[0001] This invention was made with government support under Grant No. 2015-

067017-23115 awarded by USDA/NIFA. The government has certain rights in the invention.

FIELD OF USE

[0002] The present invention relates to food additives that affect egg yolk gelation induced by freezing.

BACKGROUND OF THE INVENTION

[0003] Egg yolk, in its fluid form, is a valuable food ingredient for the manufacture of many food products. A large quantity of liquid yolk is frozen commercially for prolonged storage of up to one year (Au et al,“Determination of the Gelation Mechanism of Freeze- Thawed Hen Egg Yolk,” Journal of Agricultural andFood Chemistry 63(46): 10170-10180 (2015)). The benefits of storing egg yolk in the frozen state are prevention of microbial growth and spoilage, retention of egg yolk flavor and color, and inhibition of chemical reactions such as autoxidation of lipids and the browning reaction (Powrie,“Gelation of Egg Yolk upon Freezing and Thawing,” In Low Temperature Biology of Foodstuffs: Recent Advances in Food Science 4:319-331 (1968)). However, when yolk is frozen and stored below -6°C, an irreversible alteration in fluidity known as gelation occurs (Moran,“The Effect of Low Temperature on Hens' Eggs,” Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 98(691):436-456 (1925)). This physiological change is undesirable because of reduced yolk dispersibility in water and loss of functionality.

[0004] The mechanism for yolk gelation caused by freezing and thawing has not been fully elucidated. Regardless of the many existing proposed mechanisms, most researchers agree that ice crystal formation during freezing storage plays a fundamental role in yolk gelation. Moran,“The Effect of Low Temperature on Hens' Eggs,” Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 98(691):436-456 (1925) found that when yolk is rapidly cooled below -6°C, no significant viscosity change was seen. Lopez et al.,“Some Factors Affecting Gelation of Frozen Egg Yolk,” Journal of Milk and Food

Technology 17:334-339 (1954) and Jaax et al.,“The Effect of Pasteurization, Selected Additives and Freezing Rate on the Gelation of Frozen-Defrosted Egg Yolk,” Poultry Science 47(3):1013- 1022 (1968) found that when yolk was frozen rapidly in liquid nitrogen (-l96°C) and stored at about -20°C for periods up to 49 days, the apparent viscosities of the thawed products were lower than those of the controls frozen and stored at approximately -20°C. Additionally, Rolfe, “Characteristic of Preservation Processes as Applied to Proteinaceous Foods,” in R. A. Lawrie (Ed.) Proteins as Human hood Butterworth & Co. Ltd, pp. 107-125 (1969) stated that ice crystal formation needs to reach an extent of 81% in order for gelation to occur.

[0005] Other than rapid freezing, some other treatments have been applied to yolks to prevent gelation. Inhibition of gelation could be achieved by the addition of cryoprotective agents, proteolytic enzymes, or mechanical treatments to prevent ice crystal formation and changes in yolk’s physicochemical conditions that favor aggregation of proteins. Moran,“The Effect of Low Temperature on Hens' Eggs,” Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 98(691):436-456 (1925) was the first to report that sucrose could be used to prevent gelation of yolk. Other additives such as glucose, arabinose, galactose, glycerol, sorbitol, propylene glycol, and salt (NaCl) have also been found to be effective inhibitors of gelation (Lopez et al.,“Some Factors Affecting Gelation of Frozen Egg Yolk,” Journal ofMilk andFood Technology 17:334-339 (1954); Powrie et ah,“Gelation of Egg Yolk,” Journal of Food Science 28(l):38-46 (1963)). At low concentrations, salts were shown to stabilize the system due to electrostatic shielding of attractive forces (Hamada et ah, “Competition Between Folding, Native-State Dimerisation and Amyloid Aggregation in b- Lactoglobulin,” Journal of Molecular Biology 386(3):878-890 (2009)). Crotoxin (lecithinase A) at lmg/mL yolk and 10 mg/mL yolk used led to only 10-20% gelation compared to untreated yolk (Feeney et ah,“Effects of Crotoxin (Lecithinase A) on Egg Yolk and Yolk Constituents,” Archives of Biochemistry and Biophysics 48(1): 130-140 (1954)). The low-density lipoproteins (LDL) and high-density lipoproteins (HDL) were proposed to be attacked by the enzyme and the resultant lysophospholipoproteins had an altered solubility in water. Papain at 0.05%

concentration was also reported to inhibit gelation due to its ability to break down the proteins responsible for gelation (Lopez et ah,“Enzymic Inhibition of Gelation in Frozen Egg Yolk,” Journal ofMilk andFood Technology 18:77-80 (1955)).

[0006] The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

[0007] One aspect of the invention relates to a method for inhibiting egg yolk gelation resulting from freezing and thawing This method comprises treating egg yolk with an effective amount of a compound selected from the group consisting of amino acids, hydrolyzed proteins, hydrolyzed carboxymethyl cellulose, polyethylene glycol, and sorbitan esters, to improve the gelation properties of egg yolk.

[0008] Another aspect of the invention relates to an egg yolk-containing product having inhibited susceptibility to gelation resulting from freezing further comprising the compound selected from the group consisting of amino acids, hydrolyzed proteins, hydrolyzed

carboxymethyl cellulose, polyethylene glycol, and sorbitan esters.

