSMVVVLPDDVSGLEQ LEASITSEKLMEWTSSSIMEEKKIKVYFPHMKIEEKYNLTSVL MALGMTDLFSSSANLSGISSAEKLKVSEAVHEAFVEISEAGSE VVGSTEAGTEVTSVSEEFKADHPFLFLIKHNPTNSILFFGRCFS P PREDICTED: 96 MGSIGAASSEFCFDIFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVPFDKITASGESIESQCSTSVSVHTSLKDIF likeisoform TQITKSSDNHSLSFASRLYAEETYPILPEYLQCVKELYEGGLET X2 ISFQTAADQARELINSWIESQTNGRIKNILQPGSVDPQTEMVL [Phalacrocorax VNAIYFKGMWEKAFKDEDTQAVPFRMTEQESKPVQVMHQIG carbo] SFKVAVLASEKIKILELPYASGELSMLVLLPDDVSGLEQLETAI TFEKLMEWTSPNIMEERKIKVFLPRMKIEEKYNLTSVLMALGI TDLFSPLANLSGISSAESLKMSEAIHEAFVEISEAGSEVIGSTEA EVEVTNDPEEFRADHPFLFLIKHNPTNSILFFGRCFSP PREDICTED: 97 MGSIGAASTEFCFDVFKELKAQYVNENIFYSPMTIITALSMVY Ovalbumin- LGSKENTRAQIAKVAHFDKITGFGESIESQCGASASIQFSLKDL like FTQITKPSGNHSLSVASRIYAEETYPILPEYLECMKELYKGGLE [Merops TINFQTAANQARELINSWVERQTSGMIKNILQPSSVDSQTEMV nubicus] LVNAIYFRGLWEKAFKVEDTQATPFRITEQESKPVQMMHQIG SFKVAVVASEKIKILELPYASGRLTMLVVLPDDVSGLKQLETT ITFEKLMEWTTSNIMEERKIKVYLPRMKIEEKYNLTSVLMAL GLTDLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAGSEVVA SAEAGMDATSVSEEFRADHPFLFLIKDNTSNSILFFGRCFSP PREDICTED: 98 MGSIGAASTEFCFDVFKELKGQHVNENIFFCPLSIVSALSMVY Ovalbumin- LGARENTRAQIVKVAHFDKIAGFAESIESQCGTSVSIHTSLKD like MFTQITKPSDNYSLNFASRLYAEETYPIIPEYLQCVKELYKGG [Tauraco LETISFQTAADQAREIINSWVESQTNGMIKNILRPSSVHPQTEL erythrolophus] VLVNAVYFKGTWEKAFKDEDTQAVPFRITEQESKPVQMMYQ IGSFKVAAVTSEKMKILEVPYASGELSMLVLLPDDVSGLEQLE TAITAEKLIEWTSSTVMEERKLKVYLPRMKIEEKYNLTTVLTA LGVTDLFSSSANLSGISSAQGLKMSNAVHEAFVEIYEAGSEVV GSKGEGTEVSSVSDEFKADHPFLFLIKHNPTNSIVFFGRCFSP PREDICTED: 99 MGSIGAASTEFCFDVFKELKVHHVNENILYSPLAIISALSMVY Ovalbumin- LGAKENTRDQIDKVVHFDKITGIGESIESQCSTAVSVHTSLKD like VFDQITRPSDNYSLAFASRLYAEKTYPILPEYLQCVKELYKGG [Cuculus LETIDFQTAADQARQLINSWVEDETNGMIKNILRPSSVNPQTK canorus] IILVNAIYFKGMWEKAFKDEDTQEVPFRITEQETKSVQMMYQ IGSFKVAEVVSDKMKILELPYASGKLSMLVLLPDDVYGLEQL ETVITVEKLKEWTSSIVMEERITKVYLPRMKIMEKYNLTSVLT AFGITDLFSPSANLSGISSTESLKVSEAVHEAFVEIHEAGSEVV GSAGAGIEATSVSEEFKADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin 100 MGSIGAASTEFCLDVFKELKVQHVNENIFYSPLSIISALSMVYL [Antrostomus GARENTRAQIDKVVHFDKITGFEDSIESQCGTSVSVHTSLKDM carolinensis] FTQITKPSDNYSVGFASRLYAAETYQILPEYSQCVKELYKGGL ETINFQKAADQATELINSWVESQTNGMIKNILQPSSVDPQTQIF LVNAIYFKGMWQRAFKEEDTQAVPFRISEKESKPVQMMYQI GSFKVAVIPSEKIKILELPYASGLLSMLVILPDDVSGLEQLENAI TLEKLMQWTSSNMMEERKIKVYLPRMRMEEKYNLTSVFMA LGITDLFSSSANLSGISSAESLKMSDAVHEASVEIHEAGSEVVG STGSGTEASSVSEEFRADHPYLFLIKHNPTDSIVFFGRCFSP PREDICTED: 101 MGSIGAASTEFCFDVFKELKFQHVDENIFYSPLTIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKIAGFEETVESQCGTSVSVHTSLKD like MFAQITKPSDNYSLSFASRLYAEETYPILPEYLQCVKELYKGG [Opisthocomus LETISFQTAADQARDLINSWVESQTNGMIKNILQPSSVGPQTE hoazin] LILVNAIYFKGMWQKAFKDEDTQEVPFRMTEQQSKPVQMMY QTGSFKVAVVASEKMKILALPYASGQLSLLVMLPDDVSGLK QLESAITSEKLIEWTSPSMMEERKIKVYLPRMKIEEKYNLTSV LMALGITDLFSPSANLSGISSAESLKMSQAVHEAFVEIYEAGSE VVGSTGAGMEDSSDSEEFRVDHPFLFFIKHNPTNSILFFGRCFS P PREDICTED: 102 MGSIGPLSVEFCCDVFKELRIQHPRENIFYSPVTIISALSMVYL Ovalbumin- GARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSLKDIF like TQITKPSDNYTVGIASRLYAEEKYPILPEYLQCIKELYKGGLEP [Lepidothrix INFQTAAEQARELINSWVESQTNGMIKNILQPSSVNPETDMVL coronata] VNAIYFKGLWEKAFKDEDIQTVPFRITEQESKPVQMMFQIGSF RVAEITSEKIRILELPYASGQLSLWVLLPDDISGLEQLETAITFE NLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTSLGITDL FSSSANLSGISSAESLKVSSAFHEASVEIYEAGSKVVGSTGAEV EDTSVSEEFRADHPFLFLIKHNPSNSIFFFGRCFSP PREDICTED: 103 MGSIGTASAEFCFDVFKELKVHHVNENIFYSPLSIISALSMVYL Ovalbumin GARENTKTQMEKVIHFDKITGLGESMESQCGTGVSIHTALKD [Struthio MLSEITKPSDNYSLSLASRLYAEQTYAILPEYLQCIKELYKESL camelus ETVSFQTAADQARELINSWIESQTNGVIKNFLQPGSVDSQTEL australis] VLVNAIYFKGMWEKAFKDEDTQEVPFRITEQESRPVQMMYQ AGSFKVATVAAEKIKILELPYASGELSMLVLLPDDISGLEQLE TTISFEKLTEWTSSNMMEDRNMKVYLPRMKIEEKYNLTSVLI ALGMTDLFSPAANLSGISAAESLKMSEAIHAAYVEIYEADSEI VSSAGVQVEVTSDSEEFRVDHPFLFLIKHNPTNSVLFFGRCISP PREDICTED: 104 MGSIGAVSTEFSCDVFKELRIHHVQENIFYSPVTIISALSMIYLG Ovalbumin- ARDSTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSIKDMFT like KITKASDNYSIGIASRLYAEEKYPILPEYLQCVKELYKGGLESI [Acanthisitta SFQTAAEQAREIINSWVESQTNGMIKNILQPSSVDPQTDIVLV chloris] NAIYFKGLWEKAFRDEDTQTVPFKITEQESKPVQMMYQIGSF KVAEITSEKIKILEVPYASGQLSLWVLLPDDISGLEKLETAITFE NLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTALGITDL FSSSANLSGISSAESLKVSEAFHEAIVEISEAGSKVVGSVGAGV DDTSVSEEFRADHPFLFLIKHNPTSSIFFFGRCFSP PREDICTED: 105 MGSIGAASTEFCFDVFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVHFDKIAGFGESTESQCGTSVSAHTSLKD like[Tyto MSNQITKLSDNYSLSFASRLYAEETYPILPEYSQCVKELYKGG alba] LESISFQTAAYQARELINAWVESQTNGMIKDILQPGSVDSQTK MVLVNAIYFKGIWEKAFKDEDTQEVPFRMTEQETKPVQMMY QIGSFKVAVIAAEKIKILELPYASGQLSMLVILPDDVSGLEQLE TAITFEKLTEWTSASVMEERKIKVYLPRMSIEEKYNLTSVLIAL GVTDLFSSSANLSGISSAESLRMSEAIHEAFVETYEAGSTESGT EVTSASEEFRVDHPFLFLIKHKPTNSILFFGRCFSP PREDICTED: 106 MGSIGAASSEFCFDIFKELKVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDKVVPFDKITASGESIESQVQKIQCSTSVSVHTS likeisoform LKDIFTQITKSSDNHSLSFASRLYAEETYPILPEYLQCVKELYE X1 GGLETISFQTAADQARELINSWIESQTNGRIKNILQPGSVDPQT [Phalacrocorax EMVLVNAIYFKGMWEKAFKDEDTQAVPFRMTEQESKPVQV carbo] MHQIGSFKVAVLASEKIKILELPYASGELSMLVLLPDDVSGLE QLETAITFEKLMEWTSPNIMEERKIKVFLPRMKIEEKYNLTSV LMALGITDLFSPLANLSGISSAESLKMSEAIHEAFVEISEAGSE VIGSTEAEVEVTNDPEEFRADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin- 107 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALSMVYL like[Pipra GARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSLKDIF filicauda] TQITKPSDNYTVGIASRLYAEEKYPILPEYLQCIKELYKGGLEP ISFQTAAEQARELINSWVESQTNGIIKNILQPSSVNPETDMVLV NAIYFKGLWEKAFKDEGTQTVPFRITEQESKPVQMMFQIGSF RVAEIASEKIRILELPYASGQLSLWVLLPDDISGLEQLETAITFE NLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTSLGITDL FSSSANLSGISSAERLKVSSAFHEASMEINEAGSKVVGAGVDD TSVSEEFRVDRPFLFLIKHNPSNSIFFFGRCFSP Ovalbumin 108 MGSIGAASTEFCFDMFKELKVHHVNENIIYSPLSIISILSMVFLG [Dromaius ARENTKTQMEKVIHFDKITGFGESLESQCGTSVSVHASLKDIL novaehollandiae] SEITKPSDNYSLSLASKLYAEETYPVLPEYLQCIKELYKGSLET VSFQTAADQARELINSWVETQTNGVIKNFLQPGSVDPQTEMV LVDAIYFKGTWEKAFKDEDTQEVPFRITEQESKPVQMMYQA GSFKVATVAAEKMKILELPYASGELSMFVLLPDDISGLEQLET TISIEKLSEWTSSNMMEDRKMKVYLPHMKIEEKYNLTSVLVA LGMTDLFSPSANLSGISTAQTLKMSEAIHGAYVEIYEAGSEMA TSTGVLVEAASVSEEFRVDHPFLFLIKHNPSNSILFFGRCIFP ChainA, 109 MGSIGAASTEFCFDMFKELKVHHVNENIIYSPLSIISILSMVFLG Ovalbumin ARENTKTQMEKVIHFDKITGFGESLESQCGTSVSVHASLKDIL SEITKPSDNYSLSLASKLYAEETYPVLPEYLQCIKELYKGSLET VSFQTAADQARELINSWVETQTNGVIKNFLQPGSVDPQTEMV LVDAIYFKGTWEKAFKDEDTQEVPFRITEQESKPVQMMYQA GSFKVATVAAEKMKILELPYASGELSMFVLLPDDISGLEQLET TISIEKLSEWTSSNMMEDRKMKVYLPHMKIEEKYNLTSVLVA LGMTDLFSPSANLSGISTAQTLKMSEAIHGAYVEIYEAGSEMA TSTGVLVEAASVSEEFRVDHPFLFLIKHNPSNSILFFGRCIFPHH HHHH Ovalbumin- 110 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALSMVYL like GARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSLKDIF [Corapipo TQITKPSDNYTVGIASRLYAEEKYPILPEYLQCIKELYKGGLEP altera] ISFQTAAEQARELINSWVESQTNGMIKNILQPSAVNPETDMVL VNAIYFKGLWEKAFKDEGTQTVPFRITEQESKPVQMMFQIGS FRVAEITSEKIRILELPYASGQLSLWVLLPDDISGLEQLETAITF ENLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTSLGITD LFSSSANLSGISSAERLKVSSAFHEASMEIYEAGSKVVGSTGA GVDDTSVSEEFRVDRPFLFLIKHNPSNSIFFFGRCFSP Ovalbumin- 111 MEDQRGNTGFTMGSIGAASTEFCIDVFRELRVQHVNENIFYSP likeprotein LTIISALSMVYLGARENTRAQIDQVVHFDKIAGFGDTVESQCG [Amazona SSPSVHNSLKTVXAQITQPRDNYSLNLASRLYAEESYPILPEYL aestiva] QCVKELYNGGLETVSFQTAADQARELINSWVESQTNGIIKNIL QPSSVDPQTEMVLVNAIYFKGLWEKAFKDEETQAVPFRITEQ ENRPVQMMYQFGSFKVAXVASEKIKILELPYASGQLSMLVLL PDEVSGLEQNAITFEKLTEWTSSDLMEERKIKVFFPRVKIEEK YNLTAVLVSLGITDLFSSSANLSGISSAENLKMSEAVHEAXVE IYEAGSEVAGSSGAGIEVASDSEEFRVDHPFLFLIXHNPTNSILF FGRCFSP PREDICTED: 112 MGSIGAASTEFCIDVFRELRVQHVNENIFYSPLSIISALSMVYL Ovalbumin- GARENTRAQIDEVFHFDKIAGFGDTVDPQCGASLSVHKSLQN like VFAQITQPKDNYSLNLASRLYAEESYPILPEYLQCVKELYNEG [Melopsittacus LETVSFQTGADQARELINSWVENQTNGVIKNILQPSSVDPQTE undulatus] MVLVNAIYFKGLWQKAFKDEETQAVPFRITEQENRPVQMMY QFGSFKVAVVASEKVKILELPYASGQLSMWVLLPDEVSGLEQ LENAITFEKLTEWTSSDLTEERKIKVFLPRVKIEEKYNLTAVL MALGVTDLFSSSANFSGISAAENLKMSEAVHEAFVEIYEAGSE VVGSSGAGIEAPSDSEEFRADHPFLFLIKHNPTNSILFFGRCFSP Ovalbumin- 113 MGSIGPLSVEFCCDVFKELRIQHARDNIFYSPVTIISALSMVYL like GARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSVHTSLKDIF [Neopelma TQITKPRENYTVGIASRLYAEEKYPILPEYLQCIKELYKGGLEP chrysocephalum] ISFQTAAEQARELINSWVESQTNGMIKNILQPSSVNPETDMVL VNAIYFKGLWKKAFKDEGTQTVPFRITEQESKPVQMMFQIGS FRVAEITSEKIRILELPYASGQLSLWVLLPDDISGLEQLESAITF ENLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTSLGITD LFSSSANLSGISSAEKLKVSSAFHEASMEIYEAGNKVVGSTGA GVDDTSVSEEFRVDRPFLFLIKHNPSNSIFFFGRCFSP PREDICTED: 114 MGSIGAASAEFCVDVFKELKDQHVNNIVESPLMIISALSMVNI Ovalbumin- GAREDTRAQIDKVVHFDKITGYGESIESQCGTSIGIYFSLKDAF like TQITKPSDNYSLSFASKLYAEETYPILPEYLKCVKELYKGGLE [Buceros TISFQTAADQARELINSWVESQTNGMIKNILQPSSVDPQTEMV rhinoceros LVNAIYFKGLWEKAFKDEDTQAVPFRITEQESKPVQMMYQIG silvestris] SFKVAVIASEKIKILELPYASGQLSLLVLLPDDVSGLEQLESAIT SEKLLEWTNPNIMEERKTKVYLPRMKIEEKYNLTSVLVALGIT DLFSSSANLSGISSAEGLKLSDAVHEAFVEIYEAGREVVGSSE AGVEDSSVSEEFKADRPFIFLIKHNPTNGILYFGRYISP PREDICTED: 115 MGSIGAANTDFCFDVFKELKVHHANENIFYSPLSIVSALAMV Ovalbumin- YLGARENTRAQIDKALHFDKILGFGETVESQCDTSVSVHTSLK like DMLIQITKPSDNYSFSFASKIYTEETYPILPEYLQCVKELYKGG [Cariama VETISFQTAADQAREVINSWVESHTNGMIKNILQPGSVDPQTK cristata] MVLVNAVYFKGIWEKAFKEEDTQEMPFRINEQESKPVQMMY QIGSFKLTVAASENLKILEFPYASGQLSMMVILPDEVSGLKQL ETSITSEKLIKWTSSNTMEERKIRVYLPRMKIEEKYNLKSVLM ALGITDLFSSSANLSGISSAESLKMSEAVHEAFVEIYEAGSEVT SSTGTEMEAENVSEEFKADHPFLFLIKHNPTDSIVFFGRCMSP Ovalbumin 116 MGSIGPLSVEFCCDVFKELRIQHARENIFYSPVTIISALSMVYL [Manacus GARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSLKDIF vitellinus] TQITKPSDNYTVGIASRLYAEEKYPILPEYLQCIKELYKGGLEP ISFQTAAEQARELINSWVESQTNGMIKNILQPSSVNPETDMVL VNAIYFKGLWEKAFKDESTQTVPFRITEQESKPVQMMFQIGSF RVAEIASEKIRILELPYASGQLSLWVLLPDDISGLEQLETAITFE NLKEWTSSTKMEERKIKVYLPRMKIEEKYNLTSVLTSLGITDL FSSSANLSGISSAERLKVSSAFHEASMEIYEAGSRVVEAGVDD TSVSEEFRVDRPFLFLIKHNPSNSIFFFGRCFSP Ovalbumin- 117 MGSIGPVSTEFCCDIFKELRIQHARENIIYSPVTIISALSMVYLG like ARDNTKAQIEKAVHFDKIPGFGESIESQCGTSLSIHTSLKDILT [Empidonax QITKPSDNYTVGIASRLYAEEKYPILSEYLQCIKELYKGGLEPI traillii] SFQTAAEQARELINSWVESQTNGMIKNILQPSSVNPETDMVL VNAIYFKGLWEKAFKDEGTQTVPFRITEQESKPVQMMFQIGS FKVAEITSEKIRILELPYASGKLSLWVLLPDDISGLEQLETAITF ENLKEWTSSTRMEERKIKVYLPRMKIEEKYNLTSVLTSLGITD LFSSSANLSGISSAERLKVSSAFHEVFVEIYEAGSKVEGSTGAG VDDTSVSEEFRADHPFLFLVKHNPSNSIIFFGRCYLP PREDICTED: 118 MGSTGAASMEFCFALFRELKVQHVNENIFFSPVTIISALSMVY Ovalbumin- LGARENTRAQLDKVAPFDKITGFGETIGSQCSTSASSHTSLKD like VFTQITKASDNYSLSFASRLYAEETYPILPEYLQCVKELYKGG [Leptosomus LESISFQTAADQARELINSWVESQTNGMIKDILRPSSVDPQTKI discolor] ILITAIYFKGMWEKAFKEEDTQAVPFRMTEQESKPVQMMYQI GSFKVAVIPSEKLKILELPYASGQLSMLVILPDDVSGLEQLETA ITTEKLKEWTSPSMMKERKMKVYFPRMRIEEKYNLTSVLMA LGITDLFSPSANLSGISSAESLKVSEAVHEASVDIDEAGSEVIGS