(12) United States Patent (10) Patent No.: US 8,133,521 B2 Strohbehn Et Al

USOO8133521B2

(12) United States Patent (10) Patent No.: US 8,133,521 B2 Strohbehn et al. (45) Date of Patent: *Mar. 13, 2012

(54) METHOD OF SEPARATING COMPONENTS (56) References Cited OF TECHNICAL EGGS, EDIBLE EGGS, YOLKAND WHITES AND PRODUCTS U.S. PATENT DOCUMENTS THEREFROM 4,112,123 A 9, 1978 Roberts 4,330,446 A 5/1982 Miyosawa 5,037,661. A * 8/1991 Merchant et al...... 426/47 (75) Inventors: Ronald E. Strohbehn, Nevada, IA (US); 5,367,054 A 11/1994 Lee Jesse I. Figgins, Ames, IA (US) 5,399,331 A 3/1995 Loughrey 2005/0176122 A1 8, 2005 Lihime et al. (73) Assignee: Biova, L.L.C., Johnston, IA (US) 2006/0223986 A1 10, 2006 Chou OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 International Search Report, Biova, L.L.C., PCT/US08/50653 dated May 9, 2008, 1 page. U.S.C. 154(b) by 1100 days. Search Report for co-pending PCT/US2008/050653 listing relevant This patent is Subject to a terminal dis art, May 9, 2008. claimer. * cited by examiner (21) Appl. No.: 11/971,796 Primary Examiner — Rosanne Kosson (74) Attorney, Agent, or Firm — McKee, Voorhees & Sease, (22) Filed: Jan. 9, 2008 P.L.C. (65) Prior Publication Data (57) ABSTRACT US 2008/O166447 A1 Jul. 10, 2008 The present invention relates to methods for the separation of various components from eggs. More particularly, the present Related U.S. Application Data invention relates to methods for the separation of proteins and lipids from eggs, including technical eggs (inedible) or edible (60) Provisional application No. 60/884,145, filed on Jan. eggs, yolks or whites, which comprises cross-linking the 9, 2007. lipids of eggs with a cross-linking reagent. In an embodiment, the method includes separating the proteins from the cross (51) Int. C. linked lipids. In an embodiment, the method includes the A23. I/09 (2006.01) separation of various components associated with the cross A2.3L I/32 (2006.01) linked lipids. The methods disclosed herein allow for the C07K L/4 (2006.01) isolation of multiple different components from the egg in an C07K L/6 (2006.01) efficient, cost-effective manner without compromising the C07K L/30 (2006.01) recovery process of the components or their Subsequent util C07K L/34 (2006.01) ity in various applications or compositions. The compositions (52) U.S. C...... 426/480; 426/614; 426/442; 530/412: and isolated components obtained by the methods of the 530/417:530/420; 530/422 invention can be used in pharmaceutical, medical, nutritional, (58) Field of Classification Search ...... 426/480, cosmetic or health applications. 426/614, 442; 530/420, 422, 412, 417 See application file for complete search history. 16 Claims, 17 Drawing Sheets

Cross-Linking pH at which Reagent Cross-Linking OCCurs Separation 1 N Cross-Linked Lipids Proteins comprising albumin, OVotransferrin and small molecular weight proteins. U.S. Patent Mar. 13, 2012 Sheet 1 of 17 US 8,133,521 B2

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Technical Eggs, Edible Eggs, Egg Yokes, or Egg Whites - Incoming Solids = 1.1% dilute w/H2O Solids diluted to 2.2-3.0% 1 part eggs: 5 ports H2O %6 Nalco 111.5, pH adj. pH 6.8 w/2N HC

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FIG.3A U.S. Patent Mar. 13, 2012 Sheet 4 of 17 US 8,133,521 B2

Technical Eggs, Edible Eggs, Egg Yokes, or Egg Whites - incoming Solids = 11% dilute w/H2O Solids diluted to 2.2-3.0% 1 part eggs; 5 parts H2O %6 Nalco 111.5, pH adj. pH 6.8 w/2N HC Centrifuge

Egg Lipids Protein Supernate reconstitute 1 part stir 1 hr., mix lipid: 4 parts 0.3M ppt. thoroughly Saline (W/w)

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4 Precipitate 4 Supernate 10% adj. to pH 2.5 Acetic Acid w/HC Conc./Dialyze Stir 1 hr.

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5 Precipitate 5 Supernate pH adj. 10% pH 5.0 MeOH or EtOH NaOH 50% Centrifuge

Stir 1 hr. Mix thoroughly 7A white lipid extract 7B Supernate Reconstitute pellet in Conc./Dialyze/Spray Dry 10% Ocid

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S S. S S S US 8,133,521 B2 1. 2 METHOD OF SEPARATING COMPONENTS requiring whipping or binding of egg products. Additionally, OF TECHNICAL EGGS, EDIBLE EGGS, the Substitution of egg powders allows manufacturers to add YOLKAND WHITES AND PRODUCTS additional nutritional value to the products. In nutraceutical, THEREFROM cosmetic and pharmaceutical applications, lysozyme, avidin and ovotransferrin can be extracted from the egg albumen. CROSS-REFERENCE TO RELATED Such products can be utilized as natural antimicrobial APPLICATIONS (lysozyme), food preservatives in cheese and wine, a nutri tional ingredient in iron-fortified products (Ovotransferin), as This application claims priority under 35 U.S.C. S 119 of a well as use in pharmaceuticals. Additionally, avidin may be provisional application Ser. No. 60/884,145 filed Jan. 9. 10 used in biotechnology research for diagnostic kits. 2007, which application is hereby incorporated by reference An individual analysis of some of the eggs components in its entirety. follows, including some of particular commercial interest. Ovotransferrin is a neutral glycoprotein synthesized in the FIELD OF THE INVENTION hen Oviduct and deposited in the egg white albumen at a ratio 15 of approximately 12% of the total protein content. Ovotrans This invention relates to a process of recovery of compo ferrin is an 80 kDa matrix covalent dimmer protein observed nents from whole eggs, technical or edible eggs, yolks or at high concentration in the uterine fluid at the initial stage of whites using various physical and chemical treatments and shell mineralization and is also present in extracts from dem has particular but not exclusive application to their recovery ineralized eggshell, the sites of calcite nucleation. Northern from avian eggs. blotting and RT-PCR demonstrate that ovotransferrin is expressed in the proximal Oviduct, and at a lower magnitude BACKGROUND OF THE INVENTION in the distal oviduct. Ovotransferrin is also present in the tubular gland cells of the uterus. Ovotransferrin is also An avian egg, in general, comprises an eggshell, two egg thought to impact calcium carbonate crystals and calcite mor shell membranes, and an egg white and an egg yolk. Both the 25 phology, Suggesting that ovotransferrin has a dual role, a egg white and the egg yolk contain nutritionally physiologi protein influencing nucleation and growth of calcite crystals cally valuable components such as proteins, for instance oval and as a bacteriostatic filter to reinforce its inhibition of bumin, ovotransferrin, ovomucin, and others, as well as lip Salmonella growth in egg albumen. ids. The egg white is known as the albumen (Latin, albus, for Ovotransferrin can be used as a nutritional ingredient in “white') and is comprised of four alternating layers of thick 30 iron-fortified products such as iron Supplements, iron forti and thin albumen contain approximately 40 different pro fied mixes for instant drinks, sport bars and protein Supple teins, which are the main components of the egg white in ments and iron-fortified beverages. There is also extensive addition to water. The egg white is approximately two-thirds evidence of an antibacterial effect of ovotransferrin based on of the total eggs weight out of its shell with 90% of that iron deprivation, iron being an essential growth factor for weight coming from water. The remaining weight of the egg 35 most micro-organisms. Ovotransferrin tightly binds transi white comes from protein, trace minerals, fatty material, Vita tion metals (FeIII, CuIII, Al III) with a binding log con mins, and glucose. The U.S. large eggs white typically stant of about 15 at pH 7.0 and higher. In vivo, ovotransferrin weighs 38 grams with 3.9 grams of protein, 0.3 grams of demonstrates therapeutic properties against acute enteritis in carbohydrate and 62 milligrams of Sodium. The most pre infants (Corda, R., et al., Conalbumen in the treatment of dominant proteins and their approximate respective percent 40 acute enteritis in the infant, Int. J. Tiss. Reac. V(1), 117-123 age of composition of the albumen include the following: (1983)). Ovalbumin (54%). Ovotransferrin (12%). Ovomucoid Ovalbumin is the main protein found in egg white, making (11%), Globulins (8%) (Function-Plugs defects in mem up close to 60% of the total protein. The ovalbumin functions branes, shell), Lysozyme (3.5%). Ovomucin (1.5%), Avidin as nourishment and blocks digestive enzymes. It belongs to (0.06%), and others (10%). There are also opaque ropes of 45 the serpin Superfamily of proteins, although unlike the major egg white, called the chalazae, that hold the yolk in the center ity of serpins it is unable to inhibit any proteases. The oval of the egg. They attach the yolk's casing to the membrane bumin protein of chickens is made up of 385 amino acids, and lining the eggshell. Additionally, there is a Vitelline mem its relative molecular mass is 45kDa. It is a glycoprotein with brane, a clear casing that encloses the yolk. 4 sites of glycosylation. It is secreted from the cell, despite The egg yolk makes up about 33% of the liquid weight of 50 lacking an N-terminal leader sequence. The function of oval the egg, a majority of the calories contained in the egg, most bumin is unknown, although it is presumed to be a storage of the minerals (iron, phosphorus, calcium, thiamine, and protein. riboflavin) and virtually all of the fat soluble vitamins (A, D, Ovalbumin is useful in cases of poisoning by heavy metals E and K). The approximate composition (by weight) of the (such as iron) as a chelator to heavy metals by trapping the most prevalent fatty acids in egg yolk include: unsaturated 55 metal ions within the sulfhydryl bonds of the protein and fatty acids (Oleic acid (47%), Linoleic acid (16%), Palmi preventing the absorption of the metals into the gastrointes toleic acid (5%), Linolenic acid (2%)) and saturated fatty tinal tract and prevents poisoning. Additionally, it is an impor acids (Palmitic acid (23%), Stearic acid (4%), Myristic acid tant protein in several different areas of research. Ovalbumin (1%)). The yolk is also a source of lecithin, a common emul is commonly used in general studies of protein structure and sifier. 60 properties. It is also utilized in studies of serpin structure and Each of the various egg components has utility in a variety function (the fact that ovalbumin does not inhibit proteases of industries. Specifically, the egg component's utility spans means that by comparing its structure with that of inhibitory across the food, nutraceutical, pharmaceutical and cosmetic serpins, the structural characteristics required for inhibition industries. Egg powders are utilized in a variety of food can be determined). Ovalbumin is also used in proteomics, as applications. For example, egg yolk and whole egg powders 65 a molecular weight marker for calibrating electrophoresis and egg albumen powders are used in a variety of baked gels. Additionally, ovalbumin is utilized in immunology stud goods, mayonnaise, quiches and other food applications ies as a stimulator of allergic reactions in test Subjects. US 8,133,521 B2 3 4 Lysozyme is a 14.4kDa, 129 aminoacid residue enzyme is a protein having three isoforms (TGF-B1, TGF-B2 and that damages bacterial cell walls by catalyzing hydrolysis of TGF-B3). It is synthesized in a wide variety of tissues. The 1,4-beta-linkages between N-acetylmuramic acid and TGF-B family is part of a superfamily of proteins known as N-acetyl-D-glucosamine residues in a peptidoglycan and the transforming growth factor beta Superfamily, which between N-acetyl-D-glucosamine residues in chitodextrins. 5 includes inhibins, activin, anti-millerian hormone, bone Large amounts of lysozyme can be found in egg whites and morphogenetic protein, decapentaplegic and Vg-1. TGF-B the eggshell, as originating from the uterine fluid. Lysozyme controls proliferation, differentiation, and other functions in protein is highly concentrated in the limiting membrane cir most cell types. It can also act as a negative autocrine growth cumscribing the egg white and forms the innermost layer of factor. TGF-B induces apoptosis in numerous cell types and the shell membranes. It is also present in the shell membranes, 10 and in the matrix of the calcified shell. Lysozyme provides therefore plays a crucial role in the regulation of the cell cycle. anti-microbial protection as it digests bacterial cell walls and Insulin-like growth factors (IGFs) are polypeptides with protective structural properties to an eggshell. high sequence similarity to insulin. They are part of a com Lysozyme functions by attacking peptidoglycans (found in plex system cells use to communicate with their physiologic the cells walls of bacteria, especially Gram-positive bacteria) 15 environment, consisting of two cell-surface receptors (IGF1R and hydrolyzing the glycosidic bond that connects N-acetyl and IGF2R), two ligands (IGF-1 and IGF-2), a family of six muramic acid with the fourth carbon atom of N-acetylglu high-affinity IGF binding proteins (IGFBP 1-6), as well as cosamine. It does this by binding to the peptidoglycan mol associated IGFBP degrading enzymes, known as proteases. ecule in the binding site within the prominent cleft between its Most cells are affected by IGF-1, especially cells in muscle, two domains. This causes the Substrate molecule to adopt a cartilage, bone, liver, kidney, nerves, skin, and lungs. In addi strained conformation similar to that of the transition state. tion to the insulin-like effects, IGF-1 can also regulate cell The lysozyme binds to a hexasaccharide and then distorts the growth and development, especially in nerve cells, as well as 4th Sugar in hexasaccharide (the D ring) into a half-chair cellular DNA synthesis. IGF-2 is secreted by the brain, kid conformation. In this stressed state the glycosidic bond is ney, pancreas and muscle in mammals and birds. easily broken. The amino acid side chains glutamic acid 35 25 Transfer factors are immune messenger molecules found in (Glu35) and aspartate 52 (Asp52) have been found to be all higher animals. They are found in white blood cells, colos critical to the activity of this enzyme. Glu35 acts as a proton trum, and eggs. They transfer immunity against many patho donor to the glycosidic bond, cleaving the C-O bond in the gens that would otherwise kill the offspring of a species. They Substrate, whilst Asp52 acts as a nucleophile to generate a derive from leukocyte lysates of immune donors which can glycosyl enzyme intermediate. The glycosyl enzyme inter 30 transfer strong local and systemic cellular immunity to non mediate then reacts with a water molecule, to give the product immune recipients. Transfer factors could be utilized to of hydrolysis and leaving the enzyme unchanged. The amino immunity between compatible sources. They can be utilized acid side chains glutamic acid 35 (Glu35) and aspartate 52 by one prone to illness, i.e., colds, Sore throats, ear infections, (Asp52) have been found to be critical to the activity of this influenza, and numerous other ailments and diseases, in place enzyme. Glu35 acts as a proton donor to the glycosidic bond, 35 of conventional commercially available immune boosters, cleaving the C Obond in the substrate, whilst Asp52 acts as preventions or treatments. a nucleophile to generate a glycosyl enzyme intermediate. Sialic acids are a group of naturally occurring N- and The glycosyl enzyme intermediate then reacts with a water O-acyl derivatives of the deoxyamino Sugar neuraminic acid. molecule, to give the product of hydrolysis and leaving the Sialic acid consists of acetylated, Sulfated, methylated, and enzyme unchanged. 