[0009] Increased consumer awareness towards healthy consumption of food low in salt and sugar has motivated reexamination of this issue by applicants. With more advanced technology and new research efforts on using food additives to improve functionality, it is important to find alternative methods to inhibit gelation, without significantly altering the saltiness or sweetness of the yolk. Physical means, such as colloid milling, was introduced to destroy or“bury” the native yolk granular and plasma low density lipoprotein (LDL) surface structures responsible for gelation. Combinations of food additives, such as hydrolyzed carboxymethyl cellulose (HCMC), hydrolyzed egg white (HEW), hydrolyzed egg yolk (HEY), proline, polyethylene glycol and Tween 80, were evaluated for their effectiveness in interrupting protein association thus inhibiting gelation. These additives have never been studied for their anti-gelation effects on egg yolk, and were selected due to their high solubility in water, low freezing point, and/or the presence of a hydrophobic side chain.

[0010] The specific impact of additives on protein interactions can vary greatly and is dependent on the chemical nature, additive concentration, protein type, and pH. In the present application, the effectiveness of each additive at varying concentrations as well as mechanical treatments such as high speed mixing and colloid milling on yolk gelation reduction was systematically tested. Synergistic effects of combined treatments were also explored. With the selected additives, the mechanism of gelation prevention was further studied. It was hypothesized that since gelation may be associated with ice crystal formation which then leads to dehydration and aggregation of lipoproteins, treatments that can reduce the amount of freezable water, minimize exposure of hydrophobic sites, and/or prevent surface aggregation can prevent gelation. To prove this hypothesis, the amount of freezable water, protein surface

hydrophobicity, and lipoprotein particle size before and after freezing were evaluated and compared.

[0011] Technological advances in preventing yolk gelation during freezing and thawing were demonstrated in the present application. Gelation negatively affects yolk functionality in food formulation. Preventing gelation using 10% salt or sugar limits the application of the yolk. Novel food additives were tested to prevent gelation induced by freezing. Significant reduction (p<0.05) in gel hardness of frozen-thawed yolk (45 hours freezing at -20°C) indicated that hydrolyzed carboxymethyl cellulose (HCMC), proline, and hydrolyzed egg white and yolk (HEW and HEY) are effective gelation inhibitors. The mechanisms in which these additives prevented gelation were further studied through measuring the changes in the amount of freezable water, lipoprotein particle size, and protein surface hydrophobicity. Overall, several alternatives of gelation inhibitor were found that have great potential in replacing the use of salt or sugar in commercial operations for freezing egg yolk for shelf-life extension.

BRIEF DESCRIPTION OF DRAWINGS

[0012] Figures 1A-1D are graphs showing the effect of rotor-stator mixing speed

(Figures 1 A and 1C) and colloid milling (CM) (Figures 1B and 1D) on the hardness and particle size distribution of frozen-thawed yolk (-20°C for 45 hours). Values with different letters are significantly different (p<0.05).

[0013] Figures 2A-2B are graphs showing the effect of various additives (Figure 2A) and quantity of additives (Figure 2B) on hardness of frozen-thawed yolk (stored at -20°C for 45 hours). Values with different letters are significantly different (p<0.05).

[0014] Figures 3A-3B are graphs showing the hardness of frozen-thawed yolk treated with HCMC of different molecular weights, and frozen at -20°C for 5 days (Figure 3 A) and 1 day at 2% (w/w) concentration (Figure 3B). Values with different letters are significantly different (p<0.05).

[0015] Figures 4A-4G are graphs showing the hardness of frozen-thawed yolk (stored at -

20°C for 45 hours) treated with combinations of additives at 5% (w/w) concentration, including Proline-HCMC (Figure 4A), HEY-HCMC (Figure 4B), HEW-HCMC (Figure 4C), HCMC-sugar (Figure 4D), FICMC-salt (Figure 4E), FIEW-salt (Figure 4F), and FtEW-sugar (Figure 4G). Values with different letters are significantly different (p<0.05).

[0016] Figures 5A-5C are graphs showing the effect of colloid milling and additives on hardness (Figure 5A) and particle size distribution (Figures 5B and 5C) of yolk frozen at -20°C for 45 hours. Abbreviations are F, fresh; G, frozen-thawed; CM, colloid milled.

[0017] Figure 6 shows schematic illustrations of the proposed gelation-inhibiting mechanism by FICMC, proline, and peptide in frozen-thawed egg yolk.

[0018] Figures 7A-7C are graphs showing the particle size distribution (Figure 7A), protein surface hydrophobicity (Figure 7B), and amount of freezable water (Figure 7C) of yolk containing various additives. Abbreviations are F, fresh yolk; G, frozen-thawed yolk (stored at - 20°C for 45 hours).

DETAILED DESCRIPTION OF INVENTION

[0019] One aspect of the invention relates to a method for inhibiting egg yolk gelation resulting from freezing and thawing. This method comprises treating egg yolk with an effective amount of a compound selected from the group consisting of amino acids, hydrolyzed proteins, hydrolyzed carboxymethyl cellulose, polyethylene glycol, and sorbitan esters, to improve the gelation properties of egg yolk.

[0020] As used above, and throughout the description herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this technology belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0021] As used herein, the term "whole egg" means a mixture of egg white and yolk. The whole egg may, but does not necessarily, include egg white and egg yolk in a ratio recognized as the ratio of yolk to white in eggshells. Whole egg products can include other optional ingredients as described below.

[0022] As used herein, the term "egg yolk" means egg yolk obtained after separating the white and the yolk by breaking fresh eggs, and as such, the egg yolk is substantially free of egg white. The egg yolk can be used in the disclosed products that can comprise other optional ingredients as described below.

[0023] As used herein, the term "egg white" means egg white obtained after separating the white and the yolk by breaking fresh eggs, and as such, the egg white is substantially free of egg yolk. The egg white can be used in the disclosed products that can comprise other optional ingredients as described below.

[0024] Amino acids that can be used according to the present invention can be any natural or non-natural amino acid, including alpha amino acids, beta amino acids, gamma amino acids, L-amino acids, and D-amino acids.