TGVGTEVTSVSEEIRADHPFLFLIKHKPTNSILFFGRCFSP Hypothetical 119 MEHAQLTQLVNSNMTSNTCHEADEFENIDFRMDSISVTNTKF protein CFDVFNEMKVHHVNENILYSPLSILTALAMVYLGARGNTESQ H355_0080 MKKALHFDSITGAGSTTDSQCGSSEYIHNLFKEFLTEITRTNAT 77[Colinus YSLEIADKLYVDKTFTVLPEYINCARKFYTGGVEEVNFKTAA virginianus] EEARQLINSWVEKETNGQIKDLLVPSSVDFGTMMVFINTIYFK GIWKTAFNTEDTREMPFSMTKQESKPVQMMCLNDTENMATL PAEKMRILELPYASGELSMLVLLPDEVSGLEQIEKAINFEKLRE WTSTNAMEKKSMKVYLPRMKIEEKYNLTSTLMALGMTDLFS RSANLTGISSVENLMISDAVHGAFMEVNEEGTEAAGSTGAIG NIKHSVEFEEFRADHPFLFLIRYNPTNVILFFDNSEFTMGSIGA VSTEFCFDVFKELRVHHANENIFYSPFTVISALAMVYLGAKDS TRTQINKVVRFDKLPGFGDSIEAQCGTSANVHSSLRDILNQIT KPNDIYSFSLASRLYADETYTILPEYLQCVKELYRGGLESINFQ TAADQARELINSWVESQTSGIIRNVLQPSSVDSQTAMVLVNAI YFKGLWEKGFKDEDTQAMPFRVTEQENKSVQMMYQIGTFK VASVASEKMKILELPFASGTMSMWVLLPDEVSGLEQLETTISI EKLTEWTSSSVMEERKIKVFLPRMKMEEKYNLTSVLMAMGM TDLFSSSANLSGISSTLQKKGFRSQELGDKYAKPMLESPALTP QVTAWDNSWIVAHPAAIEPDLCYQIMEQKWKPFDWPDFRLP MRVSCRFRTMEALNKANTSFALDFFKHECQEDDDENILFSPFS ISSALATVYLGAKGNTADQMAKTEIGKSGNIHAGFKALDLEI NQPTKNYLLNSVNQLYGEKSLPFSKEYLQLAKKYYSAEPQSV DFLGKANEIRREINSRVEHQTEGKIKNLLPPGSIDSLTRLVLVN ALYFKGNWATKFEAEDTRHRPFRINMHTTKQVPMMYLRDKF NWTYVESVQTDVLELPYVNNDLSMFILLPRDITGLQKLINELT FEKLSAWTSPELMEKMKMEVYLPRFTVEKKYDMKSTLSKM GIEDAFTKVDSCGVTNVDEITTHIVSSKCLELKHIQINKKLKCN KAVAMEQVSASIGNFTIDLFNKLNETSRDKNIFFSPWSVSSAL ALTSLAAKGNTAREMAEDPENEQAENIHSGFKELMTALNKPR NTYSLKSANRIYVEKNYPLLPTYIQLSKKYYKAEPYKVNFKT APEQSRKEINNWVEKQTERKIKNFLSSDDVKNSTKSILVNAIY FKAEWEEKFQAGNTDMQPFRMSKNKSKLVKMMYMRHTFPV LIMEKLNFKMIELPYVKRELSMFILLPDDIKDSTTGLEQLEREL TYEKLSEWADSKKMSVTLVDLHLPKFSMEDRYDLKDALKSM GMASAFNSNADFSGMTGFQAVPMESLSASTNSFTLDLYKKL DETSKGQNIFFASWSIATALAMVHLGAKGDTATQVAKGPEY EETENIHSGFKELLSAINKPRNTYLMKSANRLFGDKTYPLLPK FLELVARYYQAKPQAVNFKTDAEQARAQINSWVENETESKIQ NLLPAGSIDSHTVLVLVNAIYFKGNWEKRFLEKDTSKMPFRL SKTETKPVQMMFLKDTFLIHHERTMKFKIIELPYVGNELSAFV LLPDDISDNTTGLELVERELTYEKLAEWSNSASMMKAKVELY LPKLKMEENYDLKSVLSDMGIRSAFDPAQADFTRMSEKKDLF ISKVIHKAFVEVNEEDRIVQLASGRLTGRCRTLANKELSEKNR TKNLFFSPFSISSALSMILLGSKGNTEAQIAKVLSLSKAEDAHN GYQSLLSEINNPDTKYILRTANRLYGEKTFEFLSSFIDSSQKFY HAGLEQTDFKNASEDSRKQINGWVEEKTEGKIQKLLSEGIINS MTKLVLVNAIYFKGNWQEKFDKETTKEMPFKINKNETKPVQ MMFRKGKYNMTYIGDLETTVLEIPYVDNELSMIILLPDSIQDE STGLEKLERELTYEKLMDWINPNMMDSTEVRVSLPRFKLEEN YELKPTLSTMGMPDAFDLRTADFSGISSGNELVLSEVVHKSFV EVNEEGTEAAAATAGIMLLRCAMIVANFTADHPFLFFIRHNK TNSILFCGRFCSP PREDICTED: 120 MGSIGTASTEFCFDMFKEMKVQHANQNIIFSPLTIISALSMVYL Ovalbumin GARDNTKAQMEKVIHFDKITGFGESVESQCGTSVSIHTSLKD isoformX2 MLSEITKPSDNYSLSLASRLYAEETYPILPEYLQCMKELYKGG [Apteryx LETVSFQTAADQARELINSWVESQTNGVIKNFLQPGSVDPQTE australis MVLVNAIYFKGMWEKAFKDEDTQEVPFRITEQESKPVQMMY mantelli] QVGSFKVATVAAEKMKILEIPYTHRELSMFVLLPDDISGLEQL ETTISFEKLTEWTSSNMMEERKVKVYLPHMKIEEKYNLTSVL MALGMTDLFSPSANLSGISTAQTLMMSEAIHGAYVEIYEAGR EMASSTGVQVEVTSVLEEVRADKPFLFFIRHNPTNSMVVFGR YMSP Hypothetical 121 MTSNTCHEADEFENIDFRMDSISVTNTKFCFDVFNEMKVHHV protein NENILYSPLSILTALAMVYLGARGNTESQMKKALHEDSITGG ASZ78_006 GSTTDSQCGSSEYIHNLFKEFLTEITRTNATYSLEIADKLYVDK 007 TFTVLPEYINCARKFYTGGVEEVNFKTAAEEARQLMNSWVE [Callipepla KETNGQIKDLLVPSSVDFGTMMVFINTIYFKGIWKTAFNTEDT squamata] REMPFSMTKQESKPVQMMCLNDTFNMVTLPAEKMRILELPY ASGELSMLVLLPDEVSGLERIEKAINFEKLREWTSTNAMEKKS MKVYLPRMKIEEKYNLTSTLMALGMTDLFSRSANLTGISSVD NLMISDAVHGAFMEVNEEGTEAAGSTGAIGNIKHSVEFEEFR ADHPFLFLIRYNPTNVILFFDNSEFTMGSIGAVSTEFCFDVFKE LRVHHANENIFYSPFTIISALAMVYLGAKDSTRTQINKVVRFD KLPGFGDSIEAQCGTSANVHSSLRDILNQITKPNDIYSFSLASR LYADETYTILPEYLQCVKELYRGGLESINFQTAADQARELINS WVESQTSGIIRNVLQPSSVDSQTAMVLVNAIYFKGLWEKGFK DEDTQAIPFRVTEQENKSVQMMYQIGTFKVASVASEKMKILE LPFASGTMSMWVLLPDEVSGLEQLETTISIEKLTEWTSSSVME ERKIKVFLPRMKMEEKYNLTSVLMAMGMTDLFSSSANLSGIS STLQKKGFRSQELGDKYAKPMLESPALTPQATAWDNSWIVA HPPAIEPDLYYQIMEQKWKPFDWPDFRLPMRVSCRFRTMEAL NKANTSFALDFFKHECQEDDSENILFSPFSISSALATVYLGAK GNTADQMAKVLHFNEAEGARNVTTTIRMQVYSRTDQQRLN RRACFQKTEIGKSGNIHAGFKGLNLEINQPTKNYLLNSVNQLY GEKSLPFSKEYLQLAKKYYSAEPQSVDFVGTANEIRREINSRV EHQTEGKIKNLLPPGSIDSLTRLVLVNALYFKGNWATKFEAE DTRHRPFRINTHTTKQVPMMYLSDKFNWTYVESVQTDVLEL PYVNNDLSMFILLPRDITGLQKLINELTFEKLSAWTSPELMEK MKMEVYLPRFTVEKKYDMKSTLSKMGIEDAFTKVDNCGVT NVDEITIHVVPSKCLELKHIQINKELKCNKAVAMEQVSASIGN FTIDLFNKLNETSRDKNIFFSPWSVSSALALTSLAAKGNTARE MAEDPENEQAENIHSGFNELLTALNKPRNTYSLKSANRIYVE KNYPLLPTYIQLSKKYYKAEPHKVNFKTAPEQSRKEINNWVE KQTERKIKNFLSSDDVKNSTKLILVNAIYFKAEWEEKFQAGN TDMQPFRMSKNKSKLVKMMYMRHTFPVLIMEKLNFKMIELP YVKRELSMFILLPDDIKDSTTGLEQLERELTYEKLSEWADSKK MSVTLVDLHLPKFSMEDRYDLKDALRSMGMASAFNSNADFS GMTGERDLVISKVCHQSFVAVDEKGTEAAAATAVIAEAVPM ESLSASTNSFTLDLYKKLDETSKGQNIFFASWSIATALTMVHL GAKGDTATQVAKGPEYEETENIHSGFKELLSALNKPRNTYSM KSANRLFGDKTYPLLPTKTKPVQMMFLKDTFLIHHERTMKFK IIELPYMGNELSAFVLLPDDISDNTTGLELVERELTYEKLAEW SNSASMMKVKVELYLPKLKMEENYDLKSALSDMGIRSAFDP AQADFTRMSEKKDLFISKVIHKAFVEVNEEDRIVQLASGRLTG NTEAQIAKVLSLSKAEDAHNGYQSLLSEINNPDTKYILRTANR LYGEKTFEFLSSFIDSSQKFYHAGLEQTDFKNASEDSRKQING WVEEKTEGKIQKLLSEGIINSMTKLVLVNAIYFKGNWQEKFD KETTKEMPFKINKNETKPVQMMFRKGKYNMTYIGDLETTVL EIPYVDNELSMIILLPDSIQDESTGLEKLERELTYEKLMDWINP NMMDSTEVRVSLPRFKLEENYELKPTLSTMGMPDAFDLRTA DFSGISSGNELVLSEVVHKSFVEVNEEGTEAAAATAGIMLLRC AMIVANFTADHPFLFFIRHNKTNSILFCGRFCSP PREDICTED: 122 MASIGAASTEFCFDVFKELKTQHVKENIFYSPMAIISALSMVYI Ovalbumin- GARENTRAEIDKVVHFDKITGFGNAVESQCGPSVSVHSSLKD like LITQISKRSDNYSLSYASRIYAEETYPILPEYLQCVKEVYKGGL [Mesitornis ESISFQTAADQARENINAWVESQTNGMIKNILQPSSVNPQTEM unicolor] VLVNAIYLKGMWEKAFKDEDTQTMPFRVTQQESKPVQMMY QIGSFKVAVIASEKMKILELPYTSGQLSMLVLLPDDVSGLEQV ESAITAEKLMEWTSPSIMEERTMKVYLPRMKMVEKYNLTSV LMALGMTDLFTSVANLSGISSAQGLKMSQAIHEAFVEIYEAG SEAVGSTGVGMEITSVSEEFKADLSFLFLIRHNPTNSIIFFGRCI SP Ovalbumin, 123 MGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALAMVYL partial GARDNTRTQIDKISQFQALSDEHLVLCIQQLGEFFVCTNRERR [Anas EVTRYSEQTEDKTQDQNTGQIHKIVDTCMLRQDILTQITKPSD platyrhynchos] NFSLSFASRLYAEETYAILPEYLQCVKELYKGGLESISFQTAA DQARELINSWVESQTNGIIKNILQPSSVDSQTTMVLVNAIYFK GMWEKAFKDEDTQAMPFRMTEQESKPVQMMYQVGSFKVA MVTSEKMKILELPFASGMMSMFVLLPDEVSGLEQLESTISFEK LTEWTSSTMMEERRMKVYLPRMKMEEKYNLTSVFMALGMT DLFSSSANMSGISSTVSLKMSEAVHAACVEIFEAGRDVVGSAE AGMDVTSVSEEFRADHPFLFFIKHNPTNSILFFGRWMSP PREDICTED: 124 MGSIGAASAEFCLDIFKELKVQHVNENIIFSPMTIISALSLVYL Ovalbumin- GAKEDTRAQIEKVVPFDKIPGFGEIVESQCPKSASVHSSIQDIF like NQIIKRSDNYSLSLASRLYAEESYPIRPEYLQCVKELDKEGLET [Chaetura ISFQTAADQARQLINSWVESQTNGMIKNILQPSSVNSQTEMVL pelagica] VNAIYFRGLWQKAFKDEDTQAVPFRITEQESKPVQMMQQIGS FKVAEIASEKMKILELPYASGQLSMLVLLPDDVSGLEKLESSIT VEKLIEWTSSNLTEERNVKVYLPRLKIEEKYNLTSVLAALGIT DLFSSSANLSGISTAESLKLSRAVHESFVEIQEAGHEVEGPKEA GIEVTSALDEFRVDRPFLFVTKHNPTNSILFLGRCLSP PREDICTE 125 MGSISAASGEFCLDIFKELKVQHVNENIFYSPMVIVSALSLVY D: LGARENTRAQIDKVIPFDKITGSSEAVESQCGTPVGAHISLKD Ovalbumin- VFAQIAKRSDNYSLSFVNRLYAEETYPILPEYLQCVKELYKGG like LETISFQTAADQAREIINSWVESQTDGKIKNILQPSSVDPQTKM [Apaloderma VLVSAIYFKGLWEKSFKDEDTQAVPFRVTEQESKPVQMMYQI vittatum] GSFKVAAIAAEKIKILELPYASEQLSMLVLLPDDVSGLEQLEK KISYEKLTEWTSSSVMEEKKIKVYLPRMKIEEKYNLTSILMSL GITDLFSSSANLSGISSTKSLKMSEAVHEASVEIYEAGSEASGIT GDGMEATSVFGEFKVDHPFLFMIKHKPTNSILFFGRCISP Ovalbumin- 126 MGSIGPVSTEVCCDIFRELRSQSVQENVCYSPLLIISTLSMVYI like[Corvus GAKDNTKAQIEKAIHFDKIPGFGESTESQCGTSVSIHTSLKDIF cornix TQITKPSDNYSISIARRLYAEEKYPILPEYIQCVKELYKGGLESI cornix] SFQTAAEKSRELINSWVESQTNGTIKNILQPSSVSSQTDMVLV SAIYFKGLWEKAFKEEDTQTIPFRITEQESKPVQMMSQIGTFK VAEIPSEKCRILELPYASGRLSLWVLLPDDISGLEQLETAITFEN LKEWTSSSKMEERKIRVYLPRMKIEEKYNLTSVLKSLGITDLF SSSANLSGISSAESLKVSAAFHEASVEIYEAGSKGVGSSEAGV DGTSVSEEIRADHPFLFLIKHNPSDSILFFGRCFSP PREDICTED: 127 MGSIGAASTEFCFDVFKELKVQHVNENIIISPLSIISALSMVYLG Ovalbumin- AREDTRAQIDKVVHFDKITGFGEAIESQCPTSESVHASLKETFS like QLTKPSDNYSLAFASRLYAEETYPILPEYLQCVKELYKGGLET [Calypte INFQTAAEQARQVINSWVESQTDGMIKSLLQPSSVDPQTEMIL anna] VNAIYFRGLWERAFKDEDTQELPFRITEQESKPVQMMSQIGSF KVAVVASEKVKILELPYASGQLSMLVLLPDDVSGLEQLESSIT VEKLIEWISSNTKEERNIKVYLPRMKIEEKYNLTSVLVALGITD LFSSSANLSGISSAESLKISEAVHEAFVEIQEAGSEVVGSPGPEV EVTSVSEEWKADRPFLFLIKHNPTNSILFFGRYISP PREDICTED: 128 MGSIGPVSTEVCCDIFRELRSQSVQENVCYSPLLIISTLSMVYI Ovalbumin GAKDNTKAQIEKAIHFDKIPGFGESTESQCGTSVSIHTSLKDIF [Corvus TQITKPSDNYSISIARRLYAEEKYPILQEYIQCVKELYKGGLESI brachyrhynchos] SFQTAAEKSRELINSWVESQTNGTIKNILQPSSVSSQTDMVLV SAIYFKGLWEKAFKEEDTQTIPFRITEQESKPVQMMSQIGTFK VAEIPSEKCRILELPYASGRLSLWVLLPDDISGLEQLETSITFEN LKEWTSSSKMEERKIRVYLPRMKIEEKYNLTSVLKSLGITDLF SSSANLSGISSAESLKVSAVFHEASVEIYEAGSKGVGSSEAGV DGTSVSEEIRADHPFLFLIKHNPSDSILFFGRCFSP Hypothetical 129 MLNLMHPKQFCCTMGSIGPVSTEVCCDIFRELRSQSVQENVC protein YSPLLIISTLSMVYIGAKDNTKAQIEKAIHFDKIPGFGESTESQC DUI87_082 GTSVSIHTSLKDIFTQITKPSDNYSISIASRLYAEEKYPILPEYIQ 70[Hirundo CVKELYKGGLESISFQTAAEKSRELINSWVESQTNGTIKNILQP rustica SSVSSQTDMVLVSAIYFKGLWEKAFKEEDTQTVPFRITEQESK rustica] PVQMMSQIGTFKVAEIPSEKCRILELPYASGRLSLWVLLPDDIS GLEQLETAITSENLKEWTSSSKMEERKIKVYLPRMKIEEKYNL TSVLKSLGITDLFSSSANLSGISSAESLKVSGAFHEAFVEIYEA GSKAVGSSGAGVEDTSVSEEIRADHPFLFFIKHNPSDSILFFGR CFSP Ostrich 130 EAEAGSIGTASAEFCFDVFKELKVHHVNENIFYSPLSIISALSM OVA VYLGARENTKTQMEKVIHFDKITGLGESMESQCGTGVSIHTA sequenceas LKDMLSEITKPSDNYSLSLASRLYAEQTYAILPEYLQCIKELY secreted KESLETVSFQTAADQARELINSWIESQTNGVIKNFLQPGSVDS frompichia QTELVLVNAIYFKGMWEKAFKDEDTQEVPFRITEQESRPVQM MYQAGSFKVATVAAEKIKILELPYASGELSMLVLLPDDISGLE QLETTISFEKLTEWTSSNMMEDRNMKVYLPRMKIEEKYNLTS VLIALGMTDLFSPAANLSGISAAESLKMSEAIHAAYVEIYEAD SEIVSSAGVQVEVTSDSEEFRVDHPFLFLIKHNPTNSVLFFGRC ISP Ostrich 131 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDL construct EGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAE (secretion AGSIGTASAEFCFDVFKELKVHHVNENIFYSPLSIISALSMVYL signal+ GARENTKTQMEKVIHFDKITGLGESMESQCGTGVSIHTALKD mature MLSEITKPSDNYSLSLASRLYAEQTYAILPEYLQCIKELYKESL protein) ETVSFQTAADQARELINSWIESQTNGVIKNFLQPGSVDSQTEL VLVNAIYFKGMWEKAFKDEDTQEVPFRITEQESRPVQMMYQ AGSFKVATVAAEKIKILELPYASGELSMLVLLPDDISGLEQLE TTISFEKLTEWTSSNMMEDRNMKVYLPRMKIEEKYNLTSVLI ALGMTDLFSPAANLSGISAAESLKMSEAIHAAYVEIYEADSEI VSSAGVQVEVTSDSEEFRVDHPFLFLIKHNPTNSVLFFGRCISP DuckOVA 132 EAEAGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALAM sequenceas VYLGARDNTRTQIDKVVHFDKLPGFGESMEAQCGTSVSVHSS secreted LRDILTQITKPSDNFSLSFASRLYAEETYAILPEYLQCVKELYK frompichia GGLESISFQTAADQARELINSWVESQINGIIKNILQPSSVDSQT TMVLVNAIYFKGMWEKAFKDEDTQAMPFRMTEQESKPVQM MYQVGSFKVAMVTSEKMKILELPFASGMMSMFVLLPDEVSG LEQLESTISFEKLTEWTSSTMMEERRMKVYLPRMKMEEKYN LTSVFMALGMTDLFSSSANMSGISSTVSLKMSEAVHAACVEIF EAGRDVVGSAEAGMDVTSVSEEFRADHPFLFFIKHNPTNSILF FGRWMSP Duck 133 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDL construct EGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAE (secretion AGSIGAASTEFCFDVFRELRVQHVNENIFYSPFSIISALAMVYL signal+ GARDNTRTQIDKVVHFDKLPGFGESMEAQCGTSVSVHSSLRD mature ILTQITKPSDNFSLSFASRLYAEETYAILPEYLQCVKELYKGGL protein) ESISFQTAADQARELINSWVESQTNGIIKNILQPSSVDSQTTMV LVNAIYFKGMWEKAFKDEDTQAMPFRMTEQESKPVQMMYQ VGSFKVAMVTSEKMKILELPFASGMMSMFVLLPDEVSGLEQL ESTISFEKLTEWTSSTMMEERRMKVYLPRMKMEEKYNLTSVF MALGMTDLFSSSANMSGISSTVSLKMSEAVHAACVEIFEAGR DVVGSAEAGMDVTSVSEEFRADHPFLFFIKHNPTNSILFFGRW MSP