40 lactylated derivatives and is a large family of more than 50 Lysozyme protein is abundant in the limiting membrane members. They are ubiquitously distributed in many animal that circumscribes the egg white and forms the innermost tissues and in bacteria, primarily in glycoproteins and gan layer of the shell membranes. It is also present in the shell gliosides. They are typically the terminal residues on cell membranes, and in the matrix of the calcified shell. Calcite Surface oligosaccharides. Sialic acid-rich glycoproteins bind crystals grown in the presence of purified hen lysozyme 45 selectin in humans and other organisms. Sialic acid is also exhibited altered crystal morphology. Therefore, in addition naturally occurring in eggs. to its well-known anti-microbial properties that could add to Sialic acid is useful as a pre-cursor to many anti-inflam the protective function of the eggshell during embryonic matory medications. Various pharmaceutical agents and diag development, shell matrix lysozyme may also be a structural nostic reagents for influenza viruses are used in medical protein which in soluble form influences calcium carbonate 50 fields, leading to an increased demand for larger amounts of deposition during calcification. sialic acid worldwide. Lysozyme is used in the food industry due to its ability to Arachadonic acid is an omega-6 fatty acid, unsaturated, selectively inhibit the uncontrolled growth of Clostridium essential fatty acid, found in egg yolks. Arachadonic acid is a tyrobutyricum during the maturation of cheeses. Addition desirable product, because unlike the bad fats such as Satu ally, lysozyme can be used to protect against bacterial, viral or 55 rated fatty acids and cholesterol, arachadonic acid is a good inflammatory diseases. It can be used as an aerosol for the fat, essential to stay healthy. Essential fatty acid deficiencies treatment of bronchopulmonary diseases and for its prophy can lead to reduced growth, inability to fight infections and lactic function against infectious pathogens of the buccal infertility. Although arachadonic acid deficiencies are not cavity, such as dental caries. It can further be used in droplets extremely common in the United States, they do exist in for nasal tissue protection and various therapeutic creams 60 greater prevalence throughout the world. Arachadonic acid is designed for the protection and topical reparation of certain needed to strengthen cell membrane integrity, repair cellular diseases such as Herpes and shingles, as well as the treatment and tissue damage, optimize neurological transmission and of recurrent aphthous stomatitis. Oral administration of brain function, improve heart and circulatory function and lysozyme has also been shown to have immunostimulation produce Supple, moist skin. Therefore, obtaining a source of effects in addition to antihistamine effects. 65 arachadonic acid from eggs could be used in numerous forms Various proteins are contained within eggs, including to provide Supply for the human body to use to synthesize TGF-B and IgF-1. Transforming growth factor beta (TGF-B) regulatory molecules Such as prostaglandins (hormone like US 8,133,521 B2 5 6 chemical messenger) and thromboxanes (involved in platelet degeneration, to which there is no cure and treatments are aggregation and blood clotting). currently limited, as well as decreasing the risk of cataract Lipoproteins are globular, micelle-like particles that con development. Other carotenoids, such as beta-carotene, Vita sist of a non-polar core of acylglycerols and cholesteryl min A, and vitamin E have generally been used and seen as esters, Surrounded by an amphiphilic coating consisting of 5 beneficial anti-oxidant that may slightly retard the rate of protein, phospholipid and cholesterol. Lipoproteins have macular degeneration. However, they fail to rise to the level of been classified into five broad categories on the basis of their truly effective treatments. Zeaxanthin is not a widely avail functional and physical properties: chylomicrons (which able chemical, and is not available to the public except in transport dietary lipids from intestine to tissues), very low extremely small trace quantities in mixtures of other less density lipoproteins (VLDL), intermediate density lipopro 10 beneficial carotenoids. teins (IDL), low density lipoproteins (LDL), (all of which Typical methods of separating components from avian transport triacylglycerols and cholesterol from the liver to eggs include diluting the egg yolks and whites with waterata tissues), and high density lipoproteins (HDL) (which trans pH from about 4-5 so that the lipids separate out of solution. port endogenous cholesterol from tissues to the liver). One disadvantage of this dilution technique can be its lack of Lipoprotein particles undergo continuous metabolic pro 15 efficiency since the lipids have to settle out of solution over cessing and have variable properties and compositions. Lipo night on their own. Centrifugation is not possible as it would protein densities increase without decreasing particle diam stir up the lipids and hinder the separation of proteins from the eter because the density of their outer coatings is less than that solution. Further, with this type of processing, it can be dif of the inner core. The protein components of lipoproteins are ficult to obtain a high yield of proteins. The known technique known as apolipoproteins. High concentrations of lipopro is also disadvantageous in that only one protein of the egg, teins in the diet are strongly associated with increased risk of IgY, is isolated and the rest of the egg material discarded, cardiovascular disease, including increased risk for athero rendering the process time-consuming, economically ineffi Sclerosis and its manifestations, which include hypercholes cient, and wasteful. terolemia, myocardial infarction, and thrombosis. Thus, although it is generally recognized that eggs contain Carotenoids are naturally occurring organic pigments. 25 numerous nutritionally and physiologically or otherwise Carotenoids are found in abundance (over 600 types) in valuable components, problems remain, particularly with nature and are divided into two classes: Xanthophylls and respect to recovery of Such components in efficient and cost carotenes. The Xanthophylls include lutein and Zeaxanthin, effective ways without damaging the components or compro both naturally occurring in eggs. The yellow color of chicken mising their Suitability of the components for use in nutri egg yolks is a result of ingested Xanthophylls. In fact, eggs are 30 tional, medicinal, pharmaceutical, health, cosmetic or related one of the richest natural resources containing carotenoids. USS. Carotenoids have a number of important physiological prop erties and are often formulated into a variety of therapeutic BRIEF SUMMARY OF THE INVENTION drugs or nutritional Supplements. The carotenoids, lutein and Zeaxanthin have a yellow color, 35 In one embodiment, the invention provides a method of as seen in an egg yolk, because they absorb the high-energy separating a protein from an egg mixture having lipids and radiation of the near-ultraviolet and blue light spectrum and proteins. The method includes adding a cross-linking reagent reflect the yellow/yellow-orange wavelengths. It is theorized to the egg mixture comprising lipids and proteins to obtain that since these two pigments absorb wavelengths in the high cross-linked lipids. In another aspect, the method may energy spectrum, they may help protect retinal cells in the 40 include adjusting the pH level of the egg mixture to a pH at macula against “phototoxic' damage caused by short-wave which the cross-linking reagent is functional so that cross length high-energy light radiation. Additionally, lutein and linking of the lipids occurs. The proteins are separated from Zeaxanthin are chemically very closely related to each other; the cross-linked lipids to provide a separated protein. Accord both have the exact same chemical formulae, differing only in ing to the invention, the separated proteins may be obtained their ring stereochemistry and the spatial placement of one 45 by Subjecting the egg mixture to one or membranes or filters end ring and the placement of a double bond in that end ring of various sizes to separate or further isolate proteins or Lutein is classified as a carotenoid and is one of many populations of proteins of interest. The egg mixture may nutrients naturally occurring in egg yolks, and in part pro include a technical egg, edible egg, egg yolk, an egg white, or vides the yellow color of the egg yolk. It has a variety of combination thereof. important physiological properties, including prevention of 50 In another embodiment, the invention provides a number of age-related macular degeneration, a gradual worsening of protein compositions prepared by the methods of the inven vision due to degeneration of a portion of the retina and one of tion. In one aspect, the protein compositions are substantially the leading causes of blindness, as well as decreasing the lipid-free. The protein composition may include any of the development of cataracts within the eye. Lutein assists in following proteins: albumin, ovotransferrin, beta-livetin (al protecting eyes from oxidative stress and high-energy light 55 pha-2-glycoprotein), gamma livetin (IgY), insulin-like damage. Although there are no daily recommended amounts growth factor-1 (IGF-1), IGF-1, TGF-beta, transfer factors, of lutein, there is a demand for lutein in many diets as indi lysozyme orphosvitin or combinations thereof. In yet another viduals seek to increase lutein consumption in their diets embodiment, the invention provides for a protein composi through lutein-fortified foods, Sublingual sprays and dietary tion that is at least 10%, 15%, 20%, 25%, 30%35, 40%, 45%, Supplements, including both nutraceuticals and pharmaceu 60 50%, 55%, 60%. 65%, 70%, 75%, 80%, 85%, or 90% protein ticals. Additionally, lutein is utilized within pet food markets per weight of the composition. The protein compositions may and human skin care and cosmetics. include 5%, 10%, 15%, 20%, 25%, 30%35, or 40% ovotrans Zeaxanthin is a carotenoid found in egg yolks providing its ferrin. The protein compositions may include 5%, 10%, 15%, yellow color. It can be used as a feed additive, a colorant in the 20%, 25%, 30% 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, cosmetic and food industries, and dietary Supplements. Zeax 65 75%, 80%, 85% or 90% albumin. In another aspect, the anthin has a variety of important physiological properties, protein composition may include at least 10%. 20%, 30%, including prevention and treatment of age-related macular 40% or 50% polypeptides that are about less than 100 kDa. In US 8,133,521 B2 7 8 one aspect, the protein composition includes 10%, 15%, 20%, Yet another object, feature, or advantage of the invention is 25%, 30%35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, to provide a method for the separation of proteins from lipids 80%, 85% or 90% lysozyme. of a whole egg, yolk or white that produces a protein compo The present invention provides a method for treating an sition of high purity. individual in need of protein or amino acids. The individual 5 Another object, feature, or advantage of the invention is to may be an athlete, body-builder, weight-lifter, pregnant indi provide a method for the separation of proteins from lipids of vidual, individual recovering from Surgery, trauma, or illness, a whole egg, yolk or white that produces a protein composi or a diabetic individual. In other cases, the individual is an tion that with minimal salt (ash) content of the protein com animal. The method includes administering to an animal or position. human a protein composition of the present invention. Also 10 Still another object, feature, or advantage of the invention is to provide a method for the separation of proteins from provided is a beverage, food or nutritional product including lipids of a whole egg, yolk or white that produces a protein any of the protein compositions of the present invention. composition that has biologically active proteins. Therefore it is a primary object feature or advantage of the Yet another object, feature, or advantage of the invention is present invention to improve over the state of the art. 15 to provide an efficient method for the separation of proteins A further object, feature, or advantage of the invention is to from lipids of a whole egg, yolk or white that increases the provide a novel method for the separation of proteins from a yield of the protein. whole egg, yolk or white. A further object, feature, or advantage of the invention is to Another object, feature, or advantage of the invention is to provide a method for the separation of carotenoids from lipids provide a method for the separation of proteins from a whole of a whole egg, yolk or white that produces a carotenoid egg, yolk or white that produces a protein composition that composition that contains luteins and Zeaxanthins. can be used in medical, health and cosmetic applications. Yet another object, feature, or advantage of the invention is Still another object, feature, or advantage of the invention to provide a method for the separation of carotenoids from is to provide a method for the separation of proteins from a lipids of a whole egg, yolk or white that produces a carotenoid whole egg, yolk or white that produces a protein composition 25 composition that is bioavailable. that is lipid free or substantially lipid free. Yet another object, feature, or advantage of the invention is A further object, feature, or advantage of the invention is to to provide an efficient method for the separation of caro provide a method for the separation of proteins from a whole tenoids from lipids of a whole egg, yolk or white that egg, yolk or white that produces a protein composition that increases the yield or recovery of the carotenoids. contains natural anti-microbials. 