[0025] Suitable amino acids that can be used according to the present invention include, but are not limited to, histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), valine (Val), arginine (Arg), cysteine (Cys), glutamine (Gin), glycine (Gly), proline (Pro), serine (Ser), tyrosine (Tyr), alanine (Ala), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), and selenocysteine (Sec).

[0026] As used herein, the term“hydrolyzed proteins” or“protein hydrolysates” refers to proteins that have been hydrolyzed or broken down into shorter peptide fragments and amino acids.

[0027] Suitable protein sources for hydrolyzed proteins include milk, soy, rice, meat

(e g., beef), animal, vegetable (e g., pea, potato), egg, gelatin, and fish Suitable proteins include, but are not limited to, soy based, milk based, casein protein, whey protein, rice protein, beef collagen, pea protein, potato protein and mixtures thereof. Suitable protein hydrolysates also include, but are not limited to, soy protein hydrolysate, casein protein hydrolysate, whey protein hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg hydrolysate, gelatin protein hydrolysate, a combination of animal and vegetable protein hydrolysates, and mixtures thereof.

[0028] As used herein, the term“hydrolyzed egg white protein” or“hydrolyzed egg white” refers to a product of a hydrolysis of egg white protein. Wherein the product can be fully or partially hydrolyzed. Wherein the product is chemically or enzymatically hydrolyzed.

[0029] As used herein, the term“hydrolyzed egg yolk protein” or“hydrolyzed egg yolk” refers to a product of a hydrolysis of yolk white protein. Wherein the product can be fully or partially hydrolyzed. Wherein the product is chemically or enzymatically hydrolyzed.

[0030] Hydrolyzed egg yolk protein and hydrolyzed egg white protein are short-chain peptides produced by enzymatic hydrolysis of egg white or egg yolk, wherein the short-chain peptides have MW no larger than l5kDa.

[0031] In at least one embodiment, the compound is short-chain peptides produced by enzymatic hydrolysis of egg white or egg yolk. This short-chain peptide has MW between O. lkDa and l5kDa, between 0.5kDa and l5kDa, between 0.5kDa and MkDa, between 0.5kDa and l3kDa, between 0.5kDa and l2kDa, between 0.5kDa and l lkDa, between 0.5kDa and lOkDa, between 0.5kDa and 9kDa, between 0.5kDa and 8kDa, between 0.5kDa and 7kDa, between 0.5kDa and 6kDa, between 0.5kDa and 5kDa, between 0.5kDa and 4kDa, between 0.5kDa and 3kDa, between 0.5kDa and 2kDa, between 0.5kDa and lkDa; lkDa and 15kDa, between lkDa and MkDa, between lkDa and MkDa, between lkDa and MkDa, between lkDa and l lkDa, between lkDa and lOkDa, between lkDa and 9kDa, between lkDa and 8kDa, between lkDa and 7kDa, between lkDa and 6kDa, between lkDa and 5kDa, between lkDa and 4kDa, between lkDa and 3kDa, between lkDa and 2kDa; 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and 1 lkDa, between 2kDa and lOkDa, between 2kDa and 9kDa, between 2kDa and 8kDa, between 2kDa and 7kDa, between 21<Da and 6kDa, between 2kDa and 5kDa, between 2kDa and 4kDa, between 2kDa and 3kDa; between 3kDa and l5kDa, between 3kDa and l4kDa, between 3kDa and l3kDa, between 3kDa and l2kDa, between 3kDa and l lkDa, between 3kDa and lOkDa, between 3kDa and 9kDa, between 31<Da and 8kDa, between 3kDa and 7kDa, between 3kDa and 6kDa, between 3kDa and 5kDa, between 3kDa and 4kDa; between 4kDa and l5kDa, between 4kDa and l4kDa, between 4kDa and l3kDa, between 4kDa and 12kDa, between 4kDa and 1 lkDa, between 4kDa and lOkDa, between 4kDa and 9kDa, between 4kDa and 8kDa, between 4kDa and 7kDa, between 4kDa and 6kDa, between 4kDa and 5kDa; between 5kDa and l5kDa, between 5kDa and MkDa, between 5kDa and l3kDa, between 5kDa and l2kDa, between 5kDa and 1 lkDa, between 5kDa and lOkDa, between 5kDa and 9kDa, between 5kDa and 8kDa, between 5kDa and 7kDa, between 5kDa and 6kDa; between 6kDa and l5kDa, between 6kDa and MkDa, between 6kDa and MkDa, between 6kDa and MkDa, between 6kDa and 1 lkDa, between 6kDa and lOkDa, between 6kDa and 9kDa, between 6kDa and 8kDa, between 6kDa and 7kDa; between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and 1 lkDa, between 7kDa and lOkDa, between 7kDa and 9kDa, between 7kDa and 8kDa; between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and 1 lkDa, between 8kDa and lOkDa, and between 8kDa and 9kDa.

[0032] In yet another embodiment, compound is short-chain peptides produced by enzymatic hydrolysis of egg white or egg yolk using pepsin, wherein short-chain peptides have the MW no larger than 15 kDa.