(305) Expression of rOVA in a host cell, for instance a Pichia species, a Saccharomyces species, a Trichoderma species, a Pseudomonas species may lead to an 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 sequences. For instance, expressing an OVA sequence in a Pichia species, such as Komagataella phaffii and Komagataella pastoris may lead to addition of one or more amino acids at the N-terminus or C-terminus. In some cases, four amino acids EAEA (SEQ ID NO: 53) is added to the N-terminus of the OVA sequence upon expression in a host cell as shown in SEQ ID NO:1. For example, chicken rOVA may be provided encoding SEQ ID NO: 60, and following expression and secretion, rOVA has the amino acid sequence of SEQ ID NO:61.

(306) An rOVA can be a non-naturally occurring variant of an OVA. Such variant can comprise one or more amino acid insertions, deletions, or substitutions relative to a native OVA sequence.

(307) Such a variant can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 60-133. The term sequence identity as used herein in the context of amino acid sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the preferable alignment algorithm. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

(308) Depending on the host organism used to express the rOVA, the rOVA can have a glycosylation, acetylation, or phosphorylation pattern different from wildtype OVA. For example, the rOVA herein may or may not be glycosylated, acetylated, or phosphorylated. An rOVA may have an avian, non-avian, microbial, non-microbial, mammalian, or non-mammalian glycosylation, acetylation, or phosphorylation pattern.

(309) In some cases, rOVA may be deglycosylated (e.g., chemically, enzymatically, Endo-H, PNGase F, O-Glycosidase, Neuraminidase, ?1-4 Galactosidase, ?-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 may produce a protein that is more uniform or is capable of producing a composition with less variation.

(310) An rOVA is recombinantly expressed in a host cell. As used herein, a host or host cell denotes here any protein production host selected or genetically modified to produce a desired product. Exemplary hosts include fungi, such as filamentous fungi, as well as bacteria, yeast, plant, insect, and mammalian cells. A host cell may be 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., Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium (Talaromyces) emersonii, Penicillium funiculo sum, 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. A host cell can be an organism that is approved as generally regarded as safe by the U.S. Food and Drug Administration.

(311) An rOVA protein can be recombinantly expressed in yeast, filamentous fungi or a bacterium. In some embodiments, rOVA protein is recombinantly expressed in a Pichia species (Komagataella phaffii and Komagataella pastoris), a Saccharomyces species, a Trichoderma species, a Pseudomonas species or an E. coli species.

(312) Expression of an rOVA can be provided by an expression vector, a plasmid, a nucleic acid integrated into the host genome or other means. For example, a 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.

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

(314) To aide in the amplification of the vector prior to transformation into the host microorganism, a replication origin (e) may be contained in the vector (such as PUC_ORIC and PUC (DNA2.0)). To aide in the selection of microorganism stably transformed with the expression vector, the vector may also include a selection marker (f) such as URA3 gene and Zeocin resistance gene (ZeoR). The expression vector may also contain a restriction enzyme site (g) that allows for linearization of the expression vector prior to transformation into the host microorganism to facilitate the expression vectors stable integration into the host genome. In some embodiments the expression vector may contain any subset of the elements (b), (e), (f), and (g), including none of elements (b), (e), (f), and (g). Other expression elements and vector element known to one of skill in the art can be used in combination or substituted for the elements described herein.

(315) Exemplary promoter elements (a) may include, but are not limited to, a constitutive promoter, inducible promoter, and hybrid promoter. Promoters include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, ?-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular 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, ?-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUTi), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, ?-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxin 8 (PEX8), phosphoglycerate kinase (PGK, PGK1), pho1, PHO5, PH089, phosphatidylinositol synthase (PIS1), PYK1, pyruvate kinase (pki1), RPS7, sorbitol dehydrogenase (SDH), 3-phosphoserine aminotransferase (SER1), SSA4, SV40, TEF, translation elongation factor 1 alpha (TEF1), THIl1, homoserine kinase (THR1), tpi, TPS1, triose phosphate isomerase (TPI1), XRP2, YPT1, and any combination thereof.

(316) A signal peptide (b), also known as a signal sequence, targeting signal, localization signal, localization sequence, signal peptide, transit peptide, leader sequence, or leader peptide, may support secretion of a protein or polynucleotide. Extracellular secretion of a recombinant or heterologously expressed protein from a host cell may facilitate protein purification. A signal peptide may be derived from a precursor (e.g., prepropeptide, preprotein) of a protein. Signal peptides can be derived from a precursor of a protein other than the signal peptides in native OVA. An example of secretion protein is a S. cerevisiae alpha factor pre pro sequence shown bolded and underlined in SEQ ID NO: 60.

(317) Any nucleic acid sequence that encodes OVA can be used as (c). Preferably such sequence is codon optimized for the host cell.

(318) Exemplary transcriptional terminator elements include, but are not limited to, acu-5, adh1+, alcohol dehydrogenase (ADH1, ADH2, ADH4), AHSB4m, AINV, alcA, ?-amylase, alternative oxidase (AOD), alcohol oxidase I (AOX1), alcohol oxidase 2 (AOX2), AXDH, B2, CaMV, cellobiohydrolase I (cbh1), ccg-1, cDNA1, cellular 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, ?-glucoamylase (glaA), glyceraldehyde-3-phosphate dehydrogenase (gpdA, GAP, GAPDH), phosphoglycerate mutase (GPM1), glycerol kinase (GUTi), HSP82, invl+, isocitrate lyase (ICL1), acetohydroxy acid isomeroreductase (ILV5), KAR2, KEX2, ?-galactosidase (lac4), LEU2, melO, MET3, methanol oxidase (MOX), nmt1, NSP, pcbC, PET9, peroxin 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.

(319) Exemplary selectable markers (f) may include, but are not limited to: an antibiotic resistance gene (e.g. zeocin, ampicillin, blasticidin, kanamycin, nourseothricin, chloroamphenicol, tetracycline, triclosan, ganciclovir, and any combination thereof), an auxotrophic marker (e.g. ade1, arg4, his4, ura3, met2, and any combination thereof).

(320) In one example, a vector for expression in Pichia sp. can include an AOX1 promoter operably linked to a signal peptide (alpha mating factor) that is 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.

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

(322) A recombinant protein described herein may be secreted from the one or more host cells. In some embodiments, 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 protein from cells, liquid and solid media components and other cellular products and byproducts. In some embodiments, rOVA is produced in a Pichia Sp. and secreted from the host cells into the culture media. The secreted rOVA is then separated from other media components for further use.

(323) The present disclosure contemplates modifying glycosylation of the recombinant OVA to alter or enhance one or more functional characteristics of the protein and/or its production. In some embodiments, the change in rOVA glycosylation can be due to the host cell glycosylating the rOVA. In some embodiments, rOVA has a glycosylation pattern that is not identical to a native ovalbumin (nOVA), such as a nOVA from chicken egg. In some embodiments, rOVA is treated with a deglycosylating enzyme before it is used as an ingredient in an rOVA composition, or when rOVA is present in a composition. In some embodiments, the glycosylation of rOVA is modified or removed by expressing one or more enzymes in a host cell and exposing rOVA to the one or more enzymes. In some embodiments, rOVA and the one or more enzymes for modification or removal of glycosylation are co-expressed in the same host cell.

(324) Native ovalbumin (nOVA), such as isolated from a chicken or another avian egg, has a highly complex branched form of glycosylation. The glycosylation pattern comprises N-linked glycan structures such as N-acetylglucosamine units, galactose and N-linked mannose units. See, e.g., FIG. 1A. In some cases, the rOVA for use in a herein disclosed consumable composition and produced using the methods described herein has a glycosylation pattern which is different from the glycosylation pattern of nOVA. For example, when rOVA is produced in a Pichia sp., the protein may be glycosylated differently from the nOVA and lack galactose units in the N-linked glycosylation. FIG. 1B illustrates the glycosylation patterns of rOVA produced by P. pastoris, showing a complex branched glycosylation pattern. In some embodiments of the compositions and methods disclosed herein, rOVA is treated such that the glycosylation pattern is modified from that of nOVA and also modified as compared to rOVA produced by a Pichia sp. without such treatment. In some cases, the rOVA lacks glycosylation.

(325) The molecular weight or rOVA may be different as compared to nOVA. The molecular weight of the protein may be less than the molecular weight of nOVA or less than rOVA produced by the host cell where the glycosylation of rOVA is not modified. In embodiments, the molecular weight of an rOVA may be between 40 kDa and 55 kDa. In some cases, an rOVA with modified glycosylation has a different molecular weight, such as compared to a native OVA (as produced by an avian host species) or as compared to a host cell that glycosylates the rOVA, such as where the rOVA includes N-linked mannosylation. In some cases, the molecular weight of rOVA is greater than the molecular weight of the rOVA that is completely devoid of post-translational modifications. or an rOVA that lacks all forms of N-linked glycosylation.

5. Definitions

(326) The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

(327) 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.

(328) The terms including, includes, having, has, with, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term comprising.

(329) Ranges can be expressed herein as from about or approximately one particular value, and/or to about or approximately another particular value. When such a range is expressed, another case includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another case. It will be further understood that the endpoints of each of the ranges are 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 that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 would include a range from 8.5 to 11.5. The term about or approximately also accounts for typical error or imprecision in measurement of values.

(330) Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

Definitions

(331) The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

(332) As used herein, unless otherwise indicated, the terms a, an and the are intended to include the plural forms as well as the single forms, unless the context clearly indicates otherwise.

(333) The terms comprise, comprising, contain, containing, including, includes, having, has, with, or variants thereof as used in either the present disclosure and/or in the claims, are intended to be inclusive in a manner similar to the term comprising.

(334) The term about or approximately means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, about can mean 10% greater than or less than the stated value. In another example, about can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term about should be assumed to mean an acceptable error range for the particular value.

(335) The term substantially is meant to be a significant extent, for the most part; or essentially. In other words, the term substantially may mean nearly exact to the desired attribute or slightly different from the exact attribute. Substantially may be indistinguishable from the desired attribute. Substantially may be distinguishable from the desired attribute but the difference is unimportant or negligible.

(336) Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

EXAMPLES

(337) The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Expression Constructs, Transformation, Protein Purification and Processing

(338) Two expression constructs were created for expression of OVD (SEQ ID NO: 1) in Pichia pastoris. The first construct included the Alcohol oxidase 1 (AOX1) promoter. An OVD coding sequenced was fused in-frame with the alpha mating factor signal sequence downstream of the promoter sequence. A transcriptional terminator from the AOX1 gene was placed downstream of the OVD sequence. The expression construct was placed into a Kpas-URA 3 vector.

(339) A second expression construct was created containing the methanol-inducible DAS1 promoter (ATCC No. 28485) upstream of the alpha mating factor signal sequence fused in frame with a nucleic acid sequence encoding the same OVD protein sequence as in the first expression construct. A transcriptional terminator from the AOX1 gene was placed downstream of the OVD sequence.

(340) In both expression constructs, the OVD sequence was that of chicken (Gallus gallus) having amino acid sequence of SEQ ID NO: 1.

(341) Both expression constructs were transformed into Pichia pastoris. Successful integration of the two constructs were confirmed by genomic sequencing.

(342) Fermentation: Recombinant OVD (rOVD) from each expression construct was produced in a bioreactor at ambient conditions. A seed train for the fermentation process began with the inoculation of shake flasks with liquid growth broth. The inoculated shake flasks were kept in a shaker after which the grown Pichia pastoris was transferred to a production scale reactor.

(343) The culture was grown at 30? C., at a set pH and dissolved oxygen (DO). The culture was fed with a carbon source.

(344) Secreted rOVD was purified by separating cells from the liquid growth broth, performing multiple filtration steps, performing chromatography using and drying the final protein product to produce pure rOVD powder.

Example 2: Expression Construct, Transformation, Protein Purification and Processing

(345) Three expression constructs were created for expression of a mature form of OVD (SEQ ID NO: 1) in Pichia pastoris. The first construct included the AOX1 promoter. An OVD coding sequenced was fused in-frame with the alpha mating factor signal sequence downstream of the promoter sequence (SEQ ID NO: 39). A transcriptional terminator from the AOX1 gene was placed downstream of the OVD sequence. The host cells had eleven copies of OVD, ten of which were in the hybrid promoter system, with five driven by a shortened pAOX1. The eleventh copy was driven by a full-sized pAOX1 promoter.

(346) A second expression construct was created containing a nucleic acid encoding the P. pastoris transcription factor HAC1 under the control of a strong methanol-inducible promoter. A transcriptional terminator from the AOX1 gene was placed downstream of the HAC1 sequence.

(347) A third expression construct was created encoding a fusion protein. The construct comprises a nucleic acid that encodes the first 48 residues of Pichia OCH1 protein fused to a catalytically active version of the Streptomyces coelicoflavus EndoH (SEQ ID NO.: 52) and under a strong methanol-inducible promoter, pPEX11. A transcriptional terminator from the AOX1 gene was placed downstream of the EndoH-OCH1 fusion protein sequence.

(348) The P. pastoris strain was modified to remove cytoplasmic killer plasmids and then further modified to have a deletion in the AOX1 gene. This deletion generated a methanol-utilization slow (mutS) phenotype that reduces the strain's ability to consume methanol. This base strain was transformed with the three expression constructs.

(349) Linear cassettes of methanol-inducible promoter: ScPrePro (Saccharomyces pre-pro sequence)::ovomucoid::AOX1term; linear cassettes of methanol-inducible promoter::HAC1::AOX1term; and a linear cassette of methanol-inducible promoter::EndoH-OCH1::AOX1term were introduced into the base P. pastoris strain using standard electroporation methods. FIG. 1A illustrates the vector constructs used for the expression of rOVD.

(350) Fermentation: Recombinant OVD from each expression construct was produced in a bioreactor at ambient conditions. A seed train for the fermentation process began with the inoculation of shake flasks with liquid growth broth. The inoculated shake flasks were kept in a shaker after which the grown P. pastoris was transferred to a production-scale reactor.

(351) The culture was grown at 30? C., at a set pH and dissolved oxygen (DO). The culture was fed with a carbon source.

(352) To expand production, an rOVD P. pastoris seed strain is removed from cryo-storage and thawed to room temperature. Contents of the thawed seed vials are used to inoculate liquid seed culture media in baffled flasks which were grown at 30? C. in shaking incubators. These seed flasks are then transferred and grown in a series of larger and larger seed fermenters (number to vary depending on scale) containing a basal salt media, trace metals, and glucose. Temperature in the seed reactors are controlled at 30? C., pH at 5, and DO at 30%. pH is maintained by feeding ammonia hydroxide which also acts as a nitrogen source. Once sufficient cell mass is reached, the grown rOVD P. pastoris is inoculated in a production-scale reactor containing basal salt media, trace metals, and glucose. Like in the seed tanks, the culture is also controlled at 30? C., pH 5 and 30% DO throughout the process. pH is again maintained by feeding ammonia hydroxide. During the initial batch glucose phase, the culture is left to consume all glucose and subsequently-produced ethanol. Once the target cell density is achieved and glucose and ethanol concentrations are confirmed to be zero, the glucose fed-batch growth phase is initiated. In this phase, glucose is fed until the culture reaches a target cell density. Glucose is fed at a limiting rate to prevent ethanol from building up in the presence of non-zero glucose concentrations. In the final induction phase, the culture is co-fed glucose and methanol which induces it to produce rOVD. Glucose is fed at an amount to produce a desired growth rate, while methanol is fed to maintain the methanol concentration at 1% to ensure that expression is consistently induced. Regular samples are taken throughout the fermentation process for analyses of specific process parameters (e.g., cell density, glucose/methanol concentrations, product titer, and quality). After a designated amount of fermentation time, secreted rOVD is collected and transferred for downstream processing.

(353) The rOVD products were purified by separating cells from the liquid growth broth, performing multiple filtration steps, performing chromatography, and/or drying the final protein product to produce pure rOVD powder.

(354) Post-translation modification from the OCH1-EndoH fusion protein resulted in the removal of the alpha factor pre-pro sequence. N-terminal sequencing results showed imprecise cleavage of the N-terminal pro sequence by the Pichia host post-transcription machinery fusing an additional four amino acid residues (major) or 6 amino acid residues (minor) to the N-terminus of the produced rOVD (SEQ ID NO: 37) or (SEQ ID NO:38) in comparison to the amino acid sequence of mature OVD (SEQ ID NO:1).

(355) The molecular weight of rOVD from Pichia was compared against native chicken ovomucoid (nOVD) using SDS-PAGE. The rOVD showed a difference in migration. To ascertain whether the difference in gel migration was due to differential post-translational glycosylation, deglycosylated native ovomucoid was treated with PNGase F, an enzyme that specifically deglycosylates proteins (BioLabs 2020), and compared to the rOVD sample. The deglycosylated native ovomucoid (nOVD+PNGaseF) displayed the same band patterns and molecular weight as three rOVD samples tested (FIG. 1D). The difference in glycosylation is attributed to the action of the OCH1-EndoH in the Pichia strain, such that rOVD has only the core N-acetylglucosamine unit attached to the Asn residue instead of the complex branched glycosylation (that includes mannose) of nOVD from chicken egg white (FIG. 1B and FIG. 1C).

(356) Mass spectrometry analysis of rOVD expressed in Pichia without EndoH is shown to have eight different N-glycan structures (FIG. 1C). The structures include Man9 GlcNAc2, Man9 GlcNAc2 Hex, Man9 GlcNAc2Hex2, Man9 GlcNAc2Hex3, Man9 GlcNAc2Hex4, Man9 GlcNAc2 Hex5,v Man9 GlcNAc2Hex6, and Man9 GlcNAc2 Hex7. Table 2 below shows the percentage of N-linked glycans on the rOVD sample produced without endoglycosidase treatment.

(357) TABLE-US-00003 TABLE 2 N-linked glycans from sample detected by MALDI TOF/TOF MS. Permethylated Text description of mass (m/z).sup.1 structures Percentage 2396.2 Man.sub.9GlcNAc.sub.2 5.6 2600.3 Man.sub.9GlcNAc.sub.2Hex 25.1 2804.4 Man.sub.9GlcNAc.sub.2Hex.sub.2 31.6 3008.5 Man.sub.9GlcNAc.sub.2Hex.sub.3 18.2 3212.6 Man.sub.9GlcNAc.sub.2Hex.sub.4 6.0 3416.7 Man.sub.9GlcNAc.sub.2Hex.sub.5 7.2 3620.8 Man.sub.9GlcNAc.sub.2Hex.sub.6 3.8 3824.9 Man.sub.9GlcNAc.sub.2Hex.sub.7 2.6

Example 3: Solubility and Clarity Testing at Varying rOVD Concentrations

(358) Lyophilized rOVD (from Example 2) was blended into aqueous solution (distilled water) at different concentrations and pHs. Clarity and solubility of the rOVD solutions was then assessed visually (e.g., for turbidity, precipitate, viscosity, and color) as well as by measuring absorbance at 600 nm.

(359) FIG. 2 shows the absorbance at 600 nm of deionized water compared with the absorbance at 600 nm of a solution comprising rOVD in deionized water at a protein concentration of 4.23% w/v. The rOVD solution had a pH of 4.11. The deionized water had an absorbance of 0.037 (OD600). The solution with 4.23% w/v rOVD had an absorbance of 0.047, an increase of 27%. The photo in FIG. 2 of the rOVD solution reveals a clear and colorless solution with no precipitate and no apparent viscosity changes in appearance and visual flow of liquid.

Example 4: Solubility and Clarity Testing at Varying Temperatures

(360) The aqueous 30% rOVD (w/v) samples of Example 3, at pH 4.06 or pH 6.3 were incubated at room temperature and subjected to three heat treatments: pasteurization, hot fill, and retorting. The clarity and solubility of rOVD was then assessed visually (e.g., for turbidity, precipitate, viscosity, and color) and by measuring absorbance at 600 nm.

(361) Heat treatments on each sample were executed as follows:

(362) For pasteurization, the samples were heated to 72? C. for 1 minute and then placed in an ice bath for 10 minutes. Following the ice bath, the samples were placed at room temperature and then assessed for solubility and clarity.

(363) For hot fill, the samples were heated to 85? C. for 30 seconds and then placed at room temperature for assessment of solubility and clarity.

(364) For retorting, the samples were heated to 121? C. for 15 minutes at 19 psi and then kept at room temperature for assessment of solubility and clarity.

(365) FIG. 3 shows the results for pH, absorbance and clarity of an rOVD solution comprising 30% rOVD in deionized water. The rOVD was surprisingly soluble in deionized water at 30% (w/v based on protein amount) at either pH 4.06 or pH 6.3. The photos of the rOVD solutions at both pH 4.06 and 6.3 look clear, pale green, and viscous, though less so under the pre-processing condition, which was prior to a heat treatment. It can be concluded from FIG. 3 that rOVD can remain soluble in both acidic (pH ?4.0) and slightly acidic (pH ?6) solutions at a concentration of rOVD of 30% w/v. More specifically, the 30% rOVD solution at pH 4.06 had an OD600 of 0.101 after pasteurization and an OD600 of 0.104 after hot filling. At the less acidic pH of 6.3, the OD600 of the 30% rOVD solution after pasteurization was 0.089 and after hot filling was 0.094. As such, there appeared to be greater clarity and solubility of the rOVD at higher pH values.

(366) FIG. 4 shows the photos from the pH 4.06 experiments of FIG. 3. It can be concluded from FIG. 4 that rOVD can surprisingly remain in solution following heat application. 30% w/v.

Example 5: Solubility and Clarity Testing at Varying Temperatures and pH

(367) Lyophilized rOVD (from Example 2) was blended into aqueous solution (distilled water) at concentration of 9% (w/v). Sodium citrate buffer (0.1M) was used to adjust the pH of the solutions to pH's of 2.5, 4 or 6, as shown in Table 3 below:

(368) TABLE-US-00004 TABLE 3 Composition of the citrate buffer at pH 2.5, 4 or 6 Citric acid Sodium citrate DI water (mL) (mL) (mL) pH rOVD 49.2 0.8 50 2.5 9% w/v 37 13 50 4 9% w/v 6 44 50 6 9% w/v

(369) Following pH adjustment, separate aqueous rOVD samples at each pH were incubated at room temperature and subjected to three types of heat treatments: pasteurization, hot fill and retorting (as described below). The clarity and solubility of rOVD was then assessed visually (e.g., for turbidity, precipitate, viscosity, and color) and by measuring absorbance at 600 nm.