30 A further object, feature, or advantage of the invention is to Yet another object, feature, or advantage of the invention is provide a method for the separation of fatty acids from a to provide a method for the separation of proteins from a whole egg, yolk or white to produce a lipid extract that con whole egg, yolk or white that produces a protein composition tains fatty acids. of high purity. Yet another object, feature, or advantage of the invention is 35 to provide a method for the separation of fatty acids from a Another object, feature, or advantage of the invention is to whole egg, yolk or white to produce a lipid extract that con provide a method for the separation of proteins from a whole tains fatty acids of Sialic acid and arachadonic acid. egg, yolk or white that produces a protein composition that Other objects, features, advantages and aspects of the with minimal salt (ash) content of the protein composition. present invention will become apparent to those of skill from Still another object, feature, or advantage of the invention 40 the following description. No single embodiment need is to provide a method for the separation of proteins from a exhibit all or any of the stated objects, features, advantages whole egg, yolk or white that produces a protein composition and aspects of the present invention. It should be understood, that has biologically active proteins. however, that the following description and the specific Yet another object, feature, or advantage of the invention is examples, while indicating preferred embodiments of the to provide an efficient method for the separation of proteins 45 invention, are given by way of illustration only. Various from a whole egg, yolk or white that increases the yield of the changes and modifications within the spirit and scope of the protein. disclosed invention will become readily apparent to those It is a primary object feature or advantage of the present skilled in the art from reading the following description and invention to improve over the state of the art. from reading the other parts of the present disclosure. A further object, feature, or advantage of the invention is to 50 provide a novel method for the separation of proteins, lipo BRIEF DESCRIPTION OF THE FIGURES proteins, lipids, carotenoids, and fatty acids or combinations thereof from lipids of a whole egg, yolk or white. The invention can be more fully understood from the fol Another object, feature, or advantage of the invention is to lowing detailed description and the accompanying figures. provide a method for the separation of proteins, lipoproteins, 55 FIG. 1 is a flow chart for one embodiment of separating lipids, carotenoids, and fatty acids or combinations thereof proteins and lipids from an egg. from lipids of a whole egg, yolk or white that produces a FIG. 2 is a flow chart for one embodiment of separating composition that can be used in medical, health, nutritional, proteins of various sizes from an egg. pharmaceutical and cosmetic applications. FIG. 3 is a series of flow chart for one embodiment of Still another object, feature, or advantage of the invention 60 separating various components from an egg. FIG.3A is a flow is to provide a method for the separation of proteins from chart for one embodiment of separating proteins of various lipids of a whole egg, yolk or white that produces a protein sizes from an egg. FIG.3B is a flow chart for one embodiment composition that is lipid free or substantially lipid free. of separating from cross-linked lipids of an egg, proteins of A further object, feature, or advantage of the invention is to various sizes, lipoproteins, fatty-acids, and carotenoids or provide a method for the separation of proteins from lipids of 65 combinations thereof. a whole egg, yolk or white that produces a protein composi FIG. 4 is an HPLC chromatogram for the protein compo tion that contains natural anti-microbials. sition referred to as Protein Supernatant in FIG. 3A. The US 8,133,521 B2 9 10 protein composition was obtained using the methods FIG. 15 is an HPLC chromatogram showing commercially described herein and a membrane with a nominal 3,000 available BSA (bovine serum albumin) from Fisher. molecular weight cut off (MWCO). The composition was FIG. 16 is an HPLC chromatogram showing fatty acid prepared as an aqueous sample for HPLC size exclusion content of commercially available lysozyme from Sigma. analysis. The protein composition is about 91% protein per weight of composition and comprises about 75% albuminand DETAILED DESCRIPTION about 23% ovotransferrin. FIG. 5 is an HPLC chromatogram for the protein compo The present invention now will be described more fully sition referred to as 1 Retentate in FIG. 3A. The protein hereinafter with reference to the accompanying examples, in composition was obtained using the methods described 10 which some, but not all embodiments of the invention are herein and a membrane with a 0.8 micron membrane. The shown. Indeed, the invention may be embodied in many dif ferent forms and should not be construed as limited to the composition was prepared as an aqueous sample for HPLC embodiments set forth herein; rather, these embodiments are size exclusion analysis. The protein composition is about provided so that this disclosure will satisfy applicable legal 91% protein per weight of composition and comprises about 15 requirements. Like numbers refer to like elements through 82% albumin and about 17% ovotransferrin Out. FIG. 6 is an HPLC chromatogram for the protein compo Many modifications and other embodiments of the inven sition referred to as 2 Retentate in FIG. 3A. The protein tion set forth herein will come to mind to one skilled in the art composition was obtained using the methods described to which this invention pertains, having the benefit of the herein and a membrane with a nominal 100,000 molecular teachings presented in the descriptions and the drawings weight cut off (MWCO). The composition was prepared as an herein. Therefore, it is to be understood that the invention is aqueous sample for HPLC size exclusion analysis. The pro not to be limited to the specific embodiments disclosed and tein composition is about 95% protein per weight of compo that modifications and other embodiments are intended to be sition and comprises 82% albumin and 9% ovotransferrin. included within the scope of the appended claims. Although FIG. 7 is an HPLC chromatogram for the protein compo 25 specific terms are employed herein, they are used in a generic sition referred to as 3 Retentate in FIG. 3A. The protein and descriptive sense only and not for purposes of limitation. composition was obtained using the methods described The articles “a” and “an are used herein to refer to one or herein and a membrane with a nominal 1,000 molecular more than one (i.e., to at least one) of the grammatical object weight cut off (MWCO). The composition was prepared as an of the article. By way of example, “an element’ means one or aqueous sample for HPLC size exclusion analysis. The pro 30 more than one element. The disclosure of each reference set tein composition is about 18% protein per weight of compo forth herein is incorporated herein by reference in its entirety. sition and comprises 29% albumin and 18% ovotransferrin This invention relates in part to a novel process for the and 50% of polypeptides less than 100 kDa. recovery of various components from an egg. The invention FIG. 8 is a flow chart for one embodiment of separating has particular but not exclusive application to the separation from cross-linked lipids of an egg, proteins typically associ 35 and recovery of these components from an avian egg, e.g. hen ated with lipids from an egg. Components that typically egg. As used herein, the term "egg refers to any edible egg or remain associated with the cross-linked lipids are also shown. technical egg or part thereof including, but not limited to FIG.9 is a flow chart for another embodiment of separating whole eggs, egg yolks, or egg whites or combinations thereof. from cross-linked lipids of an egg, proteins typically associ Technical eggs include eggs that are broken or otherwise not ated with lipids from an egg. Components that typically 40 fit for consumption because they include contaminants, e.g. remain associated with the cross-linked lipids are also shown. from wash water or manure. The term "egg also includes the FIG. 10 is a flow chart for one embodiment of separating egg in its various forms, for example, raw or dried. The egg from cross-linked lipids of an egg, carotenoids. may be obtained from any number of resources, including an FIG. 11 is a flow chart for one embodiment of separating egg-breaking facility. from cross-linked lipids of an egg, proteins of various sizes, 45 Referring now to FIG. 1 there is shown, in a flow-chart lipoproteins, fatty-acids, and carotenoids or combinations format, an embodiment of the present invention, a method of thereof. separating lipids of an egg mixture from proteins of the egg FIG. 12 is an HPLC chromatogram for the protein compo mixture by cross-linking the lipids to obtain cross-linked sition referred to as 4 Supernatant in FIG. 3B. The protein lipids. The present invention includes methods of separating composition was obtained using the methods described 50 the cross-linked lipids from the proteins in the egg mixture, herein and a membrane with a nominal 3,000 molecular methods of separating proteins from the egg mixture and if weight cut off (MWCO). The composition was prepared as an desired, further isolating and purifying them, as well as meth aqueous sample for HPLC size exclusion analysis. The pro ods of separating various components from the cross-linked tein composition is about 88% protein per weight of compo lipids. sition and comprises 49% albumin, 35% ovotransferrin, and 55 In one embodiment, the method may include separating 15% lysozyme. proteins from the egg mixture by adding a cross-linking FIG. 13 is an HPLC chromatogram for the protein compo reagent to the egg mixture and adjusting the pH of the egg sition referred to as 4 Supernatant in FIG. 3B. The protein mixture to optimize cross-linking. This allows for the reagent composition was obtained using the methods described to cross-link lipids present in the egg mixture. The cross herein and a membrane with a nominal 3,000 molecular 60 linked lipids may be easily removed from the solution for weight cut off (MWCO). The composition was prepared as an further processing and to facilitate the isolation of proteins aqueous sample for HPLC size exclusion analysis. The pro solubilized in the egg mixture. The solubilized proteins may tein composition is about 89% protein per weight of compo be separated from the cross-linked lipids using any Suitable sition and comprises 8% albumin, 9% ovotransferrin, and technique, and the solubilized proteins may be further iso 83% lysozyme. 65 lated or processed as described elsewhere herein. Solubilized FIG. 14 is an HPLC chromatogram showing commercially proteins initially separated from the cross-linked lipids available IgG from Sigma. include but are not limited to ovotransferrin, albumin, beta US 8,133,521 B2 11 12 livetin (alpha-2-glycoprotein), gamma livetin (IgY), insulin particular, the methods result in products and compositions like growth factor-1 (IGF-1), or phosvitin or combinations which serve as a safe source of nutritional Supplements for thereof. The methods disclosed herein may use various physi human consumption. Unlike other methods used for process cal and chemical treatments for the separation and purifica ing the egg, toxic solvents, or other unsafe materials need not tion of the proteins. be used in the methods of the invention or are not found in the In one embodiment, the invention includes separating vari resulting product, e.g. the isolated egg component or compo ous components associated with the cross-linked lipids and if sition. The various compositions or components isolated desired, further isolating and purifying them. The compo thereof may be used in any number of applications, including nents associated with the cross-linked lipids may include but but not limited to products or services in the food, nutrition, are not limited to proteins, lipoproteins, lipids, carotenoids, 10 pharmaceutical, cosmetic, health and related industries. and fatty acids, in particular TGF-beta, transfer factors, I. Cross-Linking Lipids from the Egg Mixture lysozyme, albumin, ovotransferrin, lipids Such as triglycer In one embodiment, the methods of the present invention ides, cholesterol, phospholipids, lipoproteins, such as HDL include adding a cross-linking reagent to an egg mixture (alpha lipovitellin), LDL (beta-lipovitellin), VLDL (apovitel comprising lipids and proteins to obtain cross-linked lipids. lenins), and fatty acids, such as Sialic acid, Arachadonic acid, 15 As used herein, the term “mixture” refers to a composition of or carotenoids, Such as lutein and Zeaxanthin. matter having two or more components in varying propor With respect to the separation of various components asso tions, regardless of their physical state. The term "egg mix ciated with the cross-linked lipids, the methods disclosed ture” refers to a composition of matter having two or more herein may use various physical and chemical treatments. For components in varying proportions, regardless of their physi example, embodiments of the present invention may use a cal state, one of the components being an egg. As such an "egg Solution or a solvent or a combination of Solutions and Sol mixture' includes the egg in its various parts or forms in vents to aid in the separation of a particular component from combination with a solution or a solvent to form a solution or the cross-linked lipids. As shown in FIG. 3, this may involve Suspension. For example, the egg may be raw, Solid, or dried the adjustment of the pH of the solution. In one embodiment, prior to its addition to the solution or solvent. Typically, the Subsequent to the cross-linking of the lipids, a Saline Solution 25 egg mixture is a technical egg, edible egg, whole egg, egg may be used to facilitate the release of proteins from the yolk or egg white mixed or diluted with a solution, e.g. water, cross-linked lipids. Typically, the pH of the saline solution saline solution, or a solution having a pH Suitable for cross does not need to be readjusted from the pH of the previous linking lipids of an egg. The ratio of egg to Solution in the Solution, e.g. the egg mixture having its pH optimized for mixture is typically based on a measurement of an egg solid lipid cross-linking. In another embodiment, the pH of the 30 with moisture removed using IR32 moisture analyzer (Den solution is adjusted to a pH from about 2.0 to about 4.0 to ver Instruments, Denver, Colo.). As an example, the percent facilitate the release of proteins from the cross-linked lipids. solids of technical eggs with moisture removed is about 11% In some embodiments, the pH of the solution may be adjusted and a ratio of 1 part egg to 5 parts water, a 1:5 ratio, is used. to about 2.0 to about 4.0 Subsequent to contacting the cross With respect to whole eggs, the percent solids of whole eggs linked lipids with the saline solution or subsequent to the 35 with moisture removed is about 23% and a ratio of 1 part egg initial step of cross-linking the lipids. Without wishing to be to 7-10 parts water, a 1:7 or 1:10 ratio, is used. The dilution of bound by this theory, it is believed that the change in pH the egg with the solution assists in the separation and isolation allows for the release of additional proteins by overcoming of various egg components. protein-protein interections or protein-lipids interactions. In Any Suitable