[0033] In at least one embodiment, the compound is short-chain peptides produced by enzymatic hydrolysis of egg white or egg yolk using pepsin. This short-chain peptide has MW between O. lkDa and MkDa, between 0.5kDa and MkDa, between 0.5kDa and MkDa, between 0.5kDa and MkDa, between 0.5kDa and MkDa, between 0.5kDa and l lkDa, between 0.5kDa and lOkDa, between 0.5kDa and 9kDa, between 0.5kDa and 8kDa, between 0.5kDa and 7kDa, between 0.5kDa and 6kDa, between 0.5kDa and 5kDa, between 0.5kDa and 4kDa, between 0.5kDa and 3kDa, between 0.5kDa and 2kDa, between 0.5kDa and lkDa; lkDa and MkDa, between lkDa and MkDa, between lkDa and MkDa, between lkDa and MkDa, between lkDa and l lkDa, between lkDa and lOkDa, between lkDa and 9kDa, between lkDa and 8kDa, between lkDa and 7kDa, between lkDa and 6kDa, between lkDa and 5kDa, between lkDa and 4kDa, between lkDa and 3kDa, between lkDa and 2kDa; 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and MkDa, between 2kDa and 1 lkDa, between 2kDa and lOkDa, between 2kDa and 9kDa, between 2kDa and 8kDa, between 2kDa and 7kDa, between 21<Da and 6kDa, between 2kDa and 5kDa, between 2kDa and 4kDa, between 2kDa and 3kDa; between 3kDa and l5kDa, between 3kDa and l4kDa, between 3kDa and l3kDa, between 3kDa and l2kDa, between 3kDa and l lkDa, between 3kDa and lOkDa, between 3kDa and 9kDa, between 31<Da and 8kDa, between 3kDa and 7kDa, between 3kDa and 6kDa, between 3kDa and 5kDa, between 3kDa and 4kDa; between 4kDa and l5kDa, between 4kDa and l4kDa, between 4kDa and l3kDa, between 4kDa and 12kDa, between 4kDa and 1 lkDa, between 4kDa and lOkDa, between 4kDa and 9kDa, between 4kDa and 8kDa, between 4kDa and 7kDa, between 4kDa and 6kDa, between 4kDa and 5kDa; between 5kDa and l5kDa, between 5kDa and MkDa, between 5kDa and l3kDa, between 5kDa and l2kDa, between 5kDa and 1 lkDa, between 5kDa and lOkDa, between 5kDa and 9kDa, between 5kDa and 8kDa, between 5kDa and 7kDa, between 5kDa and 6kDa; between 6kDa and l5kDa, between 6kDa and MkDa, between 6kDa and MkDa, between 6kDa and MkDa, between 6kDa and 1 lkDa, between 6kDa and lOkDa, between 6kDa and 9kDa, between 6kDa and 8kDa, between 6kDa and 7kDa; between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and MkDa, between 7kDa and 1 lkDa, between 7kDa and lOkDa, between 7kDa and 9kDa, between 7kDa and 8kDa; between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and MkDa, between 8kDa and 1 lkDa, between 8kDa and lOkDa, and between 8kDa and 9kDa.

[0034] The polyethylene glycol (PEG) used herein may be commercially available, or prepared by methods known to one skilled in the art. Typical polyethylene glycol used has a molecular weight of less than 10,000 g/mol, less than 5,000, less than 1,000, less than 500, or ranging from 100 to 400 g/mol. Suitable polyethylene glycol has a formula of: , wherein ni is an integer from 2 to 10, for instance, from 3 to 5. Exemplary polyethylene glycols are PEG200 (molecular weight of 200 g/mol) and PEG400 (molecular weight of 400 g/mol).

[0035] Sorbitan esters that can be used according to the present invention include, but are not limited to, polyoxyethylenesorbitan monolaurate (Tween 20) polyoxyethylenesorbitan monopalmitate (Tween 40), polyoxyethylenesorbitan monostearate (Tween 60),

polyoxyethylenesorbitan tristearate (Tween 65), polyoxyethylenesorbitan monooleate (Tween 80), and polyoxyethylenesorbitan trioleate (Tween 85). [0036] As used herein, the term“hydrolyzed carboxymethyl cellulose” refers to a product of a hydrolysis of carboxymethyl cellulose or a salt thereof. Wherein the product can be fully or partially hydrolyzed.

[0037] In some embodiments, hydrolyzed carboxymethyl cellulose is prepared by enzymatic hydrolysis of carboxymethyl cellulose. In some embodiments, hydrolyzed carboxymethyl cellulose is prepared by acidic hydrolysis of carboxymethyl cellulose.

[0038] In a further embodiment, the compound is selected from the group consisting of hydrolyzed carboxymethyl cellulose (HCMC), hydrolyzed egg white (HEW), hydrolyzed egg yolk (HEY), arginine, proline, polyethylene glycol, sorbitan esters, and mixtures thereof.

[0039] In yet another embodiment, the polyethylene glycol is polyethylene glycol 200

(PEG 200).

[0040] In another embodiment, the compound is a peptide with strong antioxidant activity and angiotensin I-converting enzyme (ACE) inhibitory activity or a mixture thereof.

[0041] The angiotensin converting enzyme (ACE) catalyzes the conversion of inactive angiotensin I into angiotensin II, which is a strong vasoconstrictor, so one of the current therapies used in the treatment of hypertension consists of the administration of drugs inhibiting this enzyme. Several natural inhibitors of ACE have been recently described: peptides from wine (Takayanagi et al.,“Angiotensin I Converting Enzyme-Inhibitory Peptides From Wine,” Am. J. Enol. Vitic. 50:65-68 (1999), which is hereby incorporated by reference in its entirety), soy (Wu et al.,“Hypotensive and Physiological Effect of Angiotensin Converting Enzyme Inhibitory Peptides Derived From Soy Protein on Spontaneously Hypertensive Rats,” J. Agric Food Chem. 49:501-506 (2001), which is hereby incorporated by reference in its entirety), chickpea (Pedroche et al.,“Utilisation of Chickpea Protein Isolates for Production of Peptides With Angiotensin I-Converting Enzyme (ACE)-Inhibitory Activity,” Journal of the Science of Food and Agriculture , 82:960-965 (2002), which is hereby incorporated by reference in its entirety), fish (Fujita et al.,“LKPNM: A Product-Type ACE-Inhibitory Peptide Derived From Fish Protein,” Immunopharmacology 82:960-965 (2002), which is hereby incorporated by reference in its entirety), and whey (Pihlanto-Leppalla et al.,“Angiotensine I-Conv erring Enzyme Inhibitory Properties of Whey Protein Digests: Concentration and Characterization of Active Peptides,” J. Dairy Research , 67:53-64 (2000), which is hereby incorporated by reference in its entirety).