(370) The heat treatments on each sample were executed as follows:

(371) For pasteurization, the samples were heated to 72? C. for 1 minute and then placed in an ice bath for 10 minutes. Following the ice bath, the samples were placed at room temperature and then assessed for solubility and clarity.

(372) For hot fill, the samples were heated to 85? C. for 30 seconds and then placed at room temperature for assessment of solubility and clarity.

(373) For retorting, the samples were heated to 121? C. for 15 minutes at 19 psi and then kept at room temperature for assessment of solubility and clarity.

(374) The results of visual inspection and OD600 measurements of the samples are provided in FIG. 5A and FIG. 5B.

(375) Pictures of the samples are shown in FIG. 5A. Effect of different heating treatments on absorbance (600 nm) of rOVD solution and buffer.

(376) The addition of rOVD was found to increase the absorbance of the buffer solution. The absorbance of the rOVD solution remained the same following pasteurization and hot fill (no significant difference between pH 2.5 and pH 4). The absorbance was reduced following retorting. It was surprising that at different pH's, the rOVD solution remained clear even after the heating treatments of pasteurization and hot fill. An exception was that the rOVD solution coagulated at retorting conditions at pH 4 or pH 6. These results indicate that rOVD of the present disclosure remains soluble in solution at different acidic pHs, before and after application of heat.

Example 6: Solubility and Clarity of rOVD in Commercially-Available Beverages

(377) Based on carbonation levels published in literature (Table 4, below), San Pellegrino? was selected to represent a low carbonated beverage whereas Diet Coke? was selected as a beverage with higher carbonation level. Gatorade? and Red Bull? represented the (non-carbonated) Energy Drink category. Pedialyte? was selected to study effect of electrolytes in a beverage on rOVD solubility.

(378) TABLE-US-00005 TABLE 4 Carbonation levels for various commercially-available beverages Typical carbonation levels Volume g/L Lightly Sparkling 2 4 Fruit juice carbonate 2.5 5 Lemonade 3.0-3.5 6-7 Cola 4 8 Mixer 4.5-5.0 9-10

(379) Lyophilized rOVD was blended into various drink solutions at a range of concentrations of 30-50% (% expressed as weight protein/volume). Surprisingly rOVD of the present disclosure was soluble at 30% w/v in Pedialyte?, San Pellegrino?, Diet Coke?, and Gatorade?. Red Bull?, was solubility at 26% w/v protein. At higher concentrations (e.g., >30%) rOVD exhibited solubility at some concentrations but was accompanied by a decrease in clarity and an increase in viscosity. At even higher concentrations (e.g., approaching 50% w/v), rOVD was no longer soluble and in some samples did not wet when placed into the drink solution. Results are shown in the tables below for concentrations 30% and higher (26% for Redbull?). A marked color change was seen with added rOVD for beverages that are colorless, whereas for colored beverages (e.g., Diet Coke?), little to no color change was observed with rOVD addition.

(380) TABLE-US-00006 TABLE 5 Solubility study of rOVD in San Pellegrino? Protein pH of San concentration pH of San Pellegrino? (%) Visual inspection Pellegrino? with rOVD 50 Protein powder was not completely wetted. Did not form a solution 40 Protein powder was completely wetted. Formed a thick, pale green syrup-like mixture 35 Viscous suspension, pale brown syrup-like 30 Clear solution, pale green, 6.46 5.03 viscous

(381) TABLE-US-00007 TABLE 6 Solubility study of rOVD in Diet Coke? pH of pH of Diet Protein Diet Coke? concentration Coke? with (%) Visual inspection base rOVD 50 Protein powder was not completely wetted. Did not form a solution 40 Protein powder was completely wetted. Formed a thick, pale brown syrup-like mixture 35 Viscous suspension, pale brown syrup-like 30 Clear solution, brown, viscous 3.0 3.55

(382) TABLE-US-00008 TABLE 7 Solubility study of rOVD in Gatorade? (Thirst Quencher lemon-lime) Protein pH of concentration pH of Gatorade? (%) Visual inspection Gatorade? with rOVD 50 Protein powder was not wetted much. Did not form a solution. 40 Protein powder was not completely wetted. Formed a thick, pale yellow syrup-like mixture 35 Viscous suspension, pale yellow syrup-like 30 Clear solution, 2.78 3.7 pale yellow/green, viscous

(383) TABLE-US-00009 TABLE 8 Solubility study of rOVD in Red Bull? pH of Red Protein pH of Bull? concentration Red with (%) Visual inspection Bull? rOVD 50 Protein powder was not wetted much. Did not form a solution. 40 Protein powder was not completely wetted. Formed a thick, off white syrup-like mixture. 35 Very viscous suspension, off white syrup-like. 30 Very viscous turbid pale green solution. 26 Clear solution, pale yellow/green, 3.26 3.63 viscous.

(384) TABLE-US-00010 TABLE 9 Solubility study of rOVD in Pedialyte? pH of Protein Pedialyte? concentration pH of with (%) Visual inspection Pedialyte? rOVD 50 Protein powder was not completely wetted. Did not form a solution. 40 Protein powder was completely wetted. Formed a thick, off white syrup-like mixture. 35 Viscous suspension, pale brown syrup-like. 30 Clear solution, 5.51 5.75 pale green, viscous.

(385) Pictures of the starting drinks (no rOVD) and the 30% rOVD solutions (26% for RedBull?) are shown in FIG. 6A. Absorbance results for rOVD solutions are shown in the graphs of FIG. 6B. The rOVD solutions at 30% w/v (26% w/v for RedBull?) were assessed using absorbance at 600 nm. Change in pH upon rOVD addition was dependent on the beverage composition and initial base pH.

Example 7: Production of Recombinant OVL Protein in Pichia

(386) A recombinant lysozyme (rOVL) strain was made by transforming the Pichia species Komagataella phaffii with an expression cassette containing the OVL of SEQ ID NO: 45 expressed under the control of a methanol-inducible promoter. The OVL coding sequence encoded the mature OVL protein fused to the coding sequence for the alpha factor pre-pro secretion signal from Saccharomyces cerevisiae. The rOVL strain secreted rOVL when grown in media containing methanol. The broth containing the rOVL recombinant protein was centrifuged to remove cells and the resulting supernatant was processed similar to that of rOVD, as described above.

Example 8: OVD and OVL Combinations

(387) In this example, solutions were made containing 2.5% (w/v) rOVL and nOVD at 9% (w/v). The resulting protein blend contained 21.7% rOVL and 78.3% and.

(388) The rOVL+OVD blend was then heat treated under the following conditions:

(389) Pasteurization: 72? C. for 1 minute, followed by 10 minutes in an ice bath.

(390) Hot Fill: 85? C. for 30 seconds.

(391) Retorting: 121? C. for 15 minutes at 19 psi.

(392) A control OVD sample kept at room temperature was used to mimic aseptic processing conditions. Sodium citrate buffer (0.1M) was used to adjust the pH of the test solutions as described in Table 10.

(393) TABLE-US-00011 TABLE 10 Composition of the citrate buffer at pH 2.5, 4 and 6 Citric acid Sodium citrate DI water (mL) (mL) (mL) pH 49.2 0.8 50 2.5 37 13 50 4 6 44 50 6

(394) As shown in FIG. 7 to FIG. 10 and Table 11, when the rOVL+OVD blend was heat treated by pasteurization, hot fill or retorting. The clarity/solubility of the rOVL+OVD blend, as measured by absorbance at 600 nm, remained unaffected at pH 2.5 compared to OVD control samples left at room temperature. At pH 4, the rOVL+OVD blend retained its clarity/solubility when pasteurized or hot filled. Retorting conditions produced turbidity, as measured by increased optical density (Table 11). Heat treatment at pH 6 resulted in loss of clarity for all samples.

(395) TABLE-US-00012 TABLE 11 Absorbance of samples containing rOVL and native OVD at 600 nm Pasteur- Control ization Hot Fill Retorting pH 2.5 rOVL + OVD .sup.0.043 AB 0.039 B 0.038 B .sup.0.045 AB OVD .sup.0.044 AB* .sup.0.042 AB 0.050 A .sup.0.046 AB rOVL 0.038 B 0.038 B 0.037 B 0.037 B pH 4 rOVL + OVD 0.044 C 0.065 C 0.056 C 0.154 A OVD 0.055 C 0.166 A 0.126 B 0.315 D rOVL 0.049 C 0.042 C 0.042 C 0.040 C pH 6 rOVL + OVD 0.063 E 0.610 B 0.384 C 0.898 B OVD 0.041 E 0.202 D 0.228 D 0.525 B rOVL 0.039 E 1.425 A 0.588 B white precipitate F *samples within each sub-table sharing the same letters are statistically similar (p > 0.05)

(396) As shown in FIG. 11 to FIG. 13 and Table 12, native OVL (nOVL) samples had a similar effect on OVD as seen with the recombinant OVL (rOVL). At pH 2.5, the clarity/solubility of nOVL+OVD solutions were maintained when heat treated at all three conditions (pasteurization, hot fill or retorting). The nOVL+OVD solutions maintained their clarity at pH 4, with turbidity development only under retort conditions. pH 6 was not suitable for maintaining clarity after heat treatment.

(397) TABLE-US-00013 TABLE 12 Absorbance of samples containing commercial native OVL (nOVL) and native OVD (nOVD) at 600 nm Pasteur- Control ization Hot Fill Retorting pH 2.5 nOVL + OVD .sup.0.043 AB 0.046 AB .sup.0.043 AB 0.051 B OVD .sup.0.044 AB* 0.042 AB 0.050 B .sup.0.046 AB nOVL 0.037 A 0.038 A.sup. 0.038 A 0.036 A pH 4 nOVL + OVD .sup.0.052 CD 0.073 C.sup. 0.075 C 0.174 A OVD .sup.0.055 CD* 0.166 A.sup. 0.126 B 0.315 E.sup. nOVL 0.037 D 0.042 D.sup. 0.042 D 0.044 D pH 6 nOVL + OVD 0.054 A 0.445 F.sup. 0.322 E 0.954 H OVD 0.041 A* 0.042 CD 0.228 D 0.525 G nOVL 0.041 A 0.092 B.sup. 0.178 C Coagulated *samples within each sub-table sharing the same letters are statistically similar (p > 0.05)

(398) The addition of rOVL to OVD in a sample at room temperature or heat processed increased the protein content of the sample without affecting the clarity or solubility of the sample. Thus, the addition of rOVL to OVD to a beverage increases the protein content of the beverage without affecting clarity or solubility, or sensory quality (appearance, smell, flavor and mouthfeel) either at room temperature or after heat processing.

(399) Samples were made containing recombinant OVD of the present disclosure (rOVD) at 9% (w/v), and 2.5% (w/v) rOVL. The resulting protein blend contained 78.3% rOVD and 21.7% rOVL. FIG. 14 compares solutions at room temperature and after different heat treatments at pH 2.5, 4, 6: rOVL+rOVD with rOVD control.

(400) TABLE-US-00014 TABLE 13 Absorbance of samples containing rOVL and rOVD at 600 nm Pasteur- Room Temp ization Hot Fill Retorting pH 2.5 rOVD + rOVL .sup.0.063 AB* 0.066 A 0.061 B 0.061 B rOVD 0.062 B 0.061 B 0.062 B 0.056 C pH 4 rOVD + rOVL 0.065 B* 0.109 A 0.066 B White coagulate rOVD 0.062 B 0.060 B 0.061 B White coagulate pH 6 rOVD + rOVL 0.066 C* 0.256 A 0.091 B White coagulate rOVD 0.072 C 0.056 D 0.058 D White coagulate *samples within each sub-table sharing the same letters are statistically similar (p > 0.05)

Example 9: Comparison to Whey Protein Solutions

(401) To compare results from rOVD to an alternate protein (whey), rOVD or whey proteins +were solubilized in water at a concentration of 9% (w/v). Four commercially-available whey protein isolates (WP1, WP2, WP3 and WP4) were compared to rOVD of the present disclosure. The pH was measured by Hanna Lab pH probe for each sample and the absorbance was measured by SpectroMax at 600 nm wavelength. The results are provided in Table 14. The appearance was assessed by visual inspection; the odor was assessed by sniffing test; and the flavor was assessed by taste using a panel of 3 trained personnel.

(402) TABLE-US-00015 TABLE 14 Solution characteristics of whey protein solutions (WPI) compared to rOVD solutions. WPI 1 WPI 2 WPI 3 WPI 4 rOVD pH in 8.45% 3.15 6.53 3.92 6.13 5.05 solution Absorbance at 0.039 ? 0.001 0.423 ? 0.123 0.344 ? 0.038 0.792 ? 0.016 0.0002 ? 0.000 600 nm Appearance clear, yellow cloudy, yellow slightly cloudy, white, turbid clear, yellow colorless Odor lactic acid negative dairy cow/goat shed milky odor no odor notes odor Flavor salty, lactic plastic taste, slightly acidic, neutral taste, no slight protein acid notes unpleasant odor negative dairy acidity, milk like taste & taste flavor/odor flavor

(403) Whey protein isolates (WPI 1 and WPI 3) were at a concentration of 9 g per 100 ml distilled water, adjusted to pH 2, 4, or 6. Comparative results between whey protein solutions (WPI 1 and WPI 3) and rOVD solutions at pH 2, 4 and 6 are shown in FIG. 15A and FIG. 15B. The rOVD solutions show substantially higher solution clarity as compared to whey protein solutions at the same concentrations.

Example 10: Comparison of Clarity of Various Protein Water Solutions

(404) In this example, the solubility of recombinant ovomucoid (rOVD) protein of the present disclosure was compared to the other proteins.

(405) Appropriate amounts of acidic whey protein isolates (WP1 with 90% w/w protein and WP2 with 92.7% w/w protein), nOVD85% protein content, rOVD85.6% protein content, pea protein90% protein content; and soy protein90% protein content, were blended (using vortex) with water to form 5% protein solutions.

(406) TABLE-US-00016 TABLE 15 List of Ingredients and their proportions. Pea WP1 WP2 protein Soy (neutral) (acidic) nOVD rOVD (acidic) protein (5%) (5%) (5%) (5%) (5%) (5%) Ingredient % % % % % % Protein powder 5.6 5.4 5.9 5.8 5.6 5.6 DI water 94.4 94.6 94.1 94.2 94.4 94.4 Total 100 100 100 100 100 100

(407) FIG. 16 shows examples of the various protein solutions.

(408) 100 ?l of each protein solution was aliquoted into a flat bottom, clear 96 well plate in three replicates (as shown in Table 15). The absorbance of each sample was measured at 600 nm with a plate adapter on Spectramax. Results are provided in Table 16.

(409) TABLE-US-00017 TABLE 16 Absorbance results of various protein solutions. Whey Whey protein protein pea isolated isolated protein soy (neutral) (acidic) nOVD rOVD (acidic) protein Water OD600 0.1455 0.0527 0.0432 0.0456 0.9860 0.8821 0.0355

Example 11: Comparison of Suspension Stability of Protein Fortified Solutions

(410) In this example, the feasibility of fortifying orange juice (with added calcium and vitamin D) with rOVD was determined.

(411) Orange juice (without pulp; with 350 mg Calcium, and 2.5 mcg vitamin D per serving size of 8 fluid oz) was protein fortified using nOVD, whey protein, or rOVD (86% protein content). The samples were treated as follows. The protein of interest was added at various amounts to 10 g orange juice and mixed until completely dissolved to produce a fortified orange juice. The pH of the original orange juice with no protein fortification (as a control sample) was measured and considered as a target pH. The pH of fortified orange juice samples was adjusted using 1M citric acid and/or baking soda to become close to the target pH. The protein solubility and/or precipitation was visually observed in all samples before a heat treatment. A heat treatment of 70? C. for 1 min was applied to sufficiently reduce the microbial load in orange juice. Then, the samples were immediately cooled to 4? C. for 10 minutes.

(412) The physical and suspension stability of the samples were evaluated immediately after heating process (FIG. 17A) and after 48 hours storage at 4? C. (FIG. 17B).

(413) The suspension stability of orange juice fortified with 15% of nOVD or 15% of rOVD were found to be similar to the control, which included no protein fortification. After 48 hours, orange juice fortified with 15% of whey protein had slightly formed a gel, thus a separation was not observed in this sample. rOVD at a high concentration (30%) did not precipitate and was completely soluble in the orange juice, even in the presence of 0.25 mcg vitamin D and 35 mg calcium.

(414) rOVD was also found to be heat stable and did not form a gel during pasteurization. In terms of appearance, no significant difference was observed between the control and the orange juice fortified with 15% of nOVD or with rOVD at two levels: 15% or 20%.

Example 12: Comparison of Suspension Stability of Protein-Fortified Jelly

(415) In this example, the feasibility of fortifying jelly with rOVD was evaluated.

(416) Jello? jelly was used for protein fortification using nOVD (80% protein), rOVD (86-93% protein), unflavored whey isolate proteins (87.5-92.7% protein), unflavored gelatin (92% protein). The samples were prepared as follows:

(417) Control jelly method: hot water was added to the jelly mix power and stirred for two minutes until completely dissolved. Cold water was then added to fill 2 cm of 1 oz cups, capped and then refrigerated.

(418) Fortified jelly method: Hot water was added to the jelly mix power and stirred for two minutes until completely dissolved. Cold water was gradually added to the protein powder and slowly stirred to dissolve. The dissolved jelly solution was transferred in the protein mixture and mixed completely. 2 cm of 1 oz cups were filled, capped and then refrigerated.

(419) Protein jelly formulations: List of ingredients and their proportions used in the control and other experimental jelly samples, with specific protein of interest, are presented below in Table 17.

(420) TABLE-US-00018 TABLE 17 List of Ingredients Control Whey 20% nOVD 20% rOVD 20% Ingredient % % % % Jello 15.23 11.7 11.4 11.7 Cold water 42.38 32.7 31.8 32.5 Hot water 42.38 32.7 31.8 32.5 Protein 0 22.9 25.0 23.3 Total g 100 100 100 100

(421) (* amounts of ingredient adjusted based on % protein w/w content) The textures of the jelly samples were measured using Brookfield CT3 Texture Analyzer (Table 18). From each jelly sample three readings were taken. Jellies were centrally located under the test probe and compressed to a distance of 5 mm following the test settings below. Adhesiveness (also known as stickiness) measured the energy required to separate the attractive forces between the surface of the jelly and the surface of the probe (which approximates the stickiness on a tongue, teeth, and/or palate). Hardness is the force required to compress the jelly to attain a given deformation.

(422) TABLE-US-00019 TABLE 18 Texture Analyzer Test Settings Test Compression test probe TA 5 Textural properties Hardness (g) and adhesiveness (mj) Speed 1 mm/sec Distance 5 mm Sample size cylinder shape; H: 20 mm D: 36 mm Trigger load 4.5 g

(423) In terms of adhesiveness, no statistically-significant difference between jelly fortified with 20% nOVD and the control was observed. On the other hand, the adhesiveness values for jelly with 20% of whey and rOVD proteins were significantly lower (Table 19).

(424) TABLE-US-00020 TABLE 19 Texture Analysis Results. Treatments Jelly control 20% whey protein 20% nOVD 20% rOVD Hardness (g) *53.2 ? 1.7 a 32.8 ? 3.4 b 31.1 ? 4.3 b 32.8 ? 3.4 b Adhesiveness (mj) 0.09 ? 0.03 a 0.03 ? 0.005 c 0.08 ? 0.01 ab 0.04 ? 0.02 bc Jelly pH 4.3 5.5 5 5.5 *Mean ? Std Dev; Jelly samples containing different letters for a given quantitative parameter (for example Hardness) are statistically different to each other at p < 0.05.

(425) Jelly fortified with 20% of whey, nOVD, or rOVD were significantly less hard than the control jelly.

(426) No significant difference was observed between the clarity of the control jelly and jelly fortified with 20% of rOVD (p<0.05). Jelly fortified with whey isolate protein was opaque and unclear. (FIG. 18A and FIG. 18B).

(427) The texture of Jello? fortified with 20% of whey protein was very soft and not comparable to the control. (FIG. 18C).

(428) The texture of Jello? with either 16% or 20% of hydrolyzed gelatin was rubbery, with strong bounce and resistance to deformation. In this experiment aliquoting of the samples was not possible, since the jelly set very quickly at room temperature. (FIG. 18D and FIG. 18E).

Example 13: Testing Recombinant OVD from Various Species

(429) In this example, properties of rOVD having amino acid sequences of non-chicken, avian species was evaluated.

(430) Two expression constructs were created for expression for two non-chicken rOVD (SEQ ID NO: 40 called rOVD-T (Turkey vulture) and SEQ ID NO:43 called rOVD-H (humming bird) hereafter) in Pichia pastoris and expressed, purified and processed similar to Example 2. Lyophilized rOVD samples were blended into aqueous solution (distilled water) at different concentrations and pHs. Clarity and solubility of the rOVD solutions was then assessed visually (e.g., for turbidity, precipitate, viscosity, and color) as well as by measuring absorbance at 600 nm.

(431) FIG. 19A shows protein-water samples comprising rOVD-H in deionized water at protein concentrations of 4.23%, 10%, 20% or 30% w/v. The solutions had a pH of 4.15. Like the chicken rOVD of the previous examples, FIG. 19A reveals a clear and colorless solution with no precipitate and no apparent viscosity changes in appearance and visual flow of liquid for solutions comprising up to 20% rOVD-H.