cross-linking reagent may be used so long as another embodiment, Subsequent to contacting the cross 40 it is able to cross-link lipids. As used herein, the term “cross linked lipids with a solution having a pH from about 2.0 to linking refers to aggregating lipids by hydrophobic interac about 4.0, the pH of the solution is adjusted to a pH from about tion or adsorping of lipids. As used herein, the term "cross 4.0 to about 6.0 to further facilitate the separation of the linking reagent” refers to a material that can be used to cross proteins. Without wishing to be bound by this theory, it is link a lipid, e.g. form an aggregate of lipids or adsorb lipids. contemplated that the change in pH aids in the proteins 45 Exemplary cross-linking reagents include but are not limited solubility. to cyclobetadextran, silicon dioxide, colloidal silica material, Various process steps have been disclosed and the methods Such as an acid stabilized dispersion of colloidal silica par of the invention allow a number of different products to be ticles (e.g. Nalco-1042, Nalco-1130, Nalco-1115, Nalco obtained from the egg mixture, e.g. solubilized proteins, or 1030, Nalco-1140, Nalco-2326, Nalco-1050, Nalco-1060, the cross-linked lipids or a combination of both. The present 50 and Nalco-2327, all available from Nalco Chemical Co), invention contemplates that Some process steps may be omit AEROSILTM (Degussa AG, Parsippany, N.J.), and fumed ted, where not all products need be recovered. One of the silica materials, such as Synthetic Amorphous Silicon Diox advantages of the overall process is that multiple useful prod ide, Crystalline-free fumed silica (e.g. Cab-O-Sil available ucts may be obtained due to the sequencing or order of steps, from Cabot Corporation), Wacker HDK (Wacker Chemie which reduces the amount of waste materials. In addition, the 55 AG (Ninchritz, Germany), AEROSIL 380 (Degussa AG, sequencing or order of steps also provides for Synergistic Parsippany, N.J.), Synthetic Amorphous Silicon Dioxide, results in that the time and cost of producing additional prod Crystalline-free precipitated silica (e.g. Hi-Sil available from ucts is less than producing each of the products in a separate PPG Industries), SIPERNATR-50 and SIPERNATR-180 process. In other words, only incremental process are needed available from Degussa AG, Parsippany, N.J.), and Synthetic to produce additional products. 60 Calcium Silicate Hydrate (e.g. LRATM agent available from Use of the methods disclosed herein allows for the isolation (Advanced Minerals, Goleta, Calif.). In one aspect, the cross of multiple different components from an egg in an efficient, linking reagent is self-catalyzing. cost-effective manner without compromising the recovery Any percentage of cross-linking reagent that can cross link process of the components or their Subsequent utility in Vari the lipids in the egg mixture may be used. In one aspect, the ous applications or products and compositions. Advanta 65 cross-linking reagent comprises from about 0.1% to about geously, the methods of the invention result in various prod 20% by weight of the egg mixture. Preferably, the cross ucts and compositions which are free from impurities. In linking reagent comprises from about 6% to about 10% by US 8,133,521 B2 13 14 weight of the egg mixture. In a preferred aspect, the cross less than 2.0%, less than 1.0%, or less than 0.5% of salt. The linking reagent comprises from about 3% to about 6.0% of protein compositions of the present invention may be pre Nalco-1 115 per weight of 20% egg mixture is employed. pared in any number of forms or formulations. In one aspect, In one embodiment, the method of the present invention the protein is prepared as a protein powder using any Suitable includes adjusting the pH of the egg mixture to a pH range or 5 technique, including but not limited to lyophilization, level at which the cross-linking reagent is functional, for vacuum drying, freeze drying, spray drying, drum drying, example, a pH where the cross-linking reagent can cross-link paddle-drying, Super critical fluid processing, air drying, or at least 25% of the lipids in the egg mixture. In one embodi other forms of evaporative drying. The drying step may be ment, the method of the present invention includes adjusting carried out any suitable temperature, for example, with the pH of the egg mixture to optimize cross-linking. In one 10 respect to freeze drying, a preferred temperature range is from aspect, the pH of the mixture is adjusted to a pH from about about 23° C. to about 40° C., with 27° C. being the more 4.0 to about 8.0, for example, by contacting the egg mixture preferred temperature. with an acidic Solution. The egg mixture is subjected to the In one embodiment of the present invention, the method cross-linking reagent for a sufficient time at a sufficient tem produces a protein composition that is at least 70%. 80%, perature for cross-linking of the lipids in the egg mixture to 15 85%, or 90% protein per weight of composition, substantially occur. In some cases, this may be as little as fifteen minutes. lipid-free, and contains minimal salt (ash). As used herein, the Typically, the cross-linking reaction is carried out at room term “substantially lipid-free” refers to an egg mixture or temperature. As apparent to one skilled in the art, the time, composition, the mixture or composition containing less than temperature, and the pH range at which to optimize cross 2.0%, less than 1.0%, or less than 0.5% of lipids, for example, linking may vary depending on the specific cross-linking of cholesterol, triglycerides, or phospholipids or combina reagent employed. Such variations are well within one skilled tions thereof. In one aspect, the protein composition includes in the art. An example of typical conditions include carrying but is not limited to ovotransferrin, albumin, beta-livetin (al out the cross-linking reaction for about 30 minutes to about 1 pha-2-glycoprotein), gamma livetin (IgY), insulin-like hour at a temperature of about 14°C. to about 37° C. at a pH growth factor-1 (IGF-1), or phosvitin or combinations from about 4.0 to about 8.0, more preferably from about 6.0 to 25 thereof. Advantageously, protein compositions of the present about 7.5. Any suitable acid or base may be used to adjust the invention may include ovotransferrins which act as natural pH including but not limited to acetic acid, boric acid, citric antimicrobials. In one aspect, as shown in FIG. 4, the protein acid, hydrochloric acid, lactic acid, oxalic acid, Sulfuric acid, composition comprises ovotransferrin and albumin. In one Sulfurous acid, Sodium hydroxide or potassium hydroxide. aspect, the protein composition is at least 70%, 80%, 85%, or In an embodiment of the invention, the method may 30 90% protein per weight of composition and comprises at least include separating the cross-linked lipids from the egg mix 15% or 20% ovotransferrin and greater than 70% or 75% ture. The cross-linking step facilitates the precipitation of the albumin. Protein percentage may be determined by any num cross-linked lipids from the egg mixture which allows for ber of methods, including but not limited to a Leco combus them to be easily removed by known techniques, such as tion analyzer. precipitation; sedimentation; centrifugation; decantation; 35 A. Removal of Residual Lipids from Solubilized Proteins particulate filtration; membrane filtration; temperature modi In an embodiment of the invention, the method may fication; liquid-liquid extraction; and the like and combina include Subjecting the egg mixture from which the cross tions thereof. In one aspect, centrifugation is employed to linked lipids have been removed, e.g. the Supernatant having generate a Supernatant comprising solubilized proteins from the solubilized proteins, to one or more membranes or filters the egg mixture and a precipitate comprising the cross-linked 40 to remove any residual lipids. Any Suitable size membrane or lipids. The Supernatant may be collected and the precipitate filter may be used. In one aspect, as shown in FIG.3, about an comprising the cross-linked lipids removed. The precipitate 0.8 micron to about 1.2 micron membrane or filter may be comprising the cross-linked lipids may be removed for fur employed to separate the proteins from the egg mixture to ther processing as described elsewhere herein. generate a substantially lipid-free protein composition. II. Separating the Solubilized Proteins from the Supernatant 45 The separated proteins may be concentrated and dialyzed. In an embodiment of the invention, the method may Dialyzing has the added benefit of removing salt (ash). In an include separating the Solubilized proteins from the egg mix embodiment of the present invention, the method may pro ture from which the cross-linked lipids have been removed. duce a protein composition that is least 70%, 80%, 85%, 90%, Such solubilized proteins include but are not limited to or 95% protein per weight of composition. In one aspect, the ovotransferrin, albumin, beta-livetin (alpha-2-glycoprotein), 50 composition contains minimal salt (ash). In one aspect, the gamma livetin (IgY), insulin-like growth factor-1 (IGF-1), or protein composition additionally comprises ovotransferrin, phosvitin or combinations thereof. As used herein, the term albumin, beta-livetin (alpha-2-glycoprotein), gamma livetin “albumin' is also used interchangeably with the term "oval (IgY), IGF-1, or phosvitin or combinations thereof. In one bumin' and alpha-livetin. aspect, as shown in FIG. 5, the protein composition comprises The solubilized proteins from the egg mixture may be 55 ovotransferrin and albumin. In one aspect, the protein com concentrated, for example, using a 3 kDa membrane, to pro position comprises at least about 10%, 15% or 20% ovotrans duce a protein composition in aqueous form. In an embodi ferrin and at least greater than 70%, 75% or 80% albumin. ment of the invention, as shown in FIG. 2, the method may Accordingly, in one embodiment, the present invention pro include Subjecting the concentrated proteins to one or more vides for a protein composition that is at least 70%. 80%, membranes or filters of various sizes to isolate specific pro 60 85%, 90%, or 95% protein per weight of composition and is teins or populations of proteins of a desired size or molecular at least about 10%, 15% or 20% ovotransferrin and at least weight as described elsewhere herein. The resulting protein greater than 70%, 75% or 80% albumin. Advantageously, the composition may be further purified by removing salt using method used results in a product which is free from impuri routine techniques such as dialysis. Accordingly, the protein ties. In particular, the method results in a product which composition resulting from the method of the present inven 65 serves as a safe Source of nutritional Supplement for and is tion may have minimal salt. As used herein, the term “mini suitable for human consumption. Unlike other methods used mal salt” refers to a composition oran egg mixture containing for processing the egg, toxic solvents, or other unsafe mate US 8,133,521 B2 15 16 rials need not be used in the methods of the invention or are produce IgY antibodies effective for treating various dis not found in the resulting product, e.g. the isolated egg com eases. The resulting IgYantibodies may be used for treatment ponent, or composition. Accordingly, in one embodiment, the of numerous varieties of diseases or conditions. Examples of protein composition or proteins isolated thereof may be used Such uses include, without limitation: treatment of peptic in any number of applications, including but not limited to 5 ulcers as disclosed in U.S. Pat. No. 6,797.268 to Kodama et products or services in the food, nutrition, and health indus al.; treatment of gastritis, gastric ulcers and duodenal ulcers as tries. Such applications are known in the art as well as the disclosed in U.S. Pat. No. 6,793,921 to Kodama et al.; inflam appropriate techniques for inclusion in Such application or matory bowel disease as disclosed in U.S. Pat. No. 7.261,891 compositions. to Kinket al.; and skin inflammatory diseases including pso B. Isolating Specific Proteins or Populations of Proteins from 10 riasis and eczema as disclosed in U.S. Pat. No. 7,147,854 to the Solubilized Proteins Ye. All references mentioned are herein incorporated by ref In an embodiment of the invention, as shown in FIG. 2, the erence, each in its entirety. Accordingly, in one embodiment, method may include Subjecting the egg mixture from which the method includes separating Solubilized proteins, in par the cross-linked lipids have been removed to one or more ticular IgY. from an egg of at least one hen immunized to membranes or filters of various sizes to isolate specific pro 15 produce IgY antibodies effective for treating a disease. teins or populations of proteins of a desired size or molecular In an embodiment of the invention, the method includes weight. Such proteins include but are not limited to ovotrans separating proteins that are less than 100 kiloDaltons (kDa) ferrin, albumin, beta-livetin (alpha-2-glycoprotein), gamma from the resulting egg mixture from which the cross-linked livetin (IgY), insulin-like growth factor-1 (IGF-1), or phos lipids have been removed. Depending on the type of mem vitin or combinations thereof. As appreciated by one skilled brane or filter used, for example, a membrane with a nominal in the art, the isolating step may be performed Subsequent to value, proteins having a molecular weight less than 100 kDa the removal any residual lipids. In one aspect, the method may may be isolated as well. See FIG. 3A. Such proteins include include Subjecting the Supernatant having the Solubilized pro but are not limited to ovotransferrin, albumin, beta-livetin teins to one or more membranes or filters. In an embodiment, (alpha-2-glycoprotein), gamma livetin (IgY), insulin-like the method includes separating proteins having a molecular 25 growth factor-1 (IGF-1), or phosvitin or combinations weight greater than a determined upper molecular weight thereof. A membrane or filter or chromatography column that threshold (e.g., 1 kDa, 3 kDa. 50kDa, 100kDa) and removing separates proteins less than 100kDa in molecular weight may proteins having a molecular weight below a determined lower be employed to separate such proteins from the egg mixture. molecular weight threshold (e.g., 1 kDa, 50 kDa, 100 kDa). In one aspect, the egg mixture is Subjected to a first membrane Although a 100 kDa molecular weight cut off (MWCO) 30 or filter, Such as a 0.8 uM membrane, prior to separating the membrane or filter is preferred, larger or Smaller nominal proteins less than 100 kDa in molecular weight from the egg MWCO membranes or filters may be used with the methods mixture with a second membrane or filter. of the present invention, ranging from 1 kDa, 5 kDa, 10 kDa, In one embodiment, as shown in FIG. 3A, the proteins are 15 kDa, 50 kDa, 100 kDa to 180 kDa, depending on the obtained from a permeate prepared by filtering the Superna molecular weight of the protein or population of proteins of 35 