[0042] Suitable peptides that can be used according to the present invention include peptides produced during hydrolysis of plant, animal, fungal, and microbial proteins. Plant proteins that can be used according to the present invention include, but are not limited to, canola proteins, pea proteins, cocoa proteins, soy proteins, peptides from wine, and flaxseed proteins. Animal proteins that can be used according to the present invention include, but are not limited to, chicken proteins, beef proteins, pork proteins, fish proteins, milk proteins, and egg proteins.

[0043] In at least one embodiment, the compound is a peptide(s) produced during hydrolysis egg proteins, for example peptides produced during hydrolysis of egg proteins using pepsin.

[0044] The effective amount of the compound (or combination of the compounds) can be between 1 and 20% w/w, between 1 and 15% w/w, between 1 and 10% w/w, or between 1 and 5% w/w.

[0045] In one embodiment, the effective amount of the compound is 1%, 2%, 3%, 4%,

5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0046] In one embodiment, egg yolk further comprises egg white.

[0047] In another embodiment, the method prevents gelation of egg yolk.

[0048] In another embodiment, the method prevents freeze induced egg yolk gelation.

[0049] In another embodiment, the compound is further combined with sugar or salt.

[0050] In yet another embodiment, the egg yolk is further mechanically treated.

[0051] In a further embodiment, the mechanical treatment is colloid milling or high speed mixing.

[0052] Colloid milling is used to reduce the particle size of a solid in suspension in a liquid, or to reduce the droplet size of a liquid suspended in another liquid. This is done by applying high levels of hydraulic shear to the process liquid. It is frequently used to increase the stability of suspensions and emulsions. Suitable colloidal mills useful in carrying out the method of the present invention include Charlotte Colloid Mill (Chemicolloid Lab’s Inc., Garden City Park, NY).

[0053] High speed mixing (e.g., homogenization using rotor-stator) is a mild physical treatment. Suitable homogenizers useful in carrying out the method of the present invention include Ultra-Turrax T rotor-stator homogenizer (Laboratory Supply Network, Inc., Atkinson, NH).

[0054] Gelation is thickening of egg yolk. Gelation of egg yolk can be evaluated by evaluating the changes in hardness of feeze-thawed yolk and particle distribution of the processed yolk.

[0055] According to the present invention, gelation of the egg yolk is inhibited when the hardness of the egg yolk treated with the compound according to the present invention is less than 100 g, less than 90 g, less than 80 g, less than 70 g, less than 60 g, less than 50 g, less than 40 g, less than 30 g, less than 20 g, less than 15 g, less than 10 g, less than 5 g.

[0056] Alternatively, gelation of the egg yolk is inhibited when the hardness of the egg yolk treated with the compound according to the present invention is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%.

[0057] Fresh untreated yolk particles size ranges from 2.5-50 pm. Freezing causes a shift to larger particles ranging from 2.9-955 pm. According to the present invention, gelation of the egg yolk is inhibited when particles sizes of the egg yolk treated with the compound according to the present invention shifts the particle size distribution to ranges from 0.1-100 pm, from 0.1-90 pm, from 0.1-80 pm, from 0.1-70 pm, from 0.1-60 pm, from 0.1-50 pm, from 0.1-40 pm, from 0.1-30 pm, from 0.1-20 pm, from 0.1-10 pm.

[0058] Another embodiment relates to an egg yolk-containing product of the present invention.

[0059] Another aspect of the invention relates to an egg yolk-containing product having inhibited susceptibility to gelation resulting from freezing further comprising the compound selected from the group consisting of amino acids, hydrolyzed proteins, hydrolyzed

carboxymethyl cellulose, polyethylene glycol, and sorbitan esters.

[0060] In another embodiment, the egg-yolk containing product is used in a food or beverage product.

[0061] Such a food product can include one or more food additives or foodstuffs; one or more flavorants, or flavor enhancers; one or more bitter compounds; one or more sweeteners; one or more bitterants; one or more sour flavorants; one or more salty flavorants; one or more umami flavorants; one or more plant or animal products; one or more fats, oils, or emulsions; and/or one or more probiotic bacteria or supplements.

[0062] In some embodiments composition comprising egg yolk may be used include in a food product. Such products may include a boiled egg itself, various food products using a boiled egg (for example, tartar sauce, prepared bread filling, and so on), various food products containing an egg as a component (for example, mayonnaise, dressing, pasta sauce, Japanese omelette, steamed egg custard, steamed egg hotchpotch, omelette, quiche, rolled omelette, noodles, fried rice, custard cream, pudding, cake, sponge cake, ice cream, egg (custard) tart, bread, crape, and so on). [0063] In some embodiments composition comprising egg yolk may be used include in a beverage product. Such products may include buttermilk, eggnog, coffee drinks, smoothies, and cocktails.

[0064] In some embodiments, the composition comprising egg yolk according to the present invention can include one or more flavorants. Representative flavorants include, but are not limited to, ethyl vanillin, amyl acetate, benzaldehyde, ethyl butyrate, methyl anthranilate, methyl salicylate, or fumaric acid.