(432) FIG. 19B shows protein-water samples comprising rOVD-T in deionized water at protein concentrations of 4.23%, 10% or 20% w/v. The solutions had a pH of 3.69. Like the chicken rOVD of the previous examples, FIG. 19B reveals a clear solution with no precipitate and no apparent viscosity changes in appearance and visual flow of liquid for solutions comprising up to 10% rOVD-T. At 20% the protein did not fully dissolve.

(433) The samples were incubated at room temperature and subjected to three types of heat treatments: pasteurization, hot fill, or retorting as in Example 4 or Example 5. The clarity and solubility of the various rOVD were then assessed visually (e.g., for turbidity, precipitate, viscosity, and color) as well as by measuring absorbance at 600 nm. Table 20 shows the results for pH, absorbance, and clarity of rOVD solution comprising 4.23% rOVD-H or rOVD-T solutions in buffer or water. Data in the pre-processing column was measured before any heat treatment. It was surprising that at different pH's the rOVD solutions remained clear even after extreme heating like pasteurization, hot fill, or retort. These results indicate that rOVD samples remain soluble in solution at different acidic pHs, before and after application of heat.

(434) These data show that the favorable properties disclosed above for the recombinant chicken OVD (see Example 2) are also obtainable with other recombinant OVDs.

(435) TABLE-US-00021 TABLE 20 Solubility and clarity study of rOVD-H and rOVD-T OD Pre-processing OD post OD post OD post Sample pH at RT pasteurization hot fill autoclave/retort rOVD-H + buffer 2.5 0.0569 0.0547 0.0548 0.0537 rOVD-T + buffer 0.058 0.059 0.057 0.056 rOVD-H + buffer 4 0.0546 0.0544 0.0552 0.0641 rOVD-T + buffer 0.055 0.055 0.057 0.055 rOVD-H + buffer 6 0.053 0.053 0.055 0.061 rOVD-T + buffer 0.054 0.054 0.054 0.068 rOVD-H + water 3.5-3.9 0.067 0.084 0.090 0.236 rOVD-T + water 0.097 0.106 0.116 0.219

Example 14: Comparison of Bovine Trypsin Inhibitory Activity

(436) rOVD as produced in Example 2 was utilized in this Example. The trypsin inhibition activity was compared between native OVD (nOVD) and recombinant OVD (rOVD) in a standard assay (AACC #22-40.01) using bovine trypsin. A comparison of rOVD with nOVD is shown in Table 21. One trypsin unit is arbitrarily defined as an increase of 0.01 absorbance unit at 410 nm per 10 ml of reaction mixture under the conditions of the assay. Trypsin inhibitor activity is expressed in terms of trypsin inhibitor units (TIU). Three different batches of rOVD (samples 1-3) were compared to a native chicken ovomucoid.

(437) TABLE-US-00022 TABLE 21 Comparison of trypsin inhibition activity Product Trypsin inhibition activity Sample 1 8190 TIU/g Sample 2 8180 TIU/g Sample 3 8649 TIU/g Native chicken Ovomucoid 13721 TIU/g

Example 15: Comparison of In Vitro Digestibility

(438) The in vitro digestibility of rOVD samples was measured using the Protein Digestibility Assay procedure (Megazyme, Medallion Labs). A comparison of rOVD samples with nOVD is shown in Table 22. The data demonstrates equivalent in vitro digestibility between native ovomucoid and rOVD.

(439) TABLE-US-00023 TABLE 22 Comparison in vitro digestibility Product In-vitro digestibility Sample 1 93% Sample 2 93% Sample 3 93% Native chicken Ovomucoid 92%

Example 16: Ovomucoid Specifications

(440) Based upon the characterization of the produced rOVD compositions and the properties of native chicken ovomucoid, product specifications (Table 23) and quality control specifications (Table 24) were constructed for an rOVD of the present disclosure

(441) Protein percentages were measured using AOAC 2006. See, Protein (crude) in animal feed, combustion method, 990.03. In: Official methods of analysis of AOAC International. 18th ed. Gaithersburg: ASA-SSA Inc. and AOAC 2006. Proximate Analysis and Calculations Crude Protein Meat and Meat Products Including Pet Foodsitem 80. In: Official methods of analysis Association of Analytical Communities, Gaithersburg, MD, 17th edition, Reference data: Method 992.15 (39.1.16); NFNAP; NITR; NT.

(442) Moisture percentages were measured using Association of Official Analytical Chemists. 1995. In Official Methods of Analysis.

(443) Carbohydrate percentages were measured using methods described in J AOAC Int. 2012 September-October; 95(5):1392-7.

(444) Fat by acid hydrolysis were measured using AOAC International. 2012. Official Method Fat (crude) or ether extraction in pet food. Gravimetric method, 954.02. In: Official Methods of Analysis of AOAC International, 19th ed., AOAC International, Gaithersburg, MD, USA, 2012.

(445) Standard plate count was measured using AOAC International. 2005. Aerobic plate count in foods, dry rehydratable film, method 990.12. AOAC International, 17th ed. Gaithersburg, MD. Yeast and mold counts were measured using AOAC Official Method 997.02. Yeast and Mold Counts in Foods Dry Rehydratable Film Method (Petrifilm? Method) First Action 1997 Final Action 2000 Salmonella was measured using AOAC International. 2005. Salmonella in selected foods, BAX automated system, method 2003.09. In Official methods of analysis of AOAC International, 17th ed., AOAC International, Gaithersburg, MD. Total coliform was measured using AOAC International. 2005. E. coli count in foods, dry rehydratable film, method 991.14. In Official methods of analysis of AOAC International, 17th ed. AOAC International, Gaithersburg, MD.

(446) TABLE-US-00024 TABLE 23 Specification for Ovomucoid produced by P. pastoris DFB-003 Physical properties Specification Source Yeast fermentation-derived Appearance White to off-white amorphous powder Solubility Soluble in water Chemical Properties (in powder as is) Specification Method Protein >75% AOAC 990.03.sup.1a AOAC 992. 15.sup.1b Moisture Maximum 10.0% AOAC 925.09.sup.2 Carbohydrate Maximum 20% Calculated Ash Maximum 2.0% AOAC 942.05.sup.3 Fat by Acid Hydrolysis <0.1% AOAC 954.02.sup.4 Hg <1 ppm ICP-AES.sup.5 Pb <1 ppm ICP-AES.sup.5 As <1 ppm ICP-AES.sup.5 Cd <1 ppm ICP-AES.sup.5 Microbial Properties (in powder as is) Specification Method Standard Plate Count <10000 CFU/g AOAC 990.12.sup.6 Yeast & Mold <100 CFU/g AOAC 997.02.sup.7 Salmonella Not Detected/25 g AOAC 2003.09.sup.8 E. coli Not Detected/25 g AOAC 991.14.sup.9 Total coliform ?30 CFU/g AOAC 991.14.sup.9

(447) TABLE-US-00025 TABLE 24 Quality control results for three lots of Ovomucoid produced by P. pastoris DFB-003 Analysis Parameter Specification SOL19303 SOL19317 SOL19351 Protein >75% 75.31 75.06 79.94 Protein (% dry >80% 82.2 82.5 87.8 weight powder) Moisture and <10% 8.4 9 9 Volatiles Carbohydrates, <20% 15.53 15.28 11.06 Calculated Ash <2% 0.76 0.66 <0.4 Fat by Acid <0.1% <0.10 <0.10 <0.10 Hydrolysis Arsenic (As) <1 mg/kg <0.010 <0.010 <0.010 Mercury (Hg) <1 mg/kg <0.010 <0.010 <0.010 Lead (Pb) <1 mg/kg 0.03 0.063 0.168 Cadmium (Cd) <1 mg/kg <0.010 <0.010 <0.010 Aerobic Plate <10000 CFU/g <10 <10 <10 Count Molds <100 CFU/g <10 <10 <10 Yeast <100 CFU/g <10 <10 <10 Salmonella Not Detected/ Not Not Not 25 g Detected Detected Detected Escherichia Coli Not Detected/ Not Not Not 25 g Detected Detected Detected Coliforms <10 CFU/g <10 <10 <10 Absence of source Not detected */ Not Not Not organism from mg sample detected detected detected product Absence of Not detected **/ Not Not Not encoding DNA mg sample detected detected detected from product * Limit of detection for source organism = 11 CFU/mg sample ** Limit of detection for encoding DNA = 10 femtogram

Example 17: Absence of Production Organism and DNA in rOVD Preparations

(448) rOVD powder was plated on PGA plates and if samples yielded colonies, these were re-streaked and analyzed by PCR for the presence of the Pichia organism. This procedure was applied to three lots of rOVD powder produced from the recombinant strain. No manufacturing organism was detected in any of the lots (Table 24).

(449) PCR analysis was used to confirm that no encoding pieces of recombinant DNA was present in the rOVD preparation using primers for the rOVD cassette. OVD plasmid DNA was used as a positive control, producing a 570 bp band corresponding the OVD PCR product. This band was absent in all three rOVD powder lots tested.

Example 18: Comparison Immunoreactivity

(450) Western Blot comparisons were performed on three rOVD lots using primary anti-ovomucoid antibody from rabbit (NBP1-74676 Novus) at a 1:2500 dilution. The secondary antibody used was goat anti-rabbit IgG conjugated to alkaline phosphatase (AP ab97048 Abcam). Molecular weight marker preparation used was from Bio Rad (161-0394). The comparison showed the same immunoreactivity for rOVD samples, native ovomucoid from chicken egg white (nOVD) and deglycosylated native ovomucoid (nOVD+PNGaseF) (FIG. 20).

Example 19: Fermentation and Purification of rOVD

(451) An rOVD P. pastoris seed strain was removed from cryo-storage and thawed to room temperature. Contents of the thawed seed vials were used to inoculate liquid culture media in the primary fermenter and grown at process temperature until target cell density was reached. Then, the grown rOVD P. pastoris was transferred to a production-scale reactor. The culture was grown in the production bioreactor at target fermentation conditions and fed a series of substrates. The fermentation was analyzed for culture purity at multiple times during the process.

(452) The recombinant OVD was purified by separating the cells from the liquid medium by centrifugation, followed by microfiltration. Fermentation broth was first brought to pH 3 and diluted with DI water. Cells were removed using bucket centrifugation. The collected supernatant was brought to pH 7 using sodium hydroxide and a 0.2 ?m filtration was performed followed by diafiltration with five volumes of deionized water. The permeates of the 0.2 ?m were adjusted to pH 5 and then concentrated via 5 kDa TFF membrane. The 5 kDa retentate was precipitated using 65% saturation ammonium sulfate. After salt addition, the pH was adjusted to pH 4-4.1 with phosphoric acid. The mixture was incubated with agitation at room temperature overnight. The next day, precipitates were spun down using bucket centrifugation. The rOVD precipitates were dissolved in DI water and pH adjusted to 5 using sodium hydroxide. The rOVD solution was then diafiltered and then the retentate was passed through 0.2 ?m bottle filters.

(453) A spray dryer was used to dehydrate the rOVD solution into rOVD powder.

Example 20: Hydrogen Peroxide Treatment During rOVD Purification

(454) Liquid rOVD was concentrated to 50-60 g/L using a 5 kDa TFF membrane. The rOVD solution was passed through a 0.2 ?m filter to remove microbes. Hydrogen peroxide, an oxygen-generating agent, in an amount to equal 10% volume of the solution was slowly added to the rOVD solution while stirring. The mixture was incubated with agitation and monitored to ensure color change from a dark green-brown color before treatment to a pale-yellow color after treatment. After 1.5 hours, diafiltration was performed via 5 kDa TFF membrane with 5 volumes of DI water. The rOVD in the 5 kDa diafiltration retentate was precipitated using ammonium sulfate at 65% salt saturation at room temperature. After addition of salt, the pH was adjusted to pH4-4.1 with phosphoric acid. The mixture was incubated with agitation overnight to form precipitates. The next day, the precipitates were spun down using bucket centrifugation. The precipitates were removed, dissolved in deionized water and pH adjusted to 5 using sodium hydroxide. Five kDa TFF membranes were cleaned and diafiltration was performed using volumes of DI water until a retentate conductivity of less than 2.0 mS was achieved. The retentate was passed through 0.2 ?m bottle filters. The filtered rOVD solution was then spray dried and stored.

Example 21: Reprocessed rOVD Treated with Hydrogen Peroxide

(455) OVD powder was dissolved in deionized water to 50-60 g/L and filtered through a hollow fiber 0.2 ?m tangential flow filter, then through a 0.2 ?m bottle filter. Hydrogen peroxide in an amount to provide a 10% solution was slowly stirred into the rOVD solution and incubated for thirty minutes. The treated solution was washed through a 5 kDa membrane using 5 volumes of DI water.

(456) Ammonium sulfate was slowly added to the retentate solution and the pH changed to between 4 to 4.1 using phosphoric acid. After overnight incubation with medium agitation, the solution was centrifuged, and supernatants discarded. Precipitates were collected, dissolved in DI water, and brought to pH 5 using sodium hydroxide. The protein solution was desalted with a 5 kDa membrane and filtered through a 0.2 ?m bottle filter. Then, the protein solution was spray dried to produce rOVD powder.

Example 22: Sensory Testing and Results

(457) The rOVD sample and the H.sub.2O.sub.2 reprocessed sample called RE-RC were analyzed for their sensory characteristics to determine the effects of hydrogen peroxide treatment.

(458) A solution of each dry sample was prepared with Deionized water at 4.23% w/v concentration. Both samples were presented to the panelists in the same session, monadically. Trained panelists (n=6) evaluated both the samples in terms of their appearance, smell, taste, mouthfeel and aftertaste. For each category, the panelists described the perceived attributes and then rated each attribute's intensity (Kemp et al. 2009) using the intensity rating scale (Table 25).

(459) Table 26 shows that the hydrogen peroxide-treated sample was lighter in color, and had a cleaner sensory profile, with fewer sensory attributes compared to the control sample.

(460) TABLE-US-00026 TABLE 25 Attribute Intensity Rating Scale SMELL, FLAVOR, AFTERTASTE & APPEARANCE MOUTHFEEL (Intensity APPEARANCE (Color rating for Individual (Clarity) Intensity) attributes in each category) 0 = clear 0 = no color 0 = not detected 1 = very slightly 1 = very pale 1 = very mild turbid 2 = slightly turbid 2 = pale 2 = mild 3 = mild/moderate 3 = moderate 3 = moderate turbidity intensity 4 = moderately 4 = dark 4 = strong turbid 5 = very turbid 5 = very dark 5 = very strong

(461) TABLE-US-00027 TABLE 26 Sensory evaluation results Powder Batch H.sub.2O.sub.2 Name rOVD Reprocessed RE-RC Appearance pale yellow/green (2), clear (0), very pale yellow (1), bubbly, not easy to mix clear (0), very frothy (sediments visible) Smell mild yeasty (2), mild/moderate very mild musty (1) musty (2.5), mild nutty (2) Taste mild buttermilk (2), mild/moderate very mild yeasty (1) toasted nutty (2.5), mild yeasty (2) Mouthfeel None (0) None (0) Aftertaste None (0) None (0)

Example 23: H.SUB.2.O.SUB.2 .Treated rOVD Tested for Solubility and Clarity

(462) Solubility and clarity of the control and hydrogen peroxide treated sample solutions (at 4.23% w/v) were measured in terms of optical density (A.sub.600) using a Spectrophotometer. Lower absorbance value (at 600 nm wavelength) indicates higher clarity and solubility of the sample solution.

(463) The hydrogen peroxide-treated sample had lower absorbance (Table 27) and a paler color compared to the control. This indicated that the treatment resulted in improved appearance, in terms of less intense color and clear solution. These features are illustrated in FIG. 21.

(464) TABLE-US-00028 TABLE 27 Absorbance (at 600 nm) of sample solutions (4.23% w/v) rOVD H.sub.2O.sub.2 Reprocessed RE-RC Absorbance 0.068 0.046

Example 24: Protein Bar Preparation and Testing for Hardness and Sensory Likeability

(465) Homogenous mixtures of chopped dates chopped nuts (almonds and walnuts), and cocoa was combined with a protein powder of interest as shown in in Table 28. The amount of dates and nuts was reduced in formulations that included protein powders as seen in Table 29. The dates:nuts ratio was kept at a constant 4.6 level. Egg white protein powder and nOVD were prepared at inclusion levels of 2, 8, 16 or 23% while rOVD was prepared at inclusion levels of 2, 4, 8, 12, or 16%. (Table 28 to Table 31).

(466) Half of each mixture was baked in an oven at 350 degrees F. for ten minutes. The other half of each mixture was tested as an unbaked mixture.

(467) TABLE-US-00029 TABLE 28 List of Ingredients and their proportions used in control formulation Ingredients Amount (%) Dates 78.67 Nuts 17.33 Cocoa 4 Total 100

(468) TABLE-US-00030 TABLE 29 List of Ingredients and their proportions used in egg white protein formulations 2% 8% 16% 23% 32% Ingredients protein protein protein protein protein Dates 76.67 71 63.33 56.67 48 Nuts 17 15.67 14 12.50 10.67 Cocoa 4 4 4 4 4 Protein 2.33 9.33 18.67 26.83 37.33 Total 100 100 100 100 100

(469) TABLE-US-00031 TABLE 30 List of Ingredients and their proportions used in nOVD formulations 2% 8% 16% 23% Ingredients protein protein protein protein Dates 76.67 70.67 62.33 55.25 Nuts 16.83 15.33 13.67 12 Cocoa 4 4 4 4 Protein 2.5 10 20 28.75 Total 100 100 100 100

(470) TABLE-US-00032 TABLE 31 List of Ingredients and their proportions used in rOVD formulations: 2% 4% 8% 12% 16% Ingredients protein protein protein protein protein Dates 76.71 74.95 71.14 67.28 63.61 Nuts 16.99 16.46 15.66 14.93 14 Cocoa 4 4 4 4 4 Protein 2.3 4.59 9.20 13.79 18.39 Total 100 100 100 100 100

Example 25: Protein Bar Hardness/Texture Test

(471) The textural properties of the baked and unbaked protein bars as prepared in Example 25 were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). A three-point bend test was used to snap, bend and measure the hardness of the protein bars. One sample for each protein inclusion level was analyzed. The test parameters used are shown in Table 32.

(472) The hardness results for the baked protein bars were much higher than the hardness results in the unbaked version. Within the unbaked protein bars, 8% inclusion for all protein powders resulted in similar hardness values. Hardness profile for all unbaked protein bars gradually increased as the protein inclusion rates increased. Hardness values at 16% and 23% protein inclusion were also comparable for egg white protein, native OVD and rOVD. See, Table 33 and Table 34.

(473) Egg white protein could be included up to 32% protein levels. A maximum of 23% protein inclusion levels in a protein bar, was observed for native and rOVD. Higher protein concentrations were unable to incorporate in a protein bar form.

(474) The hardness value for nOVD at 8% inclusion level was much lower than egg white protein and rOVD. However, similar hardness values were observed for all protein bar samples at an inclusion level of 16% and 23%. The baked protein bars with native and rOVD exhibited a porous crumb and hard outer shell for higher inclusion levels of 16% and 23%. Overall, 8% protein powder inclusion level was the most desirable (higher palatability and texture attributes) across all protein powders.

(475) TABLE-US-00033 TABLE 32 Test parameters used for three-point bend test to measure hardness using a CT3 Brookfield Texture Analyzer Test type Rupture test Probe TA7 blade Base Fixture TA-TPB Trigger load 5 g Correction load 30 g Test speed 3 mm/s Sample rate 30 points/sec Distance between support arms 2.5 cm Textural properties Hardness (g)

(476) TABLE-US-00034 TABLE 33 Test results for unbaked protein bar samples Hardness (g) for protein inclusion levels Control Sample (0%) 8% 12% 16% 23% 32% Egg 86.33 186.2 386.6 299.2 434.6 393.6 white protein nOVD 173.2 463.8 360 411 n/a rOVD 182.2 291.2 338.2 402.4 n/a

(477) TABLE-US-00035 TABLE 34 Test results for baked protein bar samples Hardness (g) for protein inclusion levels Sample Control (0%) 8% 12% 16% 23% 32% Egg 1193 1525.2 1490 1544.4 1506.6 1534.2 white protein nOVD 1072.8 1054.4 1506.2 1433.8 n/a rOVD 1380.4 1499 1504 1565.4 n/a

Example 26: Protein Bar Sensory Test

(478) Samples prepared as described in Example 26 were evaluated for quality descriptors by trained in-house panelists.

(479) The quality attributes tested included appearance, smell, taste/flavor, mouthfeel/texture and overall liking in a nine-point scale from 1: Dislike extremely, 2: Dislike very much, 3: Dislike moderately, 4: Dislike slightly, 5: Neither like nor dislike, 6: Like slightly, 7: Like moderately, 8: Like very much, and 9: Like extremely.

(480) TABLE-US-00036 TABLE 35 Sensory likeability results for 8% protein bar samples 8% Protein inclusion 8% Protein inclusion (Unbaked) (Baked) Egg Egg Attribute Unbaked white Baked white Likeability Control protein nOVD rOVD Control protein nOVD rOVD Appearance 8 7.5 8 6.5 9 7.5 8 6.5 Smell 9 8 9 8 9 7 9 8 Taste/Flavor 9 6.5 4 7 9 6 4 7 Texture/Mouthfeel 6 6 4.5 8 9 4 4.5 8 Overall 7 7 4 7 9 5 4 7

(481) For the control unbaked sample, panelists noted that it had a good appearance, slightly soft texture/bite but overall good taste and no unpleasant aftertaste. For the baked version, panelists liked every attribute of the sample to the highest score and gave it a perfect score.