tant obtained from the lipid-free egg mixture using a mem interest to be separated. brane that is equal to or at least 100 kDa in molecular cut off In one embodiment of the invention, as shown in FIG.3A, size. As appreciated by one skilled in the art, a Supernatant or the method includes isolating proteins that are greater than permeate comprising proteins of 100 kDa or less than 100 100 kiloDaltons (kDa). Depending on the type of membrane kDa may be filtered through one or more membranes of or filter used, for example, a membrane with a nominal value, 40 various sizes to remove impurities or isolate specific proteins. proteins having a molecular weight less than 100 kDa may be For example, as shown in FIG. 3A, the permeate may be isolated as well. See FIG. 3A. Such proteins include but are further subjected to a membrane that is 1 kDa or greater in not limited to ovotransferrin, albumin, beta-livetin (alpha-2- molecular cut off size to remove any impurities such as salt glycoprotein), gamma livetin (IgY), insulin-like growth fac and obtain a retentate comprising proteins greater than 1 kDa tor-1 (IGF-1), or phosvitin or combinations thereof. A mem 45 but 100 kDa or less in molecular size. Accordingly, the brane or filter or chromatography column may be employed method can be used to obtain a protein or population of to separate proteins greater than 100 kDa in size. In one proteins of 100 kDa or less but greater than 1 kDa. With aspect, the proteins are obtained from a retentate prepared by respect to ovotransferrin and albumin, a membrane that has filtering the Supernatant obtained from the egg mixture using the appropriate molecular weight cutoff of at least 100 kDa a membrane that is greater than 100kDa in size. Accordingly, 50 may be employed, although some variances are expected the method can be used to obtain a protein or population of when a membrane having a nominal MWCO is used. proteins greater than 100 kDa. In one aspect, the egg mixture As appreciated by one ordinarily skilled in the art, proteins may be subjected to a first membrane or filter, such as a 0.8 or populations of proteins, for example, those greater or less LM membrane, prior to separating the proteins greater than than 100 kDa, may be separated from the egg mixture or 100 kDa in molecular weight from the egg mixture with a 55 further isolated using any number of techniques, including second membrane or filter. but not limited to dialysis, precipitation, sedimentation, cen In an embodiment of the invention, the method includes trifugation, decantation, particulate filtration, membrane fil isolating IgY from the solubilized proteins using a membrane tration, Such as gel filtration using a molecular sieve, tem or filter with the appropriate MWCO. Accordingly, in one perature modification, extraction, Such as liquid-liquid embodiment, the invention provides a protein composition 60 extraction, solvent extraction, and desalting with 1:5 or 1:10 that is Substantially lipid-free, contains minimal salt, and dilution with water, chromatography, Such as anion exchange includes IgY. As the methods of the present invention are chromatography using a resin Such as diethylaminoethyl highly scaleable, large amounts of IgY antibodies may be (DEAE)-Sepharose or DIAION HPA-75 (manufactured by obtained in a commercially viable way for use in composi Mitsubishi Chemical), cation exchange chromatography tions for numerous applications. IgY, also referred to as IgY 65 using a resin such as S-Sepharose FF (manufactured by Phar antibodies, may be obtained from avian eggs laid from hens macia), hydrophobic chromatography using a resin Such as which have been immunized in various ways, including to butyl-Sepharose or phenyl-Sepharose, affinity chromatogra US 8,133,521 B2 17 18 phy, size exclusion chromatography, chemical precipitations, tional protein encoded by a particular gene), as well as frag chromatofocusing and electrophoresis such as isoelectric ments of proteins. In one aspect, the protein composition focusing, and the like and combinations thereof. The isolated includes Small molecular weight proteins that are less than proteins may be concentrated and dialyzed. Dialyzing has the 100 kDa comprise 40%, 45%, 50% or 55% of the protein added benefit of removing salt (ash). composition. As shown in FIG. 7, the protein composition If desired, the isolated protein or population of proteins, for comprises albumin and ovotransferrin. In one aspect, the example, those greater or less than 100 kDa, may be further protein composition comprises at least about 5%, 10% or purified, using for example, dialysis and/or chromatography, 15% ovotransferrin. In one aspect, the protein composition or sequential chromatographies, of anion- or cation exchange comprises at least about 20%, 25%, 30% or 35% albumin. In column chromatography using a resin of DEAE or CM, affin 10 ity chromatography using a gel of "heparin, biotin, hydroxya one aspect, the protein composition comprises at least about patite, hydrophobic chromatography using a gel of methyl 5% ovotransferrin, at least greater than 20% albuminand 18% HIC or t-butyl HIC, or affinity chromatography. See for protein per weight of composition. The protein composition example, Awade, A. C. On hen egg fractionation: application resulting from the method of the present invention may be of liquid chromatography to the isolation and purification of 15 prepared in any number of forms or formulations. The protein hen egg white and egg yolk proteins. Z. Lebensm.-Unters.- may be prepared as a protein powder using any suitable tech Forsch. (1996). 202: 1-14, hereinincorporated by reference in nique, including but not limited to lyophilization, vacuum its entirety. Dialyzing has the added benefit of removing the drying, freeze drying, spray drying, drum drying, paddle ash (or salt). The proteins may be isolated or purified for use drying, Super critical fluid processing, air drying, or other in any number of applications, including protein composi forms of evaporative drying. The drying step may be carried tions. out any suitable temperature, for example, with respect to Additional individual proteins or polypeptides, e.g. those freeze drying, a preferred temperature range is from about 23° less than 100 kDa, isolated by the methods of the present C. to about 40° C., with 27° C. being the more preferred invention may be may be determined using routine tech temperature. niques known to those ordinarily skilled in the art. Exemplary 25 Advantageously, the method used results in a protein com techniques include but are not limited to ELISA, Western blot position which is free from impurities. In particular, the analysis, 2D-PAGE (Westermeier, R.; Naven, T. Proteomics method results in a protein composition which serves as a safe in practice; Wiley-VCH: Weinheim, 2002: pp 11-97) and Source of nutritional Supplement for human consumption. automated spot identification by matrix-assisted laser desorp Unlike other methods used for processing the egg, toxic sol tion-ionization mass spectrometry (MALDI-MA) or liquid 30 vents, or other unsafe materials need not be used in the meth chromatography electrospray ionization mass spectrometry ods of the invention or are not found in the resulting protein (LC-ESIMS) (Dass, C. Structural Analysis of Proteins. In composition, e.g. the isolated egg protein, or protein compo Principles and practice of biological mass spectromety; Dass, sition. Accordingly, the protein composition or proteins iso C., Ed.; Wiley-Interscience: New York, 2001: pp 75-216). lated thereof may be used in any number of applications, In one embodiment, the method of the present invention 35 including but not limited to products or services in the food, produces a protein composition having proteins that are nutrition, and health industries. Such applications are known greater than 100 kDa. In one aspect, present invention pro in the art as well as the appropriate techniques for inclusion in duces a protein composition that is at least 5%, 10%, 15% or Such application or compositions. 20% protein per weight of the composition. In one aspect, the For example, the protein compositions of the present protein composition is substantially lipid-free. The composi 40 invention, in any Suitable form, e.g. aqueous or dried, can then tion may contain minimal salt (ash). In one aspect, the protein be applied to, admixed with and/or injected into consumable composition includes but is not limited to albumin, ovotrans products for humans or animals, including farm animals or ferrin, beta-livetin (alpha-2-glycoprotein), gamma livetin pets. Such consumable products include food or nutritive (IgY), insulin-like growth factor-1 (IGF-1), or phosvitin or additives such as animal feed, human food products, nutrition combinations thereof. As shown in FIG. 6, the protein com 45 Supplements for humans or animals, or the like. Accordingly, position comprises IgY. albuminand ovotransferrin. The pro a method of the present invention includes treating an animal tein composition may comprise at least about 5% or 10% or human in need of protein oramino acids by administering ovotransferrin. The protein composition may comprise at a protein composition of the present invention. The protein least about 70%, 75%, or 80% albumin. The protein compo compositions may be administered to an animal or human to sition may comprise at least about 9% ovotransferrin and at 50 increase their protein consumption. In some cases, the animal least greater than 80% albumin. or human may need to increase their consumption of amino In one embodiment, the method of the present invention acids or proteins. Such as an athlete, body-builder, weight produces a protein composition having proteins that are less lifter, pregnant individual, individual recovering from Sur than or equal to 100 kDa. The protein composition may be at gery, trauma, or illness, or a diabetic individual. least 70%, 80%, 85%, 90%, or 95% protein per weight of the 55 Animal feed and products according to the invention, e.g., composition. The protein composition is substantially lipid dry food pellets and treats, will be designed such that they free. The composition may contain minimal salt (ash). The contain appropriate nutrition levels for the particular animal, protein composition includes but is not limited to albumin, e.g., a dog or cow. In this regard, the design of food formula ovotransferrin, beta-livetin (alpha-2-glycoprotein), gamma tions for humans, e.g. body-builders, particular animals, e.g., livetin (IgY), insulin-like growth factor-1 (IGF-1), or phos 60 dog or cow, etc., is well known and established. There exist vitin or combinations thereof. In one aspect, the protein com accepted nutritional guidelines for optimal amounts of pro position includes Small molecular weight polypeptides that teins, fats, vitamins, minerals, fibers, that vary dependent are less than 100 kDa. As used herein, the term “polypeptide' upon whether the consumer is a human or particular animal means an unbranched chain of amino acid residues that are and the consumers age and health. It is anticipated that the covalently linked by an amide linkage between the carboxyl 65 protein compositions of the present invention can be added or group of one amino acid and the amino group of another. The incorporated to any animal or human drink, beverage, feed, term polypeptide can encompass whole proteins (i.e. a func food, product or Supplement. US 8,133,521 B2 19 20 III. Separating Proteins Associated with the Cross-Linked chromatography, chemical precipitations, chromatofocusing Lipids from the Cross-Linked Lipids and electrophoresis such as isoelectric focusing, and the like A. Using a Saline Solution to Separate Proteins Associated and combinations thereof. Accordingly, the process of the with the Cross-Linked Lipids from the Cross-Linked Lipids invention may also include isolating from the Supernatant, In one embodiment of the present invention, the method retentate, permeate, precipitate, protein or lipid composi includes separating proteins associated with the cross-linked tions, various proteins or other components of interest. lipids from the cross-linked lipids. Such proteins include In one aspect, the proteins are obtained from a Supernatant without limitation TGF-beta, transfer factors, lysozyme, prepared by centrifuging the cross-linked lipids/saline mix albumin, or ovotransferrin and the like or combinations ture and by employing standard techniques, such as concen thereof. In one embodiment, Subsequent to the cross-linking 10 trating the proteins by membrane filtration or dialysis. Dia of the lipids, a saline solution may be used to facilitate the lyzing has the added benefit of removing the salt (ash). The release of proteins from the cross-linked lipids. Typically, the Supernatant may be filtered through one or more membranes pH of the saline solution does not need to be readjusted from of a Suitable size to isolate specific components, including the pH of the previous solution. proteins such as lysozyme. In another aspect, proteins may be In one embodiment, as shown in FIG. 8, the method for 15 isolated according to the methods of the present invention, for separating proteins associated with the cross-linked lipids example, based in part on the proteins molecular weight. from the cross-linked lipids includes resuspending the cross Proteins that have a known molecular weight may be iso linked lipids, for example, the precipitate comprising the lated using a membrane or a series of membranes that have the cross-linked lipids, in a saline Solution. Any molarity of the appropriate exclusion limit for isolating the particular protein saline Solution that disrupts the cross-linked lipids-protein or proteins of interest. Accordingly, the selection of mem interactions or protein-protein interactions so that the pro brane size can be used to obtain a protein or population of teins disassociate from the cross-linked lipids or from pro proteins of a particular size (molecular weight). With respect teins associated with the cross-linked lipids may be used, for to lysozyme, albumin, and ovotransferrin, a membrane that example, a molarity that solubilizes the proteins into solution. has the appropriate molecular weight cutoff, e.g. 3-10 kDa, Without wishing to be bound by this theory, it is contemplated 25 may be employed. As appreciated by one skilled in the art, the that the saline Solution neutralizes charges of protein-protein Supernatant may be filtered through one or more membranes interactions or protein-lipid interactions or both. As appreci of various sizes to isolate specific proteins. Accordingly, the ated by one skilled in the art, solubilization of the proteins method can be used to obtain a protein or population of may be facilitated by stirring or mixing the Saline Solution proteins of a desired size or molecular weight. In another comprising the cross-linked lipids. In some instances, the 30 aspect, the proteins may be further separated using a mem solubilization of the proteins may occur in as little as one brane that has the appropriate molecular weight cutoff of at hour. Any amount of solution may be used so long as the least 50 kDa to separate ovotransferrin from lysozyme and amount is sufficient to overcome protein-protein interactions albumin. or protein-lipid interactions so that the proteins are released As one skilled in the art would appreciate, proteins of and Solubilized in the solution, for example, 4 parts saline to 35 similar molecular size, e.g. lysozyme and albumin, may be 1 part lipid (4:1) ratio may be used. In one aspect, the saline difficult to separate by membranes and other techniques may solution has a molarity (M) from about 0.1 M to about 2.0 M, be employed for such purposes. If desired, the isolated protein in a preferred range; the molarity of the Saline Solution is from or population of proteins may be further purified, using for about 0.2M to 1.0 M, more preferably the molarity of the example, chromatography such as sequential chromatogra saline solution is from about 0.2M to 0.3M. A person skilled 40 phies of anion-exchange column chromatography using a in the art may appropriately select the temperature, the resin of DEAE or CM, affinity chromatography using a gel of amount of the salt to be used, and the period of time sufficient heparin, biotin, hydroxyapatite, hydrophobic chromatogra to release to the proteins associated with the cross-linked phy using a gel of methyl HIC or t-butyl HIC. See for lipids. An example of typical conditions include carrying out example, Awade, A. C. On hen egg fractionation: application the cross-linking reaction for about 30 minutes to about 2 45 of liquid chromatography to the isolation and purification of hours at a temperature of about 14°C. to about 37°C. at a pH hen egg white and egg yolk proteins. Z. Lebensm.