[0065] In some embodiments, the composition comprising egg yolk includes one or more sweeteners, sweet flavorants, or sweet taste enhancers. Representative sweeteners, sweet flavorants, or sweet taste enhancers include but are not limited to: natural or synthetic carbohydrates or carbohydrate analogues, monosaccharides, di saccharides, oligosaccharides, and polysaccharides, rare sugars, or enriched fractions of the natural sweeteners.

[0066] In some embodiments, the composition comprising egg yolk may be combined with one or more artificial sweeteners. Such artificial sweeteners may include, but are not limited to: a sulfonyl amide sweetener, e.g., selected from saccharin, sodium cyclamate and acesulfame potassium.

[0067] In some embodiments, the composition comprising egg yolk may be combined with one or more bitterants, bitter flavor compounds, or bitterness-enhancing compounds. In some embodiments, the composition comprising egg yolk may be combined with one or more bitter compounds. Representative bitterants, bitter flavor compounds, bitterness-enhancing compounds, and bitter compounds, include but are not limited to: caffeine, denatonium benzoate, saccharin.

[0068] In some embodiments, the composition comprising egg yolk may be combined with one or more acids or sour flavorants. Representative sour flavorants include but are not limited to: ascorbic acid, benzoic acid, gallic acid, glucuronic acid, adipic acid, glutaric acid, malonic acid, succinic acid, malic acid, acetic acid, lactic acid, citric acid, tartaric acid, fumaric acid, phosphoric acid, pyrophosphoric acid, tannic acid, vinegar, lemon juice, lime juice, acidic fruit juices, and acidic fruit extracts.

[0069] In some embodiments, the composition comprising egg yolk may be combined with one or more salts or salt flavor enhancers. Representative salts or salt flavor enhancers include, but are not limited to: mineral salts, sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, sodium gluconate, sodium phosphates, glycine, L-alanine, L- valine, L-leucine, L-isoleucine, L-phenylalanine, L-tyrosine, L-glutamine, L-glutamic acid, L- asparagine, L-aspartic acid, L-serine, L-threonine, L-cysteine, L-methionine, L-proline, L-lysine, L-arginine, L-tryptophan, L-histidine, L-pyrolysine, L-pyroglutamine, L-4-trans-hydroxyproline, L-3-cis-hydroxyproline, L-homoserine, L-homocysteine, L-cystine, L-ornithine and L-citrulline, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, L-valine, L-arginine, and L-lysine.

[0070] In some embodiments, the composition comprising egg yolk may be combined with one or more umami flavor compounds or umami flavor enhancing compounds.

Representative umami flavor compounds or umami flavor enhancing compounds include but are not limited to: hydrolyzed soy protein, hydrolyzed corn protein, hydrolyzed wheat protein, anchovy, fish sauce, mushrooms, oyster sauce, soy sauce, soy extract, tamari, miso powder, miso paste, kombu, nori, seaweed, tomato, vegetable powder, vegetable extract, whey, and others.

[0071] In some embodiments, the composition comprising egg yolk may be combined with one or more plant or animal products. In some preferred embodiments, the composition comprising egg yolk may be combined with one or more plant or animal products where the plant or animal product is a culinary herb or spice. Representative culinary herbs and spices include but are not limited to: carrot, dehydrated carrot, onion, onion powder, onion flakes, onion extract, garlic, dehydrated garlic, garlic flakes, garlic powder, garlic extract, buttermilk, buttermilk powder, buttermilk solids, whey, whey powder, whey solids, milk, reduced fat milk, milk powder, or milk solids.

[0072] In some preferred embodiments, the composition comprising egg yolk may be combined with one or more fats, oils, or emulsions. Representative fats, oils, and emulsions include but are not limited to: com oil, peanut oil, soybean oil, palm oil, coconut oil, canola oil, rapeseed oil, olive oil, safflower oil, sunflower oil, sesame oil, almond oil, beech nut oil, brazil nut oil, cashew oil, flaxseed oil, hazelnut oil, mongongo nut oil, pecan oil, pine nut oil, pistachio oil, walnut oil, grapeseed oil, chicken fat, beef fat, lamb fat, animal fat, tallowate, tallow, beef tallow, bacon fat, ham fat, suet, milk fat, olestra, stearic acid, lauric acid, linoleic acid, palmitic acid, palmitoleic acid, myristic acid, goose fat, duck fat, and oil-in water or water-in oil emulsions.

[0073] In some preferred embodiments, the composition comprising egg yolk may be combined with one or more probiotic bacteria or supplements. Representative probiotic bacteria or supplements include but are not limited to: B. lactis, L. acidophilus, B. animalis, B. breve, or B. longum.

[0074] In some preferred embodiments, the composition comprising egg yolk may be combined with one or more starches, gums, starch-like plant extracts and materials and combinations thereof. Starches that can be used in accordance with the present invention include, but are not limited to cereal starch, tuber starch, any other plant starch (such as sago starch), or any combination of any of these in any proportion. Suitable cereal starches include corn starch such as instant com starch, wheat starch, rice starch, oat starch, waxy maize starch such as cook-up waxy maize starch and instant waxy maize starch, sorghum starch, waxy sorghum starch, seed starch and any combination of any of these in any proportion. Suitable tuber starches including potato starch, arrowroot starch, tapioca starch, and any combination of these in any proportion. Suitable gums include arabic gum, tragacanth gum, karaya gum, ghatti, guar gum such as instant, pre-hydrated guar gum, locust bean gum, xanthan gum, tamarine gum, agar-agar gum, furcellaran gum, gum acacia, and any combination of any of these in any proportion. Plant extracts that can be used in accordance with the present invention include, but are not limited to pectin, arabinogalacton, psyllium, quince seed, alginates, carrageenans, and any combination of these in any proportion.