(482) For the unbaked (8%) protein bars, panelists provided the following comments: the egg white protein bar tasted like tootsie roll, it was sweet and cohesive but had a dry mouthfeel; the native OVD bar was less sticky as compared to control but had a strong OVD-like, metallic and acidic taste and with a dry mouthfeel; and the rOVD bar had no acidity, was slightly less sweet but was cohesive and had a pleasant aftertaste.

(483) For the baked (8%) protein bars, panelists provided the following comments: the egg white protein bar was slightly acidic, had a cracker/toasted cereal like taste and aftertaste; the native OVD bar was harder and tacky as compared to control and was more palatable; and the rOVD bars lacked acidity, were chewy, and tacky which the panelists liked.

(484) TABLE-US-00037 TABLE 36 Sensory likeability results for 16% protein bar samples 16% Protein inclusion 16% Protein inclusion (Unbaked) (Baked) Egg Egg Attribute Unbaked white Baked white Likeability Control protein nOVD rOVD Control protein nOVD rOVD Appearance 8 7 8 5 9 5 6 5 Smell 9 5.5 7 5.5 9 2 7 5 Taste/Flavor 9 5 1 7 9 3 1 6 Texture/Mouthfeel 6 4 ?1 8 9 2 1 5 Overall 7 4 1 6.5 9 2 2 6

(485) For the baked (16%) protein bars, panelists provided the following comments: the egg white protein bar tasted toasted and bready, was whiteish and had a powdery mouthfeel; the nOVD bar was very hard and difficult to bite, looked like a hard bread, had a strong sour taste which left a burning sensation; the rOVD bar had muted sweetness, a mealy and a toasty flavor, with no acidity or aftertaste, and it was tacky but hard.

(486) Overall, rOVD bars performed better than nOVD bars and comparable to egg white protein samples in tests described in Example 25 to Example 27. A maximum of 23% protein inclusion for nOVD and rOVD seemed possible while egg white protein samples were able to go as high as 32% inclusion levels. Eight percent bars were deemed as the best inclusion levels for all the protein bars.

(487) Twelve percent rOVD bars had slight acidity in the unbaked bars, however no acidity was perceived in the baked bars. The baked bars were chewy, tacky and hard.

Example 27: rOVD Salad Dressing

(488) A salad dressing was prepared using a L5M-A homogenizer (Silverson) at ambient temperature. Emulsions were prepared by dispersing protein powder and salt into the aqueous phase (water and vinegar) and stirring at 2000 rpm for 5 minutes using General Purpose Disintegrating Head. After mixing, canola oil was added in a controlled manner and homogenized at 6000 rpm for 15 minutes using Square Hole High Shear Screen to make a stable oil-in-water emulsion.

(489) All emulsion samples were transferred into glass tubes, sealed with a plastic cap, and stored at 4 C for seven days. The stabilities of the samples were evaluated by visually monitoring the height of the visible serum separation at the bottom phase with storage time. Physical stability was monitored until no visual phase separation happened. The stability of the emulsion was expressed as: % serum=(Ht/H0)*100. H0 represents the initial emulsion height and the height of visible serum separation layer (Ht).

(490) List of ingredients and their proportions used in the control and other salad dressing samples with specific protein of interest were presented (Table 37).

(491) TABLE-US-00038 TABLE 37 List of Ingredients. Control nOVD 9% rOVD 9% Ingredient % % % Canola oil 45 45 45 Water 43.4 33.4 33.1 Vinegar 9.6 9.6 9.6 Emulsifier 0 10 10.3 Salt 2 2 2 Total 100.0 100.0 100

(492) Table 38 presents the emulsion stability of the dressings with storage time. Both nOVD and rOVD samples showed better emulsion stability compared to the Control sample that underwent phase separation during the first day of storage at 4 C. After Day 2, samples containing nOVD and rOVD did not exhibit much change in the emulsion phase separation. Higher values indicate lower emulsion stability.

(493) TABLE-US-00039 TABLE 38 Results of emulsion stability Time sample Emulsion stability % Day 1 9% rOVD 17 9% nOVD 15 Control 44 Day 2 9% rOVD 29.4 9% nOVD 18.8 Control 48.2 Day 3 9% rOVD 26 9% nOVD 21.3 Control 47.2 Day 7 9% rOVD 26 9% nOVD 21.3 Control 48.2 Day 8 9% rOVD 27.6 9% nOVD 23.3 Control 49.1 Day 9 9% rOVD 25.6 9% nOVD 23.3 Control 49.1

Example 28: rOVD Egg Wash Formation

(494) The film formation and sheen formation functionality of rOVD was evaluated in a bread application. Baking instructions: a. In a small container, mix together yeast and sugar, and add warm water (85-95 F). Let it sit for 5 min b. Add the water in a mixing bowl c. Slowly mix in flour (30 sec) until a firm dough is formed (mix for 2 min on speed 3) d. Knead dough (folding 7 times) on a lightly floured board for 30 sec, adding small sprinkles of flour only as needed e. Place dough in a greased bowl. Flip dough over inside the bowl so that the dough top is also lightly greased. Cover and let rise for 45 minutes at 80 F proofing temperature (1st proof) f. Turn dough out onto a floured board and knead out air (fold 7 times) g. Shape into mini loaf and place in a greased mini pan h. Cover and let rise for 30 minutes at room temperature (2nd proof) i. Apply appropriate wash on top of the dough balls at a 3% level. (In case of sesame seed application, apply 10 sesame seeds to each dough ball over the wash) j. Bake at 350 F for 8 minutes or until golden brown (switch the location of the bread at 4 min to achieve even baking on all samples) k. 3% wash of total bread dough weight was added on top. 25 g samples each were used (total egg wash=0.75 g). l. For samples with whole egg and commercial egg wash substitute, 0.75 g of each sample was applied to the dough surface.
The formulations used for protein of interest are shown in Table 39:

(495) TABLE-US-00040 TABLE 39 List of Ingredients and their proportions used in egg wash formulation: Egg white powder rOVD Ingredients % % DI water 90.67 91.21 Film forming 9.33 8.79 agent

(496) Retention of sesame seeds: Retention of any topping on cake, bread, bagels or other baked goods is an important factor for egg wash. Sesame seeds were used to evaluate the binding function of each film forming agent post baking. 10 sesame seeds were applied to each dough ball post the application of wash and before baking. Retention of these sesame seeds was calculated based on the amount of seeds stuck to the bread post baking.

(497) The following results were obtained:

(498) TABLE-US-00041 TABLE 40 Commercial Egg Negative egg wash Whole white Samples Control substitute egg protein rOVD Retention 0% 100% 100% 100% 100% level

(499) The control sample with no egg wash had no binding capacity for the sesame seeds and zero sesame seeds were retained on the surface post baking. However, all other film-forming agents were able to retain all 10 seeds post baking suggesting a 100% retention rate for toppings.

(500) Colorimetric assay: Individual sample pictures were analyzed for color data in the RGB spectrum using the Colorgrab application (Loomatix). Sample values were generated using a 2?2 cm cross-section taken from the center of the bread surface. RGB data was then converted to a CIELAB system using the online software www.colormine.org. CIELAB model is a color space system that expresses color in 3 values: L* for the lightness from black (0) to white (100), a* from green (?) to red (+), b* from blue (?) to yellow (+).

(501) TABLE-US-00042 TABLE 42 CIELAB results for bread post baking L* a* b* Negative Control 63.669 1.10972 25.4527 Whole egg 62.255 8.39894 45.57611 Commercial egg wash 68.349 0.04763 34.7033 substitute Egg white protein 76.831 2.58977 31.1123 rOVD 83.591 4.58532 42.2485

(502) rOVD and egg white protein samples had a higher L* value suggesting higher brightness or luminance. Control (no egg wash), commercial egg wash substitute and egg white protein samples had a low a* value suggesting lower redness or brownness as compared to Whole egg, and rOVD samples.

(503) Whole egg wash and rOVD samples also had similar b* values, suggesting similar yellow hues as compared to the other samples.

(504) Visual Inspection: The control sample looked pale, wrinkly and had no shine. The sample with egg-wash had good browning, great sheen and a smooth surface. The bake sheen sample had a smooth surface with a slight noticeable sheen. Egg white protein powder sample along with rOVD sample had a good sheen and browning.

(505) rOVD worked well as a film forming and sheen forming agent. All the sesame seeds remained on the surface post browning suggesting good film forming and binding capabilities. The visual inspection and color values suggested good sheen formation and browning as compared to other samples (FIG. 22).

Example 29: Preparation of Recombinant Ovalbumin

(506) A Gallus gallus OVA coding sequence was fused in-frame with the alpha mating factor signal sequence downstream of the promoter sequence (SEQ ID NO:60). A promoter was placed upstream of the signal sequence OVA coding sequence and a transcriptional terminator was placed downstream of the OVA sequence. The expression construct was placed into a Kpas-URA 3 vector.

(507) The expression constructs were transformed into Pichia pastoris. Successful integration was confirmed by genomic sequencing.

(508) Fermentation: Recombinant OVA was produced in a bioreactor at ambient conditions. A seed train for the fermentation process begins with the inoculation of shake flasks with liquid growth broth using 2 ml cryovials of Pichia pastoris which are stored at ?80? C. and thawed at room temperature prior to inoculation.

(509) The inoculated shake flasks were kept in a shaker at 30? C. for 24 hours, after which the grown Pichia pastoris was transferred to a production scale reactor.

(510) The culture was grown at 30? C., at a set pH and dissolved oxygen (DO). The culture was fed with a carbon source. At the end of the fermentation, the target OVA protein was harvested from the supernatant.

(511) Cell debris was removed, protein was purified and lyophilized to a dry powder. The OVA produced was used in the examples described below.

Example 30: Preparation of an Eggless Cake Using Recombinant Ovalbumin

(512) An eggless pound cake can be prepared with the following ingredients. A first ingredient composition made by mixing 2% to 5% recombinant ovalbumin and 0.05% to 0.5% sunflower lecithin. To prepare the pound cake, up to 4% of the dry first ingredient composition is added to 22-26% of unsalted butter, 20-25% of all-purpose flour, 18-26% of water, 20-25% sugar, 4-6% of sour cream, 1.2% of baking powder, 0.4% of vanilla flavor, 0.05 to 1.5% gums and starch and 0.18% of salt and all ingredients are then mixed to create a batter. For this recipe recombinant ovalbumin may be used at 2-5% and sunflower lecithin from 0.05 to 0.5%.

(513) In one example, pound cakes with rOVA and with whole egg (as a comparison) were made as follows:

(514) TABLE-US-00043 TABLE 44 Cake with rOVA+ Xanthan gum Ingredients % w/w Lecithin 0.09 All-purpose Flour 22.61 Granulated Sugar 22.61 Unsalted butter 25.63 Sour cream 5.03 Coarse salt 0.18 Baking powder 1.21 vanilla extract 0.37 rOVA 3.41 Water 18.74 Xanthan gum 0.05 Marigold yellow 0.06 Total 100.00

(515) TABLE-US-00044 TABLE 45 Control Pound Cake with whole Egg Ingredients % w/w Flour 23.34 Sugar 23.34 Whole egg 23.34 unsalted butter 23.34 Sour cream 5.19 baking powder 1.25 Vanilla 0.38 coarse salt 0.21 Total 100.00

(516) TABLE-US-00045 TABLE 46 Cake with rOVA+ Potato Starch+ Xanthan gum Ingredients % w/w Flour 20.73 Sugar 20.73 Unsalted butter 22.08 Sour cream 4.61 Coarse salt 0.16 Baking powder 1.11 Vanilla 0.34 rOVA 3.10 Potato starch 1.48 Xanthan gum 0.09 Lecithin 0.05 water 25.45 Marigold yellow 0.05 Total 100.00

(517) For each of the recipes, the batter was baked at 325? F. until cooked such time that a toothpick, when inserted at the middle of the cake, came out clean.

(518) TABLE-US-00046 TABLE 47 Results from using rOVA in pound cake compositions rOVA + rOVA + Potato Control Xanthan Starch + Egg pound gum Xanthan gum cake Cohesiveness *0.6 ? 0.02 a 0.64 ? 0.02 a 0.62 ? 0.02 a Resilience 0.31 ? 0 a 0.36 ? 0.05 a 0.32 ? 0.01 a Hardness (g) 73.9 ? 2.1 a 75.5 ? 7.5 a 75.6 ? 12.7 a Chewiness 1.48 ? 0.04 a 1.78 ? 0.3 a 1.63 ? 0.5 a (mJ) Springiness 3.41 ? 0 a 3.72 ? 0.21 a 3.48 ? 0.32 a (mm) Cake height 30.08 ? 1.4 a 30.07 ? 1.04 a 30.64 ? 1.01 a (cm) Sensory Appearance: Appearance: Appearance: open good yellow pale crust color, pores, golden crust, crumb, compact good yellow crumb good yellow crumb crumb, color, open color light brown crust. pores in crumb Texture: good Good rise like the Control, chewy, slightly dry and volume. good rise/volume Flavor: cakey, Aroma: butter, Aroma: buttery, buttery cakey cakey Aroma: cakey Flavor: buttery, Flavor: cakey, buttery cakey sweet Texture: more Texture: more moist texture moist texture than control than egg control, cake with egg, more cohesive than not as cohesive control cake with as egg control. egg. *Similar letters within each marker indicate there is no significant difference between the samples (mean ? std dev; p > 0.05)

(519) Texture qualities such as cohesiveness, resilience, hardness, chewiness and springiness were measured using a Brookfield CT3 Texture analyzer, 1500 g load cell. No significant difference was observed between the Control Egg cakes and cakes made with rOVA in terms of textural properties and cake height. The sensory properties were comparable to the Control cake made with whole egg.

(520) The rOVA in the pound cake demonstrated several functional features with utility in baked goods, as well as for other food products and ingredients. Results are shown in FIG. 24.

(521) TABLE-US-00047 TABLE 48 Functional features provided by rOVA in pound cakes Functionality Evidence Foaming Air cells formed, evident in the crumb structure (cross section photo) Whipping Air incorporation during mixing of batter, evident from air cells in crumb structure Gelling Protein coagulation upon heating. Creates structure of cake. Binding Binds with other ingredients, giving strength and structure to cake. Evident from texture and sensory measurements. Springiness Texture measurement Texturizer Provides structure while baking, evidenced by textural characteristics: chewiness, hardness, resilience, cohesiveness

Example 31: rOVA Applications in Meringue

(522) This example examined the feasibility of making meringue with rOVA in the recipe without using cream of tartar.

(523) Material: rOVA (pH: 4.12 as is), nOVA (pH: 6.06 as is), Fresh egg white (pH: 9 as is), Xanthan gum, Sodium lauryl sulfate (SLS), Cream of tartar, Granulated sugar, Flavor.

(524) Equipment: Kitchen Aid, Classic Plus, Breville BOV800XL Smart Electric Oven.

(525) Method: Separated egg white from the egg yolk carefully at the refrigerator temperature and then let egg whites get to room temperature before whipping. Egg white was used to make Control meringue sample. nOVA or rOVA was used to make test samples. Egg white or nOVA or rOVA solution (10% solution) was transferred to a mixer bowl and whipped for 30 seconds at medium speed (to obtain a homogeneous solution), then cream of tartar was added (for egg-whites only) and mixed at high speed until soft peaks form. While beating constantly, sugar was added gradually and beaten at high speed after each addition until sugar was dissolved completely. Continued mixing until a glossy and firm peak was formed and at the end, flavors were added. The soft meringue mix was transferred into the pan. An oven was preheated to 250? F., and meringues were baked for 50 minutes (or until an internal temperature of 160? F.). After cooling, meringues were stored in an airtight container.

(526) Exemplary meringue recipes using rOVA can include rOVA between 5-10%, sugar at about 26-32%, flavoring (e.g., 1-4%), water at about 59-64%, xanthan gum at about 0.01-0.5%, sodium lauryl sulfate at about 0.01-0.1% (all w/w). One such exemplary recipe, and comparison recipes with fresh egg white or with native OVA or with rOVA was constructed as shown below:

(527) TABLE-US-00048 TABLE 49 Recipes Meringue with Meringue with rOVA and fresh egg white nOVA (same recipe) Ingredients % w/w Ingredients % w/w Fresh egg white 68.19 nOVA and/or rOVA 8.14 Cream of tartar 1.14 Sugar 28.28 Sugar 28.41 Flavor 2.26 Flavor 2.26 Water 61.12 Total weight 100 Xanthan gum 0.1 SLS (Sodium lauryl sulfate) 0.1 Total weight 100

(528) TABLE-US-00049 TABLE 50 Results of meringue recipes Egg white nOVA rOVA meringue meringue meringue weight loss % *51 ? 1 b 60 ? 14.6 a 40 ? 4.5 c volume (ml) 6.9 ? 1.94a 7.82 ? 1.5a 8.05 ? 2.16a Density (g/ml) 0.1 ? 0.06 a 0.07 ? 0.01ab 0.06 ? 0.01b 1/density 9.14 13.78 16.65 fluffiness 100 150.5 182.1 *Samples with different letters across a row are significantly different (p < 0.05; mean ? std dev).

(529) Conclusion: Lowest weight loss was observed in meringue with rOVA. Furthermore, rOVA meringue indicated the highest fluffiness compared to the egg control and nOVA. Results are presented in FIG. 25.

(530) The rOVA usage in meringue demonstrates several functional features of rOVA.

(531) TABLE-US-00050 TABLE 51 Functional features provided by rOVA in meringues Functionality Evidence Foaming Increased foam capacity compared to egg white Whipping Whips easily-Reduced whipping/ whisking time compared to egg white Aeration Holds air bubbles, soft peak Fluffing Provides increased volume and fluffiness Gelling Protein coagulation upon heating, provides structure to the meringue sample

Example 32: Comparison of Foam Capacity and Foam Stability

(532) This example evaluated the foam capacity/stability and coagulation properties of rOVA and compared it to fresh whole egg, egg white and nOVA.

(533) Materials: store-bought egg, nOVA (Bioceutica), rOVA.

(534) Method: A stock solution of OVA (nOVA or rOVA) was made by mixing 0.7 g OVA in 9.3 g distilled water (total volume 10 ml). Cream of tartar was used (see Table 52 below) to adjust pH. Foam was made using a Dremel at speed 3. The time of whisking was recorded. Gel was made by heating 1 ml of sample at 72? C. for 10 min using a heat block.

(535) TABLE-US-00051 TABLE 52 pH adjustments to rOVA, nOVA and egg white compositions pH adjustment Initial Temper- Amount of cream pH after adding pH ature of tartar added (g) cream of tartar rOVA 3.86 21 0 3.86 solution nOVA 5.45 20.7 0.1 4.01 solution Fresh egg 8.57 20 2 4.64 white

(536) Results of the foam capacity and stability are shown in the Table 53 below. In this set, pH was not adjusted.
*Foam capacity %=[Initial liquid Vol. (ml)/Foam Vol. (ml)]*100
**Foam stability %=[(Initial liquid Vol. (ml)?Liquid drainage Vol. at 30 min (ml))/Initial liquid Vol. (ml)]*100

(537) TABLE-US-00052 TABLE 53 Results of foam capacity and stability Whole egg Egg white nOVA *Foam capacity % 210 ? 14.1 a 300 ? 0 b 338.5 ? 2.2 c Foam Stability % 56 ? 2.8 b 71 ? 1.4 a 59.3 ? 0.92 b time of whisking (second) >120 80 19 pH as is 7.6 9.1 5.9

(538) Conclusion: nOVA at pH 6 indicated the highest foam capacity compared to the egg white; however, its foam stability was lower than the egg white. Results are presented in FIG. 4

(539) The experiment was repeated using cream of tartar to adjust the pH.

(540) TABLE-US-00053 TABLE 54 Results of foam capacity and stability after pH adjustment using cream of tartar Egg white nOVA rOVA Foam capacity % 316.3 ? 5.3 b 457.9 ? 31.2 a 367.9 ? 2.9 b Foam Stability % 83.6 ? 6.2 a 65.1 ? 1.3 b 60.5 ? 0.7 b time of whisking (second) 64 19 32 Initial pH (as is) 8.57 5.45 3.86 Final pH (after adjusting 4.65 4.01 3.86 with cream of tartar)

(541) Conclusion: The foam capacity of nOVA after reducing pH was still higher than egg white. The foam capacity of rOVA was higher in value compared to that of fresh egg white. The whisking time for rOVA was half that required for fresh egg white. Results are shown in FIG. 27.

Example 33: Preparation of Recombinant Chicken Ovalbumin Expression Strain

(542) Expression Constructs Seven expression cassettes were created for expression of Gallus gallus OVA (SEQ ID NO: 61) in Pichia pastoris.