-Unters.- from about 4 to about 8, more preferably from a pH from Forsch. (1996). 202: 1-14, hereinincorporated by reference in about 6.0 to 7.5. its entirety. The proteins may be isolated or purified for use in Once the proteins of interest are released from the cross any number of applications. linked lipids, one skilled in the art would be able to readily use 50 In another aspect, the identification of isolated proteins standard biochemistry techniques to isolate one or more pro may be determined using routine techniques known to those teins of interest from the Saline solution. As appreciated by ordinarily skilled in the art. Exemplary techniques includebut one ordinarily skilled in the art, the solubilized proteins in the are not limited to ELISA, Western blot analysis, 2D-PAGE saline solution may be separated from cross-linked lipids (Westermeier, R.; Naven, T. Proteomics in practice; Wiley using any number of standard techniques, including but not 55 VCH: Weinheim, 2002: pp 11-97) and automated spot iden limited to dialysis, precipitation, sedimentation, centrifuga tification by matrix-assisted laser desorption-ionization mass tion, decantation, particulate filtration, membrane filtration, spectrometry (MALDI-MA) or liquid chromatography elec Such as gel filtration using a molecular sieve, temperature trospray ionization mass spectrometry (LC-ESIMS) (Dass, modification, extraction, such as liquid-liquid extraction, Sol C. Structural Analysis of Proteins. In Principles and practice vent extraction, and desalting with 1-2.5 mS for salt removal, 60 of biological mass spectrometry; Dass, C., Ed., Wiley-Inter chromatography, Such as anion exchange chromatography science: New York, 2001: pp 75-216). using a resin such as diethylaminoethyl (DEAE)-Sepharose Obtained from methods of the present invention is a protein or DIAION HPA-75 (manufactured by Mitsubishi Chemi composition comprising TGF-beta, transfer factors, cal), cation exchange chromatography using a resin Such as lysozyme, albumin, or ovotransferrin and the like or combi S-Sepharose FF (manufactured by Pharmacia), hydrophobic 65 nations thereof. In one embodiment, the protein composition chromatography using a resin Such as butyl-Sepharose or is at least about 70%, 75%, 80%, 85%, or 90% protein per phenyl-Sepharose, affinity chromatography, size exclusion weight of composition. In one aspect, as shown in FIG. 12, the US 8,133,521 B2 21 22 protein composition comprises albumin, ovotransferrin, and transfer factors or the like or combinations thereof. Without lysozyme. In one aspect, the protein composition comprises wishing to be bound by this theory it is believed that an acidic at least about 40%, 45%, 50% albumin. In one aspect, the solution with a pH from about 2.0 to about 4.0 facilitates the protein composition comprises at least about 30%, 35%, or release of proteins associated with the cross-linked lipids 40% ovotransferrin. In one aspect, the protein composition from the cross-linked lipids into the acidic solution, e.g. in comprises at least 10% or 15% lysozyme. In one aspect, the Some cases the proteins are released from carrier proteins protein composition comprises at least about 40%, 45%, 50% associated with the cross-linked lipids. In some embodi albumin and at least about 30%, 35%, or 40% ovotransferrin, ments, the cross-linked lipids are exposed to a solution that and at least 10% or 15% lysozyme. has a pH from about 2.0 to about 4.0 Subsequent to exposing In one aspect, the protein composition is substantially 10 the cross-linked lipids to the saline solution described above lipid-free. As appreciated by one ordinarily skilled in the art, or Subsequent to the initial step of cross-linking the lipids. the lipids can easily be removed using membrane filtration to In an embodiment of the invention, the separated cross generate a substantially lipid-free protein composition. The linked lipids suspended in Solution, e.g. the egg mixture, may protein product may be further processed by freeze drying, be treated with an appropriate solution to adjust the pH range concentrating or spray drying the protein product to any Suit 15 of the cross-linked lipids-solution to a pH value of from about able form. Advantageously, the method used results in a pro 2.0 to about 4.0, preferably from a pH from about 2.5 to about tein composition which is free from impurities. In particular, 3.0. In an embodiment of the invention, as shown in FIG. 3, the method results in a protein composition which serves as a the method includes resuspending a precipitate of the cross safe Source of nutritional Supplement for human consump linked lipids in an acidic solution that has a pH from about 2.0 tion. Unlike other methods used for processing the egg, toxic to about 4.0, preferably from a pH from about 2.5 to about 3.0. solvents, or other unsafe materials need not be used in the A person skilled in the art may appropriately select the tem methods of the invention or are not found in the resulting perature, the amount of the acid to be used, and the period of protein composition, e.g. the isolated egg protein, or protein time sufficient to release to the proteins associated with the composition. Accordingly, the protein composition or pro cross-linked lipids. An example of typical conditions includes teins isolated thereof may be used in any number of applica 25 carrying out the separation reaction under acidic conditions tions, including but not limited to products or services in the of a pH from about 2.0 to about 4.0, more preferably from food, nutrition, and health industries. Such applications are about 2.5 to 3.0, for about 10 minutes to about 1 hour at a known in the art as well as the appropriate techniques for temperature of about 14°C. to about 32° C. Stirring or mixing inclusion in Such application or compositions. may be employed to facilitate their release. Non-limiting For example, the protein compositions of the present 30 examples of Suitable acids include acetic acid, carbonic acid, invention, in any Suitable form, e.g. aqueous or dried, can then oxalic, phosphoric, chloroacetic, citric, formic, benzoic, suc be applied to, admixed with and/or injected into consumable cinic, propionic, hydrochloric, nitric, sulfuric, hydrotropic, products for humans or animals, including farm animals or hydrologic, perchloric, chloric, phosphoric, and Sulfurous pets. Such consumable products include food or nutritive acids or combinations thereof. additives such as animal feed, human food products, nutrition 35 In one aspect, the released proteins are obtained from the Supplements for humans or animals, or the like. Accordingly, Supernatant prepared by centrifuging the acidic solution hav a method of the present invention includes treating an animal ing a pH from about 2.0 to about 4.0 and by employing or human in need of protein or amino acids by administering standard techniques, such as concentrating the proteins and a protein composition of the present invention. The protein dialyzing the concentrated proteins. In one aspect, the Super compositions may be administered to an animal or human to 40 natant may be filtered through one or more filters or mem increase their protein consumption. In some cases, the animal branes of a Suitable size to isolate specific proteins. Such as or human may need to increase their consumption of amino lysozyme, albumin, ovotransferrin, TGF-beta and transfer acids or proteins, such as an athlete, body-builder, weight factors or the like or combinations thereof. In another aspect, lifter, pregnant individual, individual recovering from Sur proteins may be isolated according to the methods of the gery, trauma, or illness, or a diabetic individual. 45 present invention, for example, based in part on the proteins Animal feed and products according to the invention, e.g., molecular weight. In one aspect, proteins that have a known dry food pellets and treats, will be designed such that they molecular weight may be isolated using a membrane or a contain appropriate nutrition levels for the particular animal, series of membranes that have the appropriate molecular cut e.g., a dog or cow. In this regard, the design of food formula off for isolating the particular protein or proteins of interest. tions for humans, e.g. body-builders, particular animals, e.g., 50 Accordingly, the selection of membrane size can be used to dog or cow, etc., is well known and established. There exist obtain a protein or population of proteins of a particular size accepted nutritional guidelines for optimal amounts of pro (molecular weight). With respect to TGF-beta, IGF and trans teins, fats, vitamins, minerals, fibers, that vary dependent fer factors, a membrane that has the appropriate molecular upon whether the consumer is a human or particular animal weight cutoff of at least 10 kDa, 5 kDa, and 1 kDa respec and the consumers age and health. It is anticipated that the 55 tively may be employed. protein compositions of the present invention can be added or In one aspect of the invention, the released proteins are incorporated to any animal or human drink, beverage, feed, separated from the acidic solution having a pH from about 2.0 food, product or Supplement. to about 4.0 or further isolated using any number of standard B. Adjusting the pH to an Acidic Range for Separating Pro techniques, including but not limited to dialysis, precipita teins Associated with the Cross-Linked Lipids from the 60 tion, sedimentation, centrifugation, decantation, particulate Cross-Linked Lipids filtration, membrane filtration, Such as gel filtration using a In one embodiment of the present invention, the method molecular sieve, temperature modification, extraction, Such includes separating proteins associated with the cross-linked as liquid-liquid extraction, Solvent extraction, salting out and lipids from the cross-linked lipids by contacting the cross desalting with NaCL or KCl chromatography, such as anion linked lipids with an acidic solution having a pH range from 65 exchange chromatography using a resin Such as diethylami about 2.0 to about 4.0. Such proteins include but are not noethyl (DEAE)-Sepharose or DIAION HPA-75 (manufac limited to lysozyme, albumin, ovotransferrin, TGF-beta or tured by Mitsubishi Chemical), cation exchange chromatog US 8,133,521 B2 23 24 raphy using a resin Such as S-Sepharose FF (manufactured by dog or cow, etc., is well known and established. There exist Pharmacia), hydrophobic chromatography using a resin Such accepted nutritional guidelines for optimal amounts of pro as butyl-Sepharose or phenyl-Sepharose, affinity chromatog teins, fats, vitamins, minerals, fibers, that vary dependent raphy, size exclusion chromatography, chemical precipita upon whether the consumer is a human or particular animal tions, chromatofocusing and electrophoresis Such as isoelec and the consumers age and health. It is anticipated that the tric focusing, and the like and combinations thereof. The protein compositions of the present invention can be added or separated or isolated proteins may be concentrated and dia incorporated to any animal or human drink, beverage, feed, lyzed. Dialyzing has the added benefit of removing the salt food, product or Supplement. (ash). C. Adjusting the pH to about 4.0 to 6.0 to Facilitate the Obtained from methods of the present invention, for 10 Separation of Proteins Released from the Cross-Linked Lip example from the Supernatant having a pH from about 2.0 to ids about 4.0, is a protein composition comprising TGF-beta, In one embodiment of the present invention, the method transfer factors, lysozyme, albumin, or ovotransferrin and the includes facilitating the separation of proteins associated with like or combinations thereof. In one embodiment, the protein the cross-linked lipids from the cross-linked lipids by con composition is at least about 70%, 75%, 80%, 85%, or 90% 15 tacting the released proteins and/or the cross-linked lipids protein per weight of composition. In one aspect, as shown in with a solution having a pH from about 4.0 to about 6.0. Such FIG. 13, the protein composition comprises albumin, proteins include but are not limited to lysozyme, albumin, ovotransferrin, and lysozyme. In one aspect, the protein com ovotransferrin, TGF-beta or transfer factors or the like or position comprises at least about 5% albumin. In one aspect, combinations thereof. Without wishing to be bound by this the protein composition comprises at least about 5% or 10% theory, it is believed that the change in pH to about 4.0 to ovotransferrin. In one aspect, the protein composition com about 6.0 aids in the released proteins solubility, thereby prises at least 70%, 75%, 80% or 85% lysozyme. In one further facilitating the separation of proteins associated with aspect, the protein composition comprises at least about 5% the cross-linked lipids. According to the methods of the albumin, and at least about 5% ovotransferrin, and at least present invention, the step of contacting the released proteins 70% lysozyme. 