EXAMPLES

[0075] The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1 - Materials

[0076] Fresh Grade A white shell eggs were obtained from grocery stores in Ames, IA.

Eggs were stored in 4°C refrigerator at the research laboratory. Hydrolyzed carboxymethyl cellulose (HCMC), hydrolyzed egg white protein (HEWP), and hydrolyzed egg yolk protein (HEYP) were prepared with the methods described in the later sections. Arginine, proline, Tween 80, polyethylene glycol 200 (PEG 200), and other chemicals were purchased from Fisher Scientific (Hampton, NH).

Example 2 - Preparation of Hydrolyzed Carboxymethyl Cellulose (HCMC)

[0077] HCMC was prepared following the optimal conditions found by Sreenath,

“Hydrolysis of Carboxymethyl Celluloses by Cellulases,” LWT - Food Science and Technology 26(3):224-228 (1993), which is hereby incorporated by reference in its entirety. A 4% (w/w) solution of CMC (average molecular weight ~90 kDa) in deionized water was mixed overnight. The solution was heated in an incubator to 50°C before the pH was adjusted to 4.8 with 2 M hydrochloric acid solution. Cellulase DS enzyme was added at a concentration of 1% based on the CMC substrate (dry weight) and the solution was mixed for 18 hours in a shaking incubator set at 45 RPM. After the reaction, the solution was boiled for 30 minutes to deactivate the enzyme. The concentration of reducing ends of HCMC was measured using Somogyi-Nelson method (Nelson,“A Photometric Adaptation of the Somogyi Method for the Determination of Glucose,” Journal of Biological Chemistry l53(2):375-380 (1944), which is hereby incorporated by reference in its entirety). A quantification standard curve was established using serial dilutions of 1 mg/mL solution of glucose The standard solutions and samples were measured at 520 nm, and the absorbance of the CMC and HCMC samples was interpolated into the standard curve to determine the concentration of free reducing ends in both samples. The average molecular weight of the HCMC was estimated to be 2 9 kDa based on the inverse relationship between the obtained amount of reducing ends and molecular weights before and after hydrolysis.

Example 3 - Preparation of Hydrolyzed Egg White Protein (HEWP) and Hydrolyzed Egg

Yolk Protein (HEYP)

[0078] Fresh egg white and yolk were hydrolyzed using the method by Ruan et al.,

“Kinetics of Hydrolysis of Egg White Protein by Pepsin,” Czech J. Food Sci. 28(5):355-363 (2010), which is hereby incorporated by reference in its entirety. Egg yolk was defatted prior to hydrolysis by extracting lipids using the method of Folch et al.,“A Simple Method for the Isolation and Purification of Total Lipids From Animal Tissues,” J. Biol. Chem. 226(l):497-509 (1957), which is hereby incorporated by reference in its entirety. Fresh egg yolk was mixed in 2 parts of 2: 1 (v/v) chloroform -methanol solution in a shaking incubator for 30 minutes at the ambient temperature. The mixture was vacuum-filtered using No.2 Whatman paper, and the filter cake was air dried for 12 hours to remove solvent.

[0079] The egg protein was dispersed in deionized water at 10 g protein (dry weight)/L water and denatured in 90°C water bath for 15 minutes. The pH of the denatured dispersion was adjusted to 2 using 2 M hydrochloric acid solution. The hydrolysis reaction was performed for 3 hours after adding pepsin at a selected concentration, and the temperature and pH were maintained at 45°C and 2, respectively. Inactivation of pepsin was achieved by increasing the solution pH to 7 with 2 M sodium hydroxide solution. The hydrolysates were centrifuged at 4,000g for 15 minutes and the supernatant was collected and lyophilized.

Example 4 - Preparation of Frozen-Thawed Yolk Samples

[0080] Yolks were separated following the method by Powrie et al.,“Gelation of Egg

Yolk,” Journal of Food Science 28(l):38-46 (1963), which is hereby incorporated by reference in its entirety, with modifications. Fresh hen eggs were manually broken, and the yolks were carefully separated from the albumen, with the chalazae removed. Each yolk with intact vitelline membrane was rolled on a paper towel to remove any remaining albumen and chalazae adhering to the vitelline membrane. The vitelline membrane was pierced to collect the pure egg yolk in a beaker. The yolk was slowly stirred for homogeneity.

[0081] Additives at various concentrations (1-10% w/w) were added to yolk to make 50 g yolk mixtures which were stirred with a spatula before further thoroughly mixed using Ultra- Turrax ^® T rotor-stator homogenizer (Laboratory Supply Network, Inc., Atkinson, NH) at 8,000 RPM for 90 seconds. Three replicates of 10 g yolk mixtures were distributed to three Evergreen Scientific Dilution Vials (Fisher Scientific, Hampton, NH), and were stored in a -20°C freezer for 45 hours. The freezing rate was calculated to be 0.15°C/min monitored using a thermal sensor. The yolk mixtures were thawed for 4 hours at 25°C before analyzed for hardness.

[0082] To test the effect of mechanical treatments, fresh yolk was processed with a rotor- stator homogenizer and a colloid mill prior to freezing. For the rotor-stator homogenizer, fresh yolk was processed at 8,000, 13,500, and 24,000 RPM for 90 seconds. The shear rates were calculated to be 13,299, 22,443, and 39,898 s ¹ , respectively. For colloid milling, two liters of fresh yolk was run through the Charlotte Colloid Mill (Chemicolloid Lab’s Inc., Garden City Park, NY) at 0.003-inch clearance (shear rate of 18,618 s ¹ ) for three passes based on the conditions found to be optimal in lowering yolk viscosity after yolk freezing treatment (Lopez et al.,“Some Factors Affecting Gelation of Frozen Egg Yolk,” Journal of Milk and Food

Technology 17: 334-339 (1954), which is hereby incorporated by reference in its entirety). The processed fresh yolk was then used to prepare frozen-thawed yolk samples, and additives were also used to determine the effect of combining additive with mechanical treatments.