(543) TABLE-US-00054 TABLE 55 Expression Cassettes of Interest Strain Cassette Promoter Terminator Chicken GgOVA- K phaffii AOX1 K phaffii AOX1 transcriptional OVA A1 promoter terminator Chicken GgOVA- K phaffii AOX1 K phaffii AOX1 transcriptional OVA A2 promoter terminator Chicken GgOVA- K phaffii AOX1 K phaffii AOX1 transcriptional OVA A3 promoter terminator Chicken GgOVA- K pastoris DAS K phaffii AOX1 transcriptional OVA D1 promoter terminator Chicken GgOVA- K pastoris FLD1 K phaffii AOX1 transcriptional OVA F2 promoter terminator Chicken GgOVA- K pastoris FLD1 K phaffii AOX1 transcriptional OVA F3 promoter terminator Chicken HF-1 K phaffii PEX11 K phaffii AOX1 transcriptional OVA promoter terminator

(544) The first three cassettes were made to express a chicken OVA that comprises the amino acid sequence of chicken OVA (SEQ ID NO:61) fused in-frame with a nucleic acid encoding a secretion signal sequence; the expressed fusion protein has the amino acid sequence of (SEQ ID NO: 60). In each of the three cassettes, the Alcohol oxidase 1 (AOX1) promoter was placed upstream of the secretion signal sequence and a K. phaffii AOX1 transcriptional terminator was placed downstream of the OVA-encoding sequence. These cassettes were labeled GgOVA-A1, GgOVA-A2, and GgOVA-A3 and combined into a first plasmid.

(545) The fourth cassette included a chicken OVA coding sequence (which encodes SEQ ID NO: 61) fused in-frame with a nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 60) but with a dihydroxyacetone synthase (DAS2) promoter placed upstream of the secretion signal sequence and a K. phaffii AOX1 transcriptional terminator placed downstream of the OVA-encoding sequence. This construct was labeled GgOVA-D1.

(546) The fifth and sixth cassettes included the chicken OVA coding sequence (which encodes SEQ ID NO: 60) fused in-frame with a nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 61) but with a formaldehyde dehydrogenase (FLD) promote placed upstream of the secretion signal sequence and a K. phaffii AOX1 transcriptional terminator placed downstream of the OVA-encoding sequence. These cassettes were labeled GgOVA-F1 and GgOVA-F2 and were combined with GgOVA-D1 in a second plasmid.

(547) The seventh cassette included the peroxisome biogenesis (PEX11) promoter placed upstream of a Helper factor protein HAC1 coding sequence and a K. phaffii AOX1 transcriptional terminator placed downstream of the Helper factor sequence. This cassette was labeled HF-1 and was transformed into a third plasmid.

(548) The three plasmids were transformed stepwise into a background strain of Pichia pastoris. Genomic sequencing confirmed integration of the expression constructs and copy number of each construct is shown in Table 56 below.

(549) TABLE-US-00055 TABLE 56 Strain Genomic Composition Copies Strain Cassette integrated Chicken OVA GgOVA-A1 1 GgOVA-A2 1 GgOVA-A3 1 GgOVA-D1 2 GgOVA-F2 2 GgOVA-F3 2 HF-1 8

Example 34: Preparation of Recombinant Ovalbumin Expression Strains for Duck and Ostrich

(550) Expression Constructs: one cassette for expression of Anas platyrhynchos (duck) OVA and one cassette for expression of Struthio camelus (ostrich) OVA were created for expression in Pichia pastoris.

(551) TABLE-US-00056 TABLE 57 Expression cassettes of interest Strain Cassette Promoter ORF Terminator Duck ApdOVA K phaffii Duck K phaffii AOX1 OVA AOX1 OVA transcriptional promoter terminator Ostrich ScOVA K phaffii Ostrich K phaffii AOX1 OVA AOX1 OVA transcriptional promoter terminator

(552) One expression cassette was created for the expression of ostrich OVA. A nucleic acid encoding Struthio camelus OVA (SEQ ID NO: 130) was fused in-frame with a nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 131). The ostrich construct included the Alcohol oxidase 1 (AOX1) promoter placed upstream of the secretion signal sequence and a K. phaffii AOX1 transcriptional terminator was placed 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 57.

(553) One expression cassette was created for the expression of duck OVA. A nucleic acid encoding Anas platyrhynchos OVA (SEQ ID NO: 132) was fused in-frame with a nucleic acid encoding a secretion signal sequence (thereby encoding SEQ ID NO: 133). The duck cassette included the Alcohol oxidase 1 (AOX1) promoter placed upstream of the secretion signal sequence and a K. phaffii AOX1 transcriptional terminator was placed 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 58.

(554) TABLE-US-00057 TABLE 58 Strain genomic composition Copies Strain Cassette integrated Duck OVA ApdOVA 2 Ostrich OVA ScOVA 4

Example 35: Fermentation and Production of rOVA

(555) Fermentation: Strains for fermenting recombinant OVA (rOVA) were each cultured in a bioreactor at ambient conditions. A seed train for the fermentation process began with the inoculation of shake flasks with liquid growth broth. The inoculated shake flasks were kept in a shaker after which the grown P. pastoris was transferred to a production-scale reactor.

(556) To expand production, a seed vial of rOVA P. pastoris seed strain was removed from cryo-storage and thawed to room temperature. Contents of the thawed seed vials were used to inoculate liquid seed culture media in baffled flasks which were grown at 30? C. in shaking incubators. These seed flasks were then transferred and grown in a series of larger and larger seed fermenters (number to vary depending on scale) containing a basal salt media, trace metals, and glucose. Temperature in the seed reactors was controlled at 30? C., pH at 5, and dissolved oxygen (DO) at 30%. pH was maintained by feeding ammonia hydroxide, which also acted as a nitrogen source. Once sufficient cell mass was reached, the grown rOVA P. pastoris was inoculated into a production-scale reactor containing basal salt media, trace metals, and glucose.

(557) Like in the seed tanks, the culture was also controlled at 30? C., pH5 and 30% DO throughout the process. pH was again maintained by feeding ammonia hydroxide. During the initial batch glucose phase, the culture was left to consume all glucose and subsequently-produced ethanol. Once the target cell density was achieved and glucose and ethanol concentrations were confirmed to be zero, the glucose fed-batch growth phase was initiated. In this phase, glucose was fed until the culture reached a target cell density. Glucose was fed at a limiting rate to prevent ethanol from building up in the presence of non-zero glucose concentrations. In the final induction phase, the culture was co-fed glucose and methanol which induced it to produce rOVA via the pAOX promoters. Glucose was fed at an amount to produce a desired growth rate, while methanol was fed to maintain the methanol concentration at 1% to ensure that expression was consistently induced. Regular samples were taken throughout the fermentation process for analyses of specific process parameters (e.g., cell density, glucose/methanol concentrations, product titer, and quality). After a designated amount of fermentation time, secreted rOVA was collected and transferred for downstream processing.

(558) The fermentation broth containing the secreted rOVA was subjected to centrifugation at 12,000 rpm. The supernatant was clarified using microfiltration. To concentrate the protein and remove excess water, ultrafiltration at room temperature was used. An appropriately sized filter was used to retain the target rOVA while the compounds, salts, and water smaller than rOVA passed through the filter. To reduce the final salt content and conductivity in preparation for chromatography, the concentrated rOVA retentate was dialyzed at pH 3.5 until the final conductivity of the material was 1.7 mS/cm. The bulk of the purification was done using cation exchange chromatography at pH 3.5. Citrate buffer containing a high salt concentration of sodium chloride was used to elute the bound rOVA from the resin. To remove the excess salts, the eluant was finally dialyzed to make a final protein solution containing about 5-10% protein and 85-95% water. The final solution was sterilized by passing it through a 0.2 um bioburden filter. The water was evaporated using a spray dryer/lyophilizer at appropriate temperatures to produce a final powder containing about 80% protein.

Example 36: Preparation of Solubilized rOVA

(559) In this example, hydrophobic recombinant chicken rOVA was solubilized and passed through a 0.2 ?m filter.

(560) Recombinant rOVA was purified through ion exchange chromatography at pH 3.5 and was found to be insoluble. Sodium hydroxide was added to the solution to change the pH to 12.5 and solubilize the rOVA. The rOVA solution at pH 12.5 was passed through a 0.2 ?m filter. Following filtration, the pH was returned to 6.5 using hydrochloric acid and the rOVA was spray dried or lyophilized. This dried chicken rOVA was then used in the Examples below.

Example 37: Glycosylation of Gallus gallus rOVA

(561) In this example, Pichia-secreted rOVA was analyzed for glycosylation patterns.

(562) Native ovalbumin (nOVA) has two potential N-linked glycosylation sites (FIG. 23A). A single site of glycosylation at Asn-292 is found in the egg white. MALDI-TOF analysis has shown that the typical glycans on native OVA are organized as (Man)5(GlcNAc)5(Gal)1 (FIG. 23A) (Harvey et al., 2000). Analysis of glycans on rOVA showed a typical glycosylation pattern shown in (FIG. 23B).

(563) Pichia secreted chicken rOVA from the above Example was analyzed by gel electrophoresis migration and observed in three distinct forms (three white arrows pointing to rOVA in the Input lane below a) glycosylation-free, b) mono-glycosylated and c) di-glycosylated. Both the mono- and di-glycosylated glycosyl chains were cleaved from the mature rOVA protein using either of the endoglycanases EndoH or PNGaseF. Both the denatured 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 reactions (FIG. 28A).

(564) Pichia secreted chicken rOVA was subjected to standard analysis using Mass spectrometry. It was found to have five versions of N-linked Glycans (ManGlcNAc): high-mannose glycans of Man9 (?40%), Man10 (?47%) or Man11 (?13%) type of N-glycan structures (FIG. 28B).

Example 38: Comparison of Foaming Functionalities of Various Species rOVA

(565) In this example, chicken rOVA, duck rOVA and ostrich rOVA were evaluated for properties of foaming ability and foam retention.

(566) rOVA from ostrich and duck were produced, purified and lyophilized using methods similar to those set forth in Example 33 to 35. The ostrich rOVA and duck rOVA remained close to the acidic pH used for purification. Chicken rOVA was produced as set forth in Example 33 and solubilized at pH 12 before removing bioburden and returned to pH 6 before drying as set forth in Example 35.

(567) Lyophilized rOVA samples were blended into distilled water. Clarity and solubility of the rOVA solutions were then assessed visually. All samples were compared to chicken nOVA and chicken rOVA.

(568) Eleven mL of solution (7% w/v of protein) was created for each ostrich rOVA, chicken rOVA, and chicken nOVA. A 6 mL solution (7% w/v of protein) was created for duck rOVA due to limited availability of sample. Percent protein of the powders was used in the calculations to determine the amount necessary for a 7% solution. One mL of each solution was reserved before validation in a microtube for later use to test gelation. The samples were divided into 5 mL aliquots to be tested for foam capacity and stability.

(569) Each 5 mL aliquot was pipetted into a beaker and whipped using the Dremel on speed 3. After a stiff foam was achieved, the foaming time was recorded as well as the initial volume of the foam. Foam capacity was determined by measuring the initial volume of foam following the whipping and comparing against the initial volume of 5 mL. Foam Capacity (%) (volume of foam/initial volume)*100.

(570) The drainage was measured in 10 minute increments for 30 minutes to gather data for foam stability. The drained volume after 30 minutes was compared to the initial liquid volume (5 mL). Foam Stability (%): (Initial volume?drained volume)/initial volume*100.

(571) Chicken rOVA and ostrich rOVA were adjusted to pH 6 and tested again to ascertain effect of pH.

(572) Chicken nOVA quickly formed stiff white foam. Ostrich rOVA foamed after 15 seconds. Duck rOVA foamed after 20 seconds.

(573) TABLE-US-00058 TABLE 59 Foaming Parameters for rOVA in various species Foaming Foam Foam Time Capacity Stability Sample pH (s) (%) (%) Chicken nOVA 5.87 16 415 66.5 Chicken rOVA 6.49 101 257 61 Chicken rOVA 6.08 21 417 66.7 Chicken rOVA 3.5 28 472 100 Ostrich rOVA 3.7 22 490 81.5 Ostrich rOVA 5.73 55 275 58 (pH adjusted) Duck rOVA 4.3 26 400 70 Egg White 9.01 66.5 267.9 76.6

(574) Table 59 shows the results for foaming time, foaming capacity, 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 OVA from chicken, duck and ostrich generally had a similar or improved foaming capacity and foam stability as compared to egg white and these recombinant OVA proteins provided foaming capacity and foam stability between at least pH 3.5 and 6.5. Foam capacity and foam stability of rOVAs provide utility in compositions such as baked compositions.

Example 39: Comparison of Gelation of Various rOVA Species

(575) In this example, chicken, duck, and ostrich rOVA protein were evaluated for gelation properties. Gelation properties provide utility in applications such as cooked egg compositions.

(576) One mL of each OVA solution was reserved for use to test gelation. After the Dremel procedure and foaming test in Example 38 was completed, another 1 mL sample was extracted from the drained liquid (containing the 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. Samples were observed for gel formation.

(577) FIG. 29 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, ostrich 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 characteristics compared to chicken rOVA. Duck rOVA had gelation functionality close to that of natural egg white.

(578) These data showed that the favorable properties disclosed above for the recombinant chicken OVA (see Example 38) are also obtainable with recombinant OVAs from other species.

Example 40: Comparison of Foaming rOVA Solutions

(579) In this example, rOVA (chicken), solutions were compared to fresh egg white and evaluated for properties of foaming ability and foam retention.

(580) Lyophilized samples were blended into aqueous solution (distilled water) at different concentrations and pHs. Clarity and solubility of the solutions was then assessed visually for foaming ability and foaming retention.

(581) Protein solutions were created for each 4% rOVA, 7% rOVA, Fresh Egg White (12% protein), and 12% rOVA. Percent protein of the powders was used in the calculations to determine the amount necessary for each solution. 1 mL of each solution was reserved before validation in a microtube for later use to test gelation. The samples were divided into 5 mL aliquots to be tested for foam capacity and stability.

(582) Each 5 mL aliquot was pipetted into a beaker and whipped using the Dremel on speed 3. After a stiff foam was achieved, the foaming time was recorded as well as the initial volume of the foam. Foam capacity was determined by measuring the initial volume of foam following the whipping and compare against the initial volume of 5 mL. Foam Capacity (%)=(volume of foam/initial volume)*100.

(583) The drainage was measured in 10-minute increments for 30 minutes to gather data for foam stability. The drained volume after 30 minutes was compared to the initial liquid volume (5 mL). Foam Stability (%): (Initial volume?drained volume)/initial volume*100.

(584) TABLE-US-00059 TABLE 60 Foaming functionality for chicken rOVA Time Foaming Foam Spent Protein Capacity Stability Foaming Combination pH (%) (%) (s) Fresh Egg White 9.01 268 77 67 (12% protein) 4% OVA 6.05 333 57 25 7% OVA 6.03 333 66 19 12% OVA 6.05 313 69 18

(585) rOVA at 4%, 7% and 12% has greater foaming capacity, more foaming stability, and forms a foam more quickly than fresh egg white.

Example 41: Browning and Sheen Properties of rOVA

(586) In this example, the film formation properties of browning and sheen were evaluated for functionality of rOVA in a bread application. The functionality of rOVA for film formation was evaluated regarding the visual (sensory) characteristics of bread.

(587) Baking instructions: Yeast, sugar and warm water were mixed together in a small bowl and left to sit for five minutes. Flour was mixed into the yeast solution (30 seconds) until a firm dough was formed (mixed for 2 minutes at speed 3). Dough was kneaded on a floured board, placed into a greased bowl and left to rise for 45 minutes at 80? F. Dough was kneaded again, shaped into a 25 g mini loaf, and placed in a greased pan. The mini loaf was covered and allowed to rise for 30 minutes at room temperature. A volume of 0.75 g of the appropriate wash was applied to the top of the dough balls. Mini loaves were baked at 350? F. for eight minutes or until golden brown. Bread loaves' locations were switched in the oven at four minutes to achieve even baking of all samples.

(588) Lists of ingredients and their proportions used in the control bread and other samples are presented in the Table 61 below.

(589) TABLE-US-00060 TABLE 61 Bread Ingredients Ingredients % DI Water 41.77 Granulated Sugar 2.94 Bakers Yeast 1 All-Purpose Flour 53.62 Salt 0.67 Total 100.00

(590) The formulations used for protein of interest are shown in Table 62.

(591) TABLE-US-00061 TABLE 62 Ingredients used in wash formulations: Egg White Powder rOVA Ingredient % % DI water 90.67 91.30 Film forming agent 9.33 8.7

(592) Colorimetric assay: Individual sample pictures were analyzed for color data in the RGB spectrum using the Colorgrab application (Loomatix). Sample values were generated using a 2?2 cm cross-section taken from the center of the bread surface. RGB data was then converted to a CIELAB system using the online software www.colormine.org. CIELAB model is a color space system that expresses color in 3 values: L* for the lightness from black (0) to white (100), a* from green (?) to red (+), b* from blue (?) to yellow (+).

(593) TABLE-US-00062 TABLE 63 CIELAB results for bread post baking: L* a b* Negative Control 63.669 1.10972 25.4527 Whole egg 62.255 8.39894 45.57611 Commercial egg wash substitute 68.349 0.04763 34.7033 8% Egg white protein 76.831 2.58977 31.1123 8% rOVA 80.135 3.24212 31.53948

(594) rOVA and egg white protein samples had a higher L* value suggesting higher brightness or luminance. Control (no egg wash), commercial egg wash substitute and egg white protein samples had a low a* value suggesting lower redness or brownness as compared to whole egg, and rOVA samples. 8% egg white protein and rOVA samples also had similar b* values, suggesting similar yellow hues as compared to the other samples.

(595) Visual Inspection: The control sample looked pale, wrinkly and had no shine. The sample with whole egg had good browning, great sheen and a smooth surface. The commercial egg wash substitute sample had a smooth surface, slight noticeable sheen but lacked on browning. nOVA samples had good brown, smooth skin but lacked shine/sheen. Similarly, for rOVA samples, it had good browning, smooth skin but lacked shine/sheen. Photographs of the samples are shown in FIG. 30. In conclusion, rOVA was able to form a film comparable to a commercial egg wash substitute and nOVA.

Example 42: Adhesive Properties of rOVA

(596) In this example, rOVA was evaluated for the film formation property of adhesiveness functionality in a bread application creating a uniform film to aid addition of toppings (e.g., sesame seeds).

(597) Retention of sesame seeds: Retention of any topping on cake, bread, bagels or other baked goods is an intended consequence of an egg wash. Sesame seeds were used to evaluate the toping retention function of each film forming agent after baking.

(598) Dough balls and protein of interest were prepared as Example 41. Ten sesame seeds were applied to each dough ball after the application of wash and before baking. Retention of these sesame seeds was calculated based on the amount of seeds stuck to the bread after baking.

(599) The following results were obtained: The control sample with no egg wash had no binding capacity for the sesame seeds and zero sesame seeds were retained on the surface after baking. All other film-forming agents retained all 10 seeds post baking suggesting a 100% retention rate for toppings.

(600) TABLE-US-00063 TABLE 64 Retention levels of sesame seeds Egg white Negative Commercial Whole protein rOVA Samples Control egg wash egg (EWP) 8% Retention 0% 100% 100% 100% 100% level

Example 43: Combined Proteins rOVA Emulsions

(601) In this example, the emulsification functionality of recombinant proteins individually and in combination was observed in a salad dressing application.

(602) Lists of ingredients and their proportions used in the control dressing and other samples are presented in the Table 65 below.

(603) TABLE-US-00064 TABLE 65 List of Ingredients Ingredients for Salad dressing Canola oil DI water Vinegar Proteins of interest to be tested: nOVA-90% Protein content rOVA-92% Protein Content Egg white protein powder-85.71% Protein content

(604) Water, vinegar and protein of interest were combined in a mixer for 30 seconds. Oil was gradually added for 30 seconds and mixed for an additional 2.5 minutes. Samples were prepared without vinegar to test the emulsification capabilities of the proteins at neutral pH. pH of the solutions was adjusted using 1N sodium hydroxide. The emulsion was homogenized with a L5M-A homogenizer (Silverson) Square Hole shear head mixer for 9 minutes at 4000 rpm at ambient temperature.

(605) All emulsion samples were transferred into glass tubes, sealed with a plastic cap, 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 at the bottom phase with storage time. Physical stability was monitored for 3 days at both ambient and refrigerated conditions. The stability of the emulsion was expressed as: Creaming Index (CI)=(Ht/H0)*100. Where (H0) represents the initial emulsion height and the height of visible serum separation layer (Ht).

(606) List of ingredients and their proportions used in the control and other salad dressing samples with specific protein of interest are presented in Table 67.

(607) TABLE-US-00065 TABLE 66 List of Ingredients Acidic pH Egg white protein Neutral pH (EWP) nOVA rOVA Negative Egg white rOVA Negative 8% 8% 8% control protein 8% 8% control Ingredient % % % % % % % Canola oil 30 30 30 30 30 30 30 Water 54.67 55.11 55.30 64 60.67 61.30 70 Vinegar 6 6 6 6 0 0 0 Emulsifier 9.33 8.89 8.70 0 9.33 8.70 0 Total 100 100 100 100 100 100 100

(608) TABLE-US-00066 TABLE 67 Creaming Index Acidic pH Neutral pH 8% 8% 8% Negative 8% 8% Negative EWP nOVA rOVA control EWP rOVA control Day 0 0 0 0 40 0 0 40 Day 1 40 50 5 60 Ambient Day 1 40 50 5 90 Refrigerated Day 2 40 50 10 70 Ambient Day 2 40 50 10 90 Refrigerated Day 3 40 50 15 70 38 41 39 Ambient Day 3 40 50 15 90 38 40 43 Refrigerated

(609) Acidic pH results: On day 0, all samples except the negative control showed good emulsification properties. Thereafter, the samples were stored in ambient temperature or refrigerated temperatures to monitor stability. Samples with egg white protein (EWP) had a slight yellow appearance and separated on day 1 for both conditions of storage. Control samples separated immediately on day 1 for both conditions of storage. Eight percent nOVA also exhibited emulsion breakage on day 1, however, recombinant OVA exhibited good emulsion properties with only minimally noticeable separation. The emulsion remained equally stable until day 3 without any further separation observed. Overall, 8% rOVA performed significantly better than 8% nOVA. rOVA also exhibited better emulsion stability than EWP. Photographs of the samples are shown in FIG. 31A.