25 or the cross-linked lipids or combinations thereof with a In one aspect, the protein composition is substantially solution having a pH from about 4.0 to about 6.0 may be lipid-free. As appreciated by one ordinarily skilled in the art, performed after any number of steps, depending on the pro the lipids can easily be removed using membranes or filters to teins that are desired to be separated. In one embodiment, generate a Substantially lipid-free protein composition. In one Subsequent to contacting the cross-linked lipids with the aspect, the protein composition comprises processing the iso 30 saline solution as described elsewhere herein, the released lated protein or a combination of proteins of interest by freeze proteins or the cross-linked lipids or combinations thereofare drying, concentrating or spray drying the protein to any suit contacted with a solution that has a pH from about 4.0 to about able form. Advantageously, the method used results in a pro 6.0. In one embodiment, Subsequent to contacting the cross tein composition which is free from impurities. In particular, linked lipids with the solution having a pH from about 2.0 to the method results in a protein composition which serves as a 35 4.0 as described elsewhere herein, the released proteins or the safe Source of nutritional Supplement for human consump cross-linked lipids or combinations thereof are contacted tion. Unlike other methods used for processing the egg, toxic with a solution that has a pH from about 4.0 to about 6.0. In solvents, or other unsafe materials need not be used in the one embodiment, Subsequent to contacting the egg mixture methods of the invention or are not found in the resulting that has its pH optimized for cross-linking of the lipids as protein composition, e.g. the isolated egg protein, or protein 40 described elsewhere herein, the released proteins or the cross composition. Accordingly, the protein composition or pro linked lipids or combinations thereof are contacted with a teins isolated thereof may be used in any number of applica solution that has a pH from about 4.0 to about 6.0. As apparent tions, including but not limited to products or services in the to one ordinarily skilled, the pH ranges of the various solu food, nutrition, and health industries. Such applications are tions or mixtures, e.g. the Saline Solution, the Solution with a known in the art as well as the appropriate techniques for 45 pH from about 2.0 to about 4.0, or the egg mixture having a inclusion in Such application or compositions. pH adjusted to a pH optimized for lipid cross-linking, may be For example, the protein compositions of the present treated with an appropriate Solution to adjust the pH range of invention, in any Suitable form, e.g. aqueous or dried, can then the solutions or mixtures to a pH of from about 4.0 to about be applied to, admixed with and/or injected into consumable 6.0. A person skilled in the art may appropriately select the products for humans or animals, including farm animals or 50 temperature, the amount of the acid or base to be used, and the pets. Such consumable products include food or nutritive period of time sufficient to release to the proteins associated additives such as animal feed, human food products, nutrition with the cross-linked lipids. An example of typical conditions Supplements for humans or animals, or the like. Accordingly, includes carrying out the separation reaction under conditions a method of the present invention includes treating an animal of a pH from about 4.0 to about 6.0, more preferably from or human in need of protein or amino acids by administering 55 about 4.5 to 5.0, for about 15 minutes to about 1 hour, pref a protein composition of the present invention. The protein erably from about 30 minutes to about 1 hour, at room tem compositions may be administered to an animal or human to perature. Stirring or mixing may be employed to facilitate increase their protein consumption. In some cases, the animal their separation or solubility. Non-limiting examples of Suit or human may need to increase their consumption of amino able acids and bases include acetic acid, carbonic acid, oxalic, acids or proteins, such as an athlete, body-builder, weight 60 phosphoric, chloroacetic, citric, formic, benzoic, succinic, lifter, pregnant individual, individual recovering from Sur propionic, hydrochloric, nitric, Sulfuric, hydrotropic, hydro gery, trauma, or illness, or a diabetic individual. logic, perchloric, chloric, phosphoric, Sulfurous acids, Animal feed and products according to the invention, e.g., Sodium hydroxide or potassium hydroxide. dry food pellets and treats, will be designed such that they In one aspect, the released proteins in the acidic Solution contain appropriate nutrition levels for the particular animal, 65 may be subjected to a basic solution to achieve a pH range of e.g., a dog or cow. In this regard, the design of food formula the solution from about 4.0 to about 6.0, preferably from tions for humans, e.g. body-builders, particular animals, e.g., about 4.5 to about 5.0, to solubilize proteins. The released US 8,133,521 B2 25 26 proteins may be isolated prior to contact with the basic Solu C. Onhen egg fractionation: application of liquid chromatog tion or the pH of the solution they are in may be adjusted to a raphy to the isolation and purification of hen egg white and pH from about 4.0 to about 6.0. egg yolk proteins. Z. Lebensm.-Unters.-Forsch. (1996). 202: In one aspect of the invention, the proteins are obtained 1-14, herein incorporated by reference in its entirety. Dialyz from a Supernatant prepared by centrifuging the solution hav 5 ing has the added benefit of removing the ash (or salt). The ing a pH range from about 4.0 to about 6.0 and by employing proteins may be isolated or purified for use in any number of standard techniques, such as concentrating the proteins and applications, including protein compositions. dialyzing the concentrated proteins. Dialyzing has the added Obtained from methods of the present invention, e.g. the benefit of removing the salt (ash). In one aspect, the Superna solution having a pH from about 4.0 to about 6.0, is a sub tant may be filtered through one or more membranes of a 10 stantially lipid-free protein composition comprising Suitable size to isolate specific proteins, such as lysozyme, lysozyme, albumin, ovotransferrin, TGF-beta and transfer albumin, ovotransferrin, TGF-beta and transfer factors or the factors or the like or combinations thereof present in the egg. like or combinations thereof. In another aspect, proteins may In one aspect, the protein composition comprising aparticular be isolated according to the methods of the present invention, protein or a combination of proteins may be further processed for example, based in part on the proteins molecular weight. 15 by freeze drying, concentrating or spray drying the protein to In one aspect, proteins that have a known molecular weight any Suitable form. may be isolated using a membrane or a series of membranes A protein composition may be obtained from the acidic that have the appropriate molecular cut off for isolating the Supernatant. In one aspect, the protein composition comprises particular protein or proteins of interest. Accordingly, the lysozyme, albumin, ovotransferrin, TGF-beta and transfer selection of membrane size can be used to obtain a protein or factors or the like or combinations thereof. In one aspect, as population of proteins of a particular size (molecular weight). shown in FIG. 13, the protein composition comprises With respect to TGF-beta, IGF and transfer factors, a mem lysozyme, albumin, and ovotransferrin. In one aspect, the brane that has the appropriate molecular weight cutoff of at composition is at least 70%, 75%, 80%, 85% or 90% protein least 10 kDa, 5 kDa, and 1 kDa respectively may be per weight of composition, comprises at least 5% or 10% employed. 25 album, at least 5% or 10% ovotransferrin, and at least 70%, In one aspect, the released proteins, such as lysozyme, 80% or 85% lysozyme. In one aspect, the protein composition albumin, ovotransferrin, TGF-beta and transfer factors or the is substantially lipid-free. As appreciated by one ordinarily like or combinations thereof, may be isolated from the lipids skilled in the art, the lipids can easily be removed using in the solution having a pH range from about 4.0 to about 6.0 membrane filtration to generate a lipid-free protein composi using any number of standard techniques, including but not 30 tion. In one aspect, the protein composition comprises pro limited to dialysis, precipitation, sedimentation, centrifuga cessing the isolated protein or a combination of proteins of tion, decantation, particulate filtration, membrane filtration, interest by freeze drying, concentrating or spray drying the Such as gel filtration using a molecular sieve, temperature protein to any Suitable form. modification, extraction, such as liquid-liquid extraction, Sol Advantageously, the method used results in a protein com vent extraction, salting out and desalting with NaCL or KCl, 35 position which is free from impurities. In particular, the chromatography, Such as anion exchange chromatography method results in a protein composition which serves as a safe using a resin such as diethylaminoethyl (DEAE)-Sepharose Source of nutritional Supplement for human consumption. or DIAION HPA-75 (manufactured by Mitsubishi Chemi Unlike other methods used for processing the egg, toxic sol cal), cation exchange chromatography using a resin Such as vents, or other unsafe materials need not be used in the meth S-Sepharose FF (manufactured by Pharmacia), hydrophobic 40 ods of the invention or are not found in the resulting protein chromatography using a resin Such as butyl-Sepharose or composition, e.g. the isolated egg protein, or protein compo phenyl-Sepharose, affinity chromatography, size exclusion sition. Accordingly, the protein composition or proteins iso chromatography, chemical precipitations, chromatofocusing lated thereof may be used in any number of applications, and electrophoresis such as isoelectric focusing, and the like including but not limited to products or services in the food, and combinations thereof. Accordingly, the process of the 45 nutrition, and health industries. Such applications are known invention may also include isolating from the Supernatant, in the art as well as the appropriate techniques for inclusion in retentate, permeate, precipitate, protein or lipid composi Such application or compositions. tions, various proteins or other components of interest. For example, the protein compositions of the present In another aspect, the identification of isolated proteins invention, in any Suitable form, e.g. aqueous or dried, can then may be determined using routine techniques known to those 50 be applied to, admixed with and/or injected into consumable ordinarily skilled in the art. Exemplary techniques includebut products for humans or animals, including farm animals or are not limited to ELISA, Western blot analysis, 2D-PAGE pets. Such consumable products include food or nutritive (Westermeier, R.; Naven, T. Proteomics in practice; Wiley additives such as animal feed, human food products, nutrition VCH: Weinheim, 2002: pp 11-97) and automated spot iden Supplements for humans or animals, or the like. Accordingly, tification by matrix-assisted laser desorption-ionization mass 55 a method of the present invention includes treating an animal spectrometry (MALDI-MA) or liquid chromatography elec or human in need of protein oramino acids by administering trospray ionization mass spectrometry (LC-ESIMS) (Dass, a protein composition of the present invention. The protein C. Structural Analysis of Proteins. In Principles and practice compositions may be administered to an animal or human to of biological mass spectrometry; Dass, C. Ed., Wiley-Inter increase their protein consumption. In some cases, the animal science: New York, 2001: pp 75-216). 60 or human may need to increase their consumption of amino If desired, the isolated protein or population of proteins acids or proteins. Such as an athlete, body-builder, weight may be further purified, using for example, dialysis and/or lifter, pregnant individual, individual recovering from Sur chromatography, or sequential chromatographies, of anion gery, trauma, or illness, or a diabetic individual. or cation exchange column chromatography using a resin of Animal feed and products according to the invention, e.g., DEAE or CM, affinity chromatography using agel of heparin, 65 dry food pellets and treats, will be designed such that they biotin, hydroxyapatite, hydrophobic chromatography using a contain appropriate nutrition levels for the particular animal, gel of methyl HIC ort-butyl HIC. See for example, Awade, A. e.g., a dog or cow. In this regard, the design of food formula US 8,133,521 B2 27 28 tions for humans, e.g. body-builders, particular animals, e.g., ent to one ordinarily skilled, the pH ranges of the various dog or cow, etc., is well known and established. There exist solutions, e.g. the solution with a pH from about 2.0 to about accepted nutritional guidelines for optimal amounts of pro 4.0 may be treated with an appropriate Solution to adjust the teins, fats, vitamins, minerals, fibers, that vary dependent pH range of the solution to a pH of from about 4.0 to about upon whether the consumer is a human or particular animal 6.0. A person skilled in the art may appropriately select the and the consumers age and health. It is anticipated that the temperature, the amount of the acid or base to be used, and the protein compositions of the present invention can be added or period of time sufficient to release to the proteins associated incorporated to any animal or human drink, beverage, feed, with the cross-linked lipids. food, product or Supplement. According to the invention, the white lipid extract may be V. Separating the Triglycerides, Cholesterol, Phospholipids, 10 isolated from an acidic solution having a pH from about 2.0 to Lipoproteins, Fatty Acids from the Cross-Linked Lipids 4.0 as shown in FIG. 9, or a basic solution as shown in FIG. In an embodiment of the present invention, the method 11, using standard techniques, such as Such as precipitation, includes separating a white lipid extract comprising triglyc sedimentation, centrifugation, decantation, particulate filtra erides, cholesterol, phospholipids, lipoproteins. Such as HDL tion, membrane filtration, temperature modification, liquid (alpha lipovitellin), LDL (beta-lipovitellin), VLDL (apovitel 15 liquid extraction, and the like and combinations thereof. lenin), and fatty acids, such as Sialic acid, Arachadonic acid or In one aspect of the invention, as shown in FIG. 11, the the like or combinations thereof from the cross-linked lipids method includes centrifuging the solution having a pH from by contacting the cross-linked lipids with an acidic Solution about 4.0 to about 6.0, more preferably having a pH from having a pH range from about 2.0 to about 4.0. Without about 4.5 to about 5.0, to generate a Supernatant comprising wishing to be bound by this theory it is believed that an acidic solubilized proteins as described elsewhere herein and a pre solution with a pH from about 2.0 to about 4.0 facilitates the cipitate comprising lipids, referred to herein as a white lipid separation of triglycerides, cholesterol, phospholipids, lipo due to its color. In one aspect, the white lipid may be trans proteins, such as HDL (alpha lipovitellin), LDL (beta-lipovi ferred to a liquid or solution to form an extract. The white tellin), VLDL (apovitellenin), and fatty acids, such as sialic lipid may be resuspended into any Suitable solution that does acid, Arachadonic acid or the like or combinations thereof 25 not destroy the lipids depending on the intended purpose for from the cross-linked lipids into the acidic solution. In some the white lipid extract or triglycerides, cholesterol, phospho embodiments, the cross-linked lipids are exposed to a solu lipids, lipoproteins, such as HDL (alpha lipovitellin), LDL tion that has a pH from about 2.0 to about 4.0 subsequent to (beta-lipovitellin), VLDL (apovitellenin), and fatty acids, exposing the cross-linked lipids to the saline Solution Such as Sialic acid, Arachadonic acid or the like isolated described above or subsequent to the initial step of cross 30 therefrom. As an example, the fatty acids may be solubilized linking the lipids. into any suitable solution for further use in any number of In an embodiment of the invention, the separated cross applications. In one aspect, the white lipid is resuspended into linked lipids Suspended in Solution, e.g. the egg mixture, may a basic solution, for example, an approximately 5% NaOH be treated with an appropriate solution to adjust the pH range solution with a pH from about 9.5 to about 10.5 to solubilize of the cross-linked lipids-solution to a pH value of from about 35 the fatty acids into the basic solution. In one aspect, the white 2.0 to about 4.0, preferably from a pH from about 2.5 to about lipid is resuspended into an acidic Solution, for example, an 3.0. In an embodiment of the invention, as shown in FIG. 3, approximately 10% acetic acid with a pH from about 2.5 to the method includes resuspending a precipitate of the cross about 3.5 to solubilize the fatty acids into the basic solution. linked lipids in an acidic solution that has a pH from about 2.0 The use of an acid solution also has the added benefit of being to about 4.0, preferably from a pH from about 2.5 to about 3.0. 