(610) Neutral pH results: Emulsion stability of rOVA was comparable to egg white proteins on day 0 and 3. Neither rOVA, nor egg white proteins were able to maintain emulsion stability over three days in refrigerated form or at ambient temperature. Photographs of the samples are shown in FIG. 31B.

Example 44: Foaming Functionality

(611) In this example, the foaming functionality of rOVA was observed in an alcohol-based drink (e.g., such as a Whiskey Sour which includes a foaming agent).

(612) Bourbon whisky, fresh lemon juice, simple syrup, and protein of interest were combined in a cocktail shaker and shaken for 15 seconds. Ice was added to the cocktail shaker and the mixture shaken for another 15 seconds. Shaken mixture was poured into a glass and observed.

(613) Formulations: Control formulation included natural egg white. The negative formulation was prepared without any egg white.

(614) TABLE-US-00067 TABLE 68 List of ingredients and the formulations Ingredient Ounces mL Bourbon Whiskey 2 59 Fresh Lemon Juice 0.75 22.125 Simple syrup 0.5 14.75 Egg white 0.5 14.75 Total 3.75 110.625

(615) The proteins of interest were used to substitute the natural egg white protein and the following formulations were used:

(616) TABLE-US-00068 TABLE 69 Protein formulation 7% 12% Ingredients rOVA rOVA rOVA 8.40 14.41 Water 91.60 85.59 Total 100 100

(617) The pH of the rOVA solutions was adjusted to pH 6 (with 1M NaOH) to provide optimal foaming performance.

(618) Original recipe used 0.5 oz egg white and the same proportion was used for recombinant protein testing. rOVA at 7% and 12% foamed well but no significant difference was observed between the two levels.

(619) Photographs of craft cocktails prepared with the samples are shown in FIG. 32.

Example 45: Burger Binding

(620) In this example, texture analysis was used to observe hardness attributes along with cohesiveness, springiness and chewiness of both raw and cooked vegan burgers made with rOVA and other binding agents.

(621) The objective of this example was to evaluate the binding functionality of rOVA. Parameters such as appearance (how well the burger held together), textural aspects such as cohesiveness, springiness, chewiness and hardness were evaluated and compared against egg white, nOVA and commercially used non-protein binder.

(622) Materials: Dry base 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)), Binding agent/Protein of interest. Wet ingredients: Canola oil, coconut oil, Water. Binding agents of interest to be tested: Natural egg white protein (NEW), Methylcellulose (MC), nOVA 90% Protein content, rOVA (chicken) 92% Protein Content.

(623) Mixing: Extruded soy protein 1 was mixed with ?rd amount of water for 2.5 min. The remaining extrudated samples and water were combined with the previous mix for another 7.5 min. The blend was chilled in the freezer for 10 minutes. The binding agent was added and mixed in for 30 seconds. Canola and coconut oil blend was added and mixed for 30 seconds. The mixture was chilled in the freezer for 5 minutes, then molded into 5 g burger forms and frozen.

(624) Cooking: The frozen burger samples were thawed in the refrigerator to a 4? C. internal temperature. The samples were cooked on a griddle set at 350? F. for 5-6 min until an internal temperature of 165? F. was reached.

(625) Formulations: List of ingredients and their proportions used in the control and other experimental burger samples, with specific protein of interest, are presented below in Table 70.

(626) TABLE-US-00069 TABLE 70 List of Ingredients. Control- Natural Methylcellulose Egg White nOVA rOVA Ingredients % % % % Extruded soy protein 1 5 5 5 5 Extruded soy protein 2 13 13 13 13 Extruded soy protein 3 8 8 8 8 Binding agent 0.7 25 5 5 Canola oil 12 12 12 12 Coconut oil 6.5 6.5 6.5 6.5 Water 54.8 30.5 50.5 50.5 Total 100.00 100.00 100.00 100.00

(627) Texture Analysis: Texture analysis was performed to analyze the attributes of vegan burgers against the control. Texture analysis was used to quantify hardness attributes along with cohesiveness, springiness and chewiness.

(628) The textural properties of vegan burgers were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). The test parameters were used are presented in Table 29.

(629) TABLE-US-00070 TABLE 71 Test parameters used for three-point bend test to measure hardness of vegan burgers using a CT3 Brookfield Texture Analyzer Test type Texture Profile Analysis (TPA) Probe TA52 (Mohrs shear blade) Base Fixture TA-Base Fixture Target type Distance Target value 5 mm Trigger load 15 g Test speed 0.5 mm/s Post test speed 4.5 mm/s Textural properties Hardness 1 (g), Hardness 2 (g), Cohesiveness, Springiness, Chewiness Average Sample 25 mm *12.5 mm dimensions (Diameter*Height)

(630) The frozen samples were thawed in the refrigerator to a 4? C. internal temperature and tested for raw binding. The thawed samples were also cooked and used to measure the cooked binding values.

(631) Findings for raw binding: In terms of hardness, rOVA was significantly higher than methylcellulose and natural egg white and no difference was observed between nOVA and rOVA. All the samples were similar in terms of cohesiveness and springiness. rOVA exhibited significantly more chewiness than methylcellulose and natural egg white. Results are presented in Table 72.

(632) Table 72: Texture (TPA) results for raw binding in terms of hardness, cohesiveness, springiness and chewiness. Data that does not share the same letter within a specific attribute is significantly different from each other (p<0.05). The results were averaged over n=3.

(633) TABLE-US-00071 TABLE 72 Texture (TPA) results for raw binding in vegan burgers Sample Hardness 1 (g) Hardness 2 (g) Cohesiveness Springiness Chewiness methylcellulose 58.27 ? 10.17 (a) 40.53 ? 9.59 (a) 0.12 ? 0.07 (a) 0.42 ? 0.05 (a) 3.0 ? 1.56 (a) 0.7% natural egg 45.27 ? 9.45 (a) 33.20 ? 5.02 (a) 0.21 ? 0.03 (a) 0.34 ? 0.1 (a) 3.33 ? 1.26 (ab) white 25% nOVA 5% 81 ? 4.39 (ab) 44.27 ? 6.45 (a) 0.18 ? 0.01 (a) 0.46 ? 0.04 (a) 6.93 ? 1.12 (bc) rOVA 5% 145.07 ? 52.85 (b) 62.80 ? 21.70 (a) 0.13 ? 0.01 (a) 0.47 ? 0.04 (a) 8.23 ? 1.86 (c)

(634) Findings for cooked binding: rOVA exhibited significantly higher hardness values than methylcellulose and natural egg white. All the samples were similar to each other in terms of cohesiveness. For springiness, methylcellulose samples exhibited significantly lower values than natural egg white, nOVA and rOVA. Both nOVA and rOVA samples exhibited higher values chewiness values than methylcellulose. Results are presented in Table 31.

(635) Table 73: Texture (TPA) results for cooked binding in terms of hardness, cohesiveness, springiness and chewiness. Data that does not share the same letter within a specific attribute is significantly different from each other (p<0.05). The results are averaged over n=3.

(636) TABLE-US-00072 TABLE 73 Texture (TPA) results for cooked binding in vegan burgers Sample Hardness 1 (g) Hardness 2 (g) Cohesiveness Springiness Chewiness Methylcellulose 281.73 ? 154.7 (a) 215.80 ? 161.84 (a) 0.37 ? 0.07 (a) 0.69 ? 0.05 (a) 76.03 ? 55.15 (a) 0.7% Natural egg 390.33 ? 158.15 (a) 304.27 ? 55.83 (a) 0.57 ? 0.11 (a) 0.80 ? 0.03 (b) 178.07 ? 65.85 (ab) white 25% nOVA 5% 617.07 ? 197.49 (ab) 464.07 ? 135.33 (ab) 0.56 ? 0.08 (a) 0.81 ? 0.05 (b) 285.5 ? 104.72 (bc) rOVA 5% 922.0 ? 96.71 (b) 1712.33 ? 78.23 (b) 0.51 ? 0.08 (a) 0.86 ? 0.02 (b) 398.13 ? 44.37 (c)

Example 46: Egg White Patty

(637) In this example, the suitability of inclusion of native and recombinant protein OVA in an egg white patty application as an example of cooked egg systems was evaluated. Parameters such as nutritional value of fresh egg white when substituted by OVA and effects on texture (in terms of functionality) and appearance were evaluated.

(638) TABLE-US-00073 TABLE 74 List of ingredients used to prepare egg white patties Ingredients Dry base ingredients: Gellan gum (LT100-Modernist pantry), baking powder (Trader Joe's), salt (The spice club), Sodium Alginate (CP Kelco), Psyllium (CFF) Wet ingredients: Coconut oil, canola oil (Crisco), tapioca syrup (Ciranda), pineapple yellow AET color (Sensient), water Proteins of interest to be tested: Natural egg white nOVA (Neova Technologies)-90% Protein content rOVA (Clara Foods: 008USU_CW)-86.1% Protein Content

(639) Mixing: The dry ingredients from Table 74, except sodium alginate were mixed together. The tapioca syrup, sodium alginate and lemon-yellow color were blended separately in water. All ingredients were mixed with oil and vortexed till all ingredients are dissolved. The mixture was allowed to equilibrate by allowing to stand for 10 minutes.

(640) Cooking: A griddle was used to cook the samples. The griddle was set to 250? F. and ? inch diameter ring molds were used to cook samples. The molds were sprayed with oil and the mixture was poured into the molds. ? ice cubes were added to the molds to generate steam. The patties were allowed to cook and another ice cube was added. The patties were cooked for 5 minutes and the lid was opened. The ring molds with the cooked samples to serving plates.

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

(642) TABLE-US-00074 TABLE 75 Test parameters used for TPA test to measure textural properties of patty: Test type TPA Test parameters 50% deformation Probe TA4 (38 mm diameter cylinder) Base Fixture Base fixture Trigger load 5 g Test speed 2 mm/s Textural properties Hardness (g), Adhesiveness, Cohesiveness, Chewiness, Gumminess Sample dimension (Height) * ~12 mm * 12 mm (Diameter)
Results:

(643) TABLE-US-00075 TABLE 76 Texture Analyzer results Sample/ Hardness Hardness Attribute 1 (g) 2 (g) Adhesiveness Fracturability Cohesiveness Gumminess Chewiness Natural 726.3 ? 652 ? 0.375 ? 726.7 ? 0.765 ? 555.1 ? 33.75 ? egg 6.65 a 15.56 a 0.11 a 7.21 a 0.05 a 44.55 a 0.05 ab white nOVA 817.6 ? 761.3 ? 0.315 ? 817.6 ? 0.71 ? 583.55 ? 50.95 ? 174.51 a 171.54 a 0.02 a 174.51 a 0.01 a 133.86 a 9.40 a rOVA 869.9 ? 747.1 ? 0.185 ? 869.9 ? 0.55 ? 484.65 ? 25.53 ? 58.12 a 50.49 a 0.16 a 58.12 a 0.04 a 3.46 a 3.82 b Data that does not share the same letter for a given attribute is significantly different from each other (p < 0.05)
Data that does not share the same letter for a given attribute is significantly different from each other (p<0.05)

(644) Findings: All the samples, natural egg white, nOVA and rOVA were statistically similar in terms of hardness, adhesiveness, fracturability, cohesiveness and gumminess. For chewiness, natural egg white patty was similar to nOVA and rOVA individually, however, nOVA and rOVA were statistically different from each other. nOVA had higher chewiness values as compared to other samples. Overall, OVA protein, in both native and recombinant form, provides a good substitute to natural egg white in a non-animal patty (cooked egg application). rOVA liquid formulation was thicker in viscosity than nOVA sample and egg white sample. Results are shown in FIG. 34.

Example 47: Meringue

(645) The functionality of rOVA in a meringue food system compared to fresh egg white was evaluated in this example.

(646) Material:

(647) rOVA (Lyo 008; pH: 6.7 as is) Fresh egg white (pH: 9 as is) Sugar (C&H Sugar, Pure Cane, Granulated white) Xanthanpre hydrated TicaxanTic Gums TEC (Triethyl Citrate) SLS (Sodium lauryl sulfate) Kitchen Aid, Classic Plus Breville BOV800XL Smart Electric Oven

(648) Method: Egg white was separated from the egg yolk carefully at the refrigerator temperature and let egg whites get to the room temperature before whipping. rOVA powder, SLS, Xanthan gum and TEC were reconstituted in DI water at the room temperature. The mixture was whipped for 30 seconds at speed 5 (to obtain a homogeneous solution), then mixed at speed 8 until soft peaks formed. While beating constantly, sugar was added gradually and beat at high speed after each addition until sugar was dissolved before adding the next. Mixing was continued until a glossy and firm peak was formed. Oven (Breville BOV800XL Smart Electric Oven) was heated to 200? F.; meringues were baked for 70 minutes (or until light and crisp but not brown. After cooling, meringues were stored in an airtight container. Whipping time to produced firm foam for each protein solution was recorded.

(649) TABLE-US-00076 TABLE 77 Formulations rOVA8.3% + SLS + rOVA8.3% + TEC + Fresh egg white Xanthan gum Xanthan gum Percentage Percentage Percentage Ingredients % Ingredients % Ingredients % Fresh egg 70.6 rOVA 9.5 rOVA 9.5 white Sugar 29.4 Sugar 29 Sugar 29 Water 61.3 Water 61.3 Xanthan 0.1 Xanthan 0.1 gum gum SLS 0.1 TEC 0.048 Total 100 Total 100 Total 100 weight weight weight

(650) TABLE-US-00077 TABLE 78 Physical parameters of meringues Fresh rOVA 8.3% + SLS + rOVA 8.3% + TEC + Parameter egg white Xanthan gum Xanthan gum weight loss % * 60 ? 2 60 ? 1.1 58 ? 2.5 volume (ml) 7 ? 1.5 7.3 ? 1.5 7.9 ? 2 foam density 0.19 0.2 0.22 (g/ml) Meringue 0.056 ? 0.014 0.074 ? 0.02 0.064 ? 0.018 density (g/ml) * Average ? standard deviation (n = 6)

(651) Findings: rOVA produces meringue that is comparable to fresh egg white sample in terms of physical parameters. The appearance of rOVA meringues were visually better than fresh egg white controls. The ridges were more well defined in rOVA meringue and the samples were whiter compared to the fresh egg white control. Results are shown in FIG. 35.

Example 48: Effect of pH on Gelation Characteristics

(652) The effects of different pH conditions on the gelation characteristics of rOVA compositions in comparison to fresh egg white was evaluated in this example.

(653) TABLE-US-00078 TABLE 79 Materials: Ingredients DI water, 1N Hydrochloric acid, 1N Sodium hydroxide, 3N Sodium hydroxide Proteins of interests rOVA (008USU_CW-86.1% protein content) Egg white protein (Modernist pantry-85.71% protein content)
Method:

(654) 7% protein solution was prepared for both rOVA and egg white protein

(655) Based on the native pH, the pH of the solution was adjusted to pH 3, 4, 5, 6 with 1N HCl pH was also adjusted to the alkaline spectrum of pH 7, 8, 9, 10, 11 and 12 with microliter amounts of 1N and 3N sodium hydroxide

(656) All solutions were gelled at 85? C. for 5 min and then cooled at room temperature

(657) All the gels/solutions were taken out and evaluated visually for gel characteristics

(658) TABLE-US-00079 TABLE 80 Results: pH was recorded as follows before any pH adjustments: Sample pH 7% EWP 6.98 7% rOVA 6.82

(659) Findings: Egg white protein exhibited gelling properties at all pH's while forming firm gels at pH 4-10. The solutions for both EWP and rOVA at pH 11 and pH 12 were clear liquids, however, only EWP gelled into clear gels, while rOVA remained in solution at pH 11 and 12. rOVA 7% solutions gelled at pH 6, 7, 8 and 9. Dramatic increase in viscosity was observed for rOVA solutions at pH 5 and lower. All EWP gels had a strong egg-like smell, while for rOVA, only solutions/gels for pH 9-12 had an egg-like smell. pH 3.5-8 for rOVA did not have any characteristic smell properties. EWP and rOVA both gelled at pH 6-9; however, EWP gels were stronger and firmer than rOVA gels. Overall, although EWP exhibited better gelling properties than rOVA over a broader pH spectrum, it came with the presence of a strong egg-like smell. rOVA provided gelling properties in the pH 6-8 range and provided sensory neutrality (e.g., no smell). At pH 8 and 9, rOVA provided clear firm gel which can have unique value proposition in embodiments requiring transparent visual appearance.

Example 49: Protein Bars

(660) rOVA was used as a protein source in a protein bar application and compared to eff white proteins and nOVA.

(661) Preparation Instructions:

(662) In a small mixer, dates, nuts were chopped/blended. Dates, nuts, cocoa and the protein of interest were added in a mixing bowl till a homogenous mixture was formed. The mixture was split into two equal parts and one part was tested as the unbaked version. The other half was baked in an oven at 350 F for 10 minutes.

(663) TABLE-US-00080 TABLE 81 List of Ingredients and their proportions used in control formulation: Amount Ingredients (%) Dates 78.53 Nuts 17.47 Cocoa 4 Total 100

(664) For formulations with inclusion of protein powders, the dates and nuts inclusion was reduced, however keeping the dates: nuts ratio constant at a 4.5 level.

(665) TABLE-US-00081 TABLE 82 List of Ingredients and their proportions used in egg white protein formulations: 4% 8% 12% 16% 23% Ingredients protein protein protein protein protein Dates 74.73 70.93 67.13 63.27 56.62 Nuts 16.60 15.73 14.87 14.07 12.54 Cocoa 4 4 4 4 4 Protein powder 4.67 9.33 14 18.67 26.84 Total 100 100 100 100 100

(666) TABLE-US-00082 TABLE 83 List of Ingredients and their proportions used in nOVA formulations: 4% 8% 12% 16% 23% Ingredients protein protein protein protein protein Dates 75.02 71.25 67.67 64.01 57.72 Nuts 16.54 15.86 13.33 14.20 12 Cocoa 4 4 4 4 4 Protein powder 4.45 8.89 15 17.78 28.75 Total 100 100 100 100 100

(667) TABLE-US-00083 TABLE 84 List of Ingredients and their proportions used in rOVA formulations: 4% 8% 12% 16% 23% Ingredients protein protein protein protein protein Dates 74.60 70.66 66.76 62.86 55.92 Nuts 16.6 15.73 14.83 13.93 12.52 Cocoa 4 4 4 4 4 Protein powder 4.8 9.60 14.40 19.21 27.57 Total 100 100 100 100 100

(668) Texture analysis: The textural properties of the protein bar (baked and unbaked) were measured using a CT3 Brookfield Texture Analyzer (1500 g load cell). A three point bend test was used to snap, bend and measure the hardness of the protein bar. One sample for each protein inclusion level was analyzed. The following test parameters were used:

(669) TABLE-US-00084 TABLE 85 Test parameters used for three-point bend test to measure hardness of crackers using a CT3 Brookfield Texture Analyzer Test type Rupture test Probe TA7 blade Base Fixture TA-TPB Trigger load 5 g Correction load 30 g Test speed 3 mm/s Sample rate 30 points/sec Distance between support arms 2.5 cm Textural properties Hardness (g)

(670) TABLE-US-00085 TABLE 86 Texture analysis test results for unbaked protein bar samples: (n = 1) Hardness (g) for protein inclusion levels Sample Control (0%) 4% 8% 12% 16% 23% Egg white 113.9 168.8 319.2 422.8 475 597.8 protein nOVA 204.8 231 408 420.05 443.8 rOVA 182 222.6 314.4 418 689.8

(671) TABLE-US-00086 TABLE 87 Texture analysis test results for baked protein bar samples: (n = 1) Hardness (g) for protein inclusion levels Sample Control (0%) 4% 8% 12% 16% 23% Egg white 902.7 1499.6 1484 1561 1553.4 1609.4 protein nOVA 1505.4 1523.8 1542.2 1585 1662.8 rOVA 1485.2 1530 1561 1522.4 1552.8

(672) For the unbaked samples, the control sample with no protein had the lowest hardness values. For all the proteins of interest, EWP, nOVA and rOVA, hardness values increased with increasing protein content. Egg white protein samples had higher hardness values than nOVA and rOVA samples at 8, 12, 16 and 23%. nOVA samples had minimal increase in hardness from 12-23% protein inclusion. nOVA and rOVA sample hardness was comparable at 4, 8, 12 and 16%. However, rOVA had a much higher hardness value for 23% protein inclusion.

(673) Overall, the hardness of the baked samples was much higher than the unbaked samples. The control sample had the lowest hardness. All the samples with protein inclusions were much harder even at lower protein inclusion rates. The upper threshold limit (load cell) for the TA unit is 1500 g. All the baked protein samples reached the threshold value making it difficult to identify subtle differences between the samples. nOVA and rOVA sample hardness was comparable at 4, 8, 12 and 16% for both, unbaked and baked protein bar. Photos are shown in FIG. 36.

(674) While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

INCORPORATION BY REFERENCE

(675) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

(676) Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.