40 an anti-microbial, which, therefore, also makes it a good A person skilled in the art may appropriately select the tem choice for a storage Solution. In particular, an acetic acid perature, the amount of the acid or base to be used, and the Solution advantageously has been shown to stabilize lipids. period of time sufficient to release or solubilize to the com Alternately, the white lipid may be resuspended into a solu ponents, e.g. fatty acids, associated with the cross-linked tion that has a neutral pH, for example, one that is suitable for lipids. An example of typical conditions includes carrying out 45 cosmetic purposes. the separation reaction under acidic conditions of a pH from Obtained is a white lipid extract composition or precipitate about 2.0 to about 4.0 for about 10 minutes to about 1 hour at comprising triglycerides, cholesterol, phospholipids, lipo a temperature of about 14° C. to about 32° C. Stirring or proteins such as HDL (alpha lipovitellin), LDL (beta-lipovi mixing may be employed to facilitate their release. Non tellin), VLDL (apovitellenins), and fatty acids, such as sialic limiting examples of Suitable acids Suitable for adjusting the 50 acid, Arachadonic acid or the like or combinations thereof. pH include acetic acid, carbonic acid, oxalic, phosphoric, The white lipid extract, white lipid precipitate, or triglycer chloroacetic, citric, formic, benzoic, Succinic, propionic, ides, cholesterol, phospholipids, lipoproteins such as HDL hydrochloric, nitric, Sulfuric, hydrotropic, hydrologic, per (alpha lipovitellin), LDL (beta-lipovitellin), VLDL (apovitel chloric, chloric, phosphoric, and Sulfurous acids or combina lenins), and fatty acids, Such as Sialic acid, Arachadonic acid tions thereof. 55 or the like or combinations thereofisolated therefrom may be In one embodiment of the present invention, the method used in any number of applications, including but not limited includes facilitating the separation of a white lipid extract to products or services in the food, nutrition, and health indus comprising triglycerides, cholesterol, phospholipids, lipo tries. Such applications are known in the art as well as the proteins, such as HDL (alpha lipovitellin), LDL (beta-lipovi appropriate techniques for inclusion in Such application or tellin), VLDL (apovitellenin), and fatty acids, such as sialic 60 compositions. acid, Arachadonic acid or the like or combinations thereof VI. Separating the Carotenoids from the Cross-Linked Lipids from the cross-linked lipids by adjusting the pH of the solu In an embodiment of the present invention, the method tion from about 2.0 to about 4.0 to a pH from about 4.0 to includes separating carotenoids. Such as lutein and Zeaxan about 6.0, more preferably from about 4.5 to about 5.0. With thin, from the cross-linked lipids. According to the invention, out wishing to be bound by this theory, it is believed that the 65 the method includes separating the cross-linked lipids com change in pH to about 4.0 to about 6.0 aids in the separation prising carotenoids from an acidic Solution with a pH from ofa white lipid extract from the cross-linked lipids. As appar about 2.0 to about 4.0, preferably from an acidic solution with US 8,133,521 B2 29 30 from about 2.5 to about 3.0. In one aspect, the separation may In one aspect, a carotenoid composition comprises caro be accomplished by Standard techniques, including but not tenoids obtained from an egg using the methods of the present limited to precipitation, sedimentation, centrifugation, invention. In one aspect, the carotenoids comprise luteins and decantation, particulate filtration, membrane filtration, chro Zeaxanthins. Advantageously, the carotenoids and caro matography, and the like and combinations thereof. In one tenoids of the present invention are bioavailable since they are aspect, the carotenoids are obtained by centrifuging the cross substantially lipid-free. linked lipids from the acidic solution to obtain a precipitate. The carotenoids, carotenoid composition, and Zeaxanthin In some embodiments, the cross-linked lipids are con or lutein thereof may be used in any number of applications, tacted with a solution that has a pH from about 2.0 to about 4.0 including but not limited to products or services in the food, 10 nutrition, pharmaceutical and health industries. Such appli Subsequent to contacting the cross-linked lipids with the cations are known in the art as well as the appropriate tech saline solution described above or subsequent to the initial niques for inclusion in Such application or compositions. step of cross-linking the lipids. In one aspect, the cross-linked Examples of carotenoids in pharmaceutical uses include, lipids may be subjected to a saline Solution as described without limitation: treatment of cancer Such as disclosed in elsewhere herein and as shown in FIGS. 10-11, prior to being 15 U.S. Pat. No. 5,811,119 to Mehta et al.; age-related macular subjected to an acidic solution that adjusts the pH of the saline degeneration such as disclosed in U.S. Pat. No. 6,329,432 to solution to a pH from about 2.0 to about 4.0, preferably from Norman et al. U.S. Pat. No. 6,218,436 to Howard et al.: a pH from about 2.5 to about 3.0. As appreciated by one hypertriglyceridaemia as disclosed in U.S. Pat. No. 5,811,119 ordinarily skilled in the art, the solution may be centrifuged to to Klor et al., inflammatory disorders such as disclosed in separate the cross-linked lipids comprising the carotenoids U.S. Pat. No. 5,886,053 to Schmutzler et al.; and skin damage from other components, e.g. proteins released into Solution at due to exposure to sunlight such as disclosed in U.S. Pat. No. the various steps of the method. 5,804,168 to Murad. Carotenoids may also be used as feed In an embodiment of the present invention, the method Supplements for pigmenting certain parts and products of includes resuspending the precipitate of cross-linked lipids poultry, fish and crustacea, as disclosed in U.S. Pat. No. comprising the carotenoids in a polar solvent to release the 25 5,605,699 to Bernhard et al., U.S. Pat. No. 5,849,345 to Giger carotenoids from the cross-linked lipids. As used herein, the et al., U.S. Pat. No. 7,220,874 to Cordona et al. All references term polar solvent includes solvents that are miscible with mentioned are herein incorporated by reference, each in its water, including but not limited to water, an alcohol Such as entirety. It would be highly advantageous to have a source of ethanol or methanol, caprylic acid, and the like. Any polar feed supplement for pigmentation in poultry which can be Solvent that solubilizes any of the carotenoids in the egg may 30 derived from eggs, a poultry product. Carotenoids used in be used. The percentage of polar solvent used may be about poultry feed is generally obtained from other sources such as 50-100%. Any percentage or form of polar solvent may be marigolds. Using the process of the present invention, would used so long as it is able to solubilize one or more of the allow a poultry and egg producer to have its own source of carotenoids. Such as lutein and Zeaxanthin or combinations carotenoids for feed supplement and thereby decrease reli thereof into the solvent. A person skilled in the art may 35 ance on Suppliers of feed Supplements. Accordingly, in an appropriately select the temperature, the percent of the Sol embodiment of the present invention, the method includes vent to be used, and the period of time sufficient to release to feeding the carotenoids to poultry. the carotenoids from the cross-linked lipids. An example of Therefore methods and resulting compositions related to typical conditions includes carrying out the separation reac the recovery of components from whole eggs, technical or tion for about 30 minutes to about 1 hour at a room tempera 40 edible eggs, yolks or whites have been disclosed. Various ture. Mixing or stirring of the precipitate of cross-linked embodiments, alternatives, and options have been discussed lipids comprising the carotenoids in the solvent may facilitate throughout this disclosure. The present invention is not to be the release or solubility of the carotenoids. limited to specific embodiments presented herein. In an embodiment of the present invention, the method includes separating from the polar solvent carotenoids, for 45 We claim: example, lutein, Zeaxanthin and the like, or combinations 1. A method of separating protein from an egg mixture, thereof. The carotenoids, including but not limited to luteins wherein the egg mixture comprises lipids and solubilized and Zeaxanthins, may be separated from the polar solvent proteins, the method comprising the steps of: using any Suitable technique. The carotenoids may be sepa (a) adding a cross-linking reagent to the egg mixture com rated from the Solvent by centrifuging the resuspended pre 50 prising lipids and Solubilized proteins to obtain cross cipitate/polar solvent mixture to obtain a Supernatant and linked lipids; extracting the colorful carotenoids therefrom. Such tech (b) adjusting the pH level of the egg mixture to a pH at niques are well within the knowledge of one ordinarily skilled which the cross-linking reagent is functional so that in the art, see, for example, Tyzkowski and Hamilton, Poultry cross-linking of lipids occurs; and Sci. 70(3):651654 (1991), and U.S. Pat. Nos. 7,173,145 and 55 (c) separating the proteins from the cross-linked lipids to 6,909,021, herein incorporated by reference in its entirety, provide a separated protein fraction. describing the extraction of carotenoids, such as lutein and 2. The method of claim 1 wherein the suitable pH for Zeaxanthin. Standard techniques that may also be used cross-linking to occur is from about 6.0 to about 8.0. include to separate or further isolated the carotenoids include 3. The method of claim 1 wherein the egg mixture com but are not limited to precipitation, sedimentation, centrifu 60 prises a technical egg, an edible egg, an egg yolk, or an egg gation, decantation, Solvent extraction, Such as Super critical white, or combination thereof. fluid extraction or liquid-liquid extraction, Saponification, 4. The method of claim 1 wherein the cross-linking reagent chromatography, chemical precipitations, multiple solvents, is selected from the group consisting of beta-cyclodextrin, blends of chloroform, or fatty acids, like oleic acid or caprylic silicon dioxide, colloidal silica material, fumed silica mate acid, or mixtures thereof, temperature modification, particu 65 rial, and synthetic calcium silicate hydrate. late filtration, membrane filtration, chromatography, and the 5. The method of claim 4 wherein the colloidal silica mate like or combinations thereof. rial is an acid stabilized dispersion of colloidal silica particles. US 8,133,521 B2 31 32 6. The method of claim 4 wherein the fumed silica material 12. The method of claim 1, wherein the separated protein is selected from the group consisting of synthetic amorphous fraction comprises proteins of greater than 100 kDa in silicon dioxide, crystalline-free fumed silica, crystalline-free molecular weight. precipitated silica. 13. The method of claim 12 wherein proteins greater than 7. The method of claim 1, wherein the separating step of (c) 100 kDa in molecular weight are separated using a membrane comprises dialysis, precipitation, sedimentation, centrifuga that has a molecular weight cutoff of at least 100kDa to obtain tion, decantation, particulate filtration, membrane filtration, a retentate protein mixture comprising proteins that are temperature modification, extraction, desalting, chromatog greater than 100 kDa in molecular weight and a permeate raphy, chemical precipitations, chromatofocusing, electro protein mixture comprising proteins that are less than 100 10 kDa in molecular weight. phoresis, or combinations thereof. 14. The method of claim 13 further comprising separating 8. The method of claim 1, further comprising the step of proteins that are less than 100 kDa in molecular weight centrifuging the protein fraction made in step (c) and recov obtained from the permeate protein mixture but greater than 1 ering a Supernatant that comprises solubilized proteins. kDa in molecular weight by Subjecting the permeate to a 9. The method of claim 8 wherein separating the proteins 15 membrane that has a molecular weight cutoff of at least 1 from the Supernatant is performed using membrane filtration kDa. to obtain a retentate protein mixture. 15. The method of claim 1, further comprising drying the 10. The method of claim 9 wherein the membrane used to separated protein. recover the proteins from the Supernatant has a molecular cut 16. The method of claim 15, wherein the drying step is off weight of at least 0.8 micron. selected from the group consisting of spray-drying, freeze 11. The method of claim 1 wherein the proteins comprise drying, spray-freeze drying, paddle-drying, drum drying, lyo ovotransferrin, albumin, beta-livetin (alpha-2-glycoprotein), philization, vacuum drying, or air drying. gamma livetin (IgY), insulin-like growth factor-1 (IGF-1), or phosvitin or combinations thereof. k k k k k