Example 5 - Texture Analysis of Frozen-Thawed Yolk Samples

[0083] Hardness tests were carried out using a TA.XTPlus Texture Analyzer (Stable

Micro Systems, United Kingdom) with a load cell of 50 kg. A penetration distance of 10 mm and a speed of 1 mm/sec were performed using a cylindrical probe (TA-10) to characterize the frozen-thawed yolk gels of 10 g kept in 20 mL vials. A trigger force of 5 g was applied, and the maximum positive force recorded corresponds to hardness. The mean of three replicates was reported.

Example 6 - Quantification of Freezable Water in Selected Yolk Samples

[0084] Content of freezable water was determined following the differential scanning calorimetry (DSC) method reported by Au et al.,“Determination of the Gelation Mechanism of Freeze-Thawed Hen Egg Yolk,” Journal of Agricultural and Food Chemistry 63(46): 10170- 10180 (2015), which is hereby incorporated by reference in its entirety. DSC was performed on fresh yolk and yolk mixtures containing various additives. Exothermic and endothermic transition heats of 10-15 mg sample in aluminum hermetic pans with sealed lids were measured in four replicates. Scanning conditions were modified from Wakamatu et al.,“On Sodium Chloride Action in the Gelation Process of Low Density Lipoprotein (LDL) from Hen Egg Yolk,” Journal of Food Science 48(2):507-5 l2 (1983), which is hereby incorporated by reference in its entirety. Each sample was held for 1 min at 20°C, cooled to -50°C at l°C/min rate and held at that temperature for 1 min, then heated from -50°C to 20°C at l0°C/min rate.

[0085] Melting temperature (T _m ) and the heat of fusion, or change in enthalpy (DH), of exothermic and endothermic peaks were obtained. The amount of freezable water in yolk was calculated following the method by Wakamatu et al.,“On Sodium Chloride Action in the Gelation Process Of Low Density Lipoprotein (LDL) from Hen Egg Yolk,” Journal of Food Science 48(2):507-512 (1983), which is hereby incorporated by reference in its entirety. The exothermic or endothermic heat was divided by the corresponding heat of fusion of pure water (242.88 J/g for cooling and 320.62 J/g for heating). Freezable water content was reported as the average of the exothermic and endothermic freezable water values per gram of solid.

Example 7 - Particle Size Analysis

[0086] Particle size distributions of fresh and frozen-thawed yolk were measured by laser diffraction (LD) method using Malvern Mastersizer 2000 particle size analyzer with Hydro 2000 MU large volume wet sample dispersion unit (Malvern Instruments, Inc., Worchestershore, UK) following the method by Au et al.,“Determination of the Gelation Mechanism of Freeze- Thawed Hen Egg Yolk,” Journal of Agricultural and Food Chemistry 63(46): l0170-10180 (2015), which is hereby incorporated by reference in its entirety. All samples were diluted at a sample: deionized water ratio of 1 : 1.5 (v/v) and mixed for 1.5 hours on a stir plate until being homogeneous. Diluted samples were added dropwise to a 1 L beaker of deionized water within the wet sample dispersion unit. Measurements were made in triplicate when obscurations of 10-15% were reached. Two refractive indices (RI) were used: 1.33 (water/background), and 1.42 (yolk/sample) (Kralik et al.,“Effects of Dietary Selenium Source and Storage on Internal Quality of Eggs,” Acta Veter inaria Brno 78(2):219-222 (2009), which is hereby incorporated by reference in its entirety). Example 8 - Protein Surface Hydrophobicity

[0087] Protein surface hydrophobicity (So) of the fresh and frozen-thawed yolk mixtures was determined using l-anilino-8-naphthalene sulfonate (ANS) as a hydrophobic probe (Wu et al.,“Hydrophobicity, Solubility, and Emulsifying Properties of Soy Protein Peptides Prepared by Papain Modification and Ultrafiltration,” Journal of the American Oil Chemists' Society 75(7):845-850 (1998), which is hereby incorporated by reference in its entirety). The protein was serially diluted with deionized water to obtain protein concentrations ranging from 0 000675 to 0.01925%. Twenty microliters of ANS (8.0 mM in 0.1 M phosphate buffer, pH 7.0) were added to 4 mL of the diluted lipoprotein dispersion. The fluorescence intensity (FI) of the protein was measured in duplicates using Synergy™ H4 Microplate Reader (BioTek, Winooski, VT). Excitation and emission wavelengths were 390 and 470 nm, respectively. The FI reading was standardized by adjusting the spectrofluorometer reading for 10 pL of ANS in 5 mL methanol to 80% of full scale. The slope of the plots of FI vs. percentage of protein

concentration was calculated by least square linear regression and used as the surface hydrophobicity.

Example 9 - Statistical Analysis

[0088] Statistical analysis was performed with JMP Pro 13, statistical software from

Statistical Analysis System (SAS) Institute Inc. (Cary, NC). One-way analysis of variance (ANOVA) tests were conducted, and significance of difference (p<0.05) was calculated using Tukey’s HSD (honest significant difference) test.

Example 10 - Effect of Mechanical Treatments on Yolk Gelation

[0089] The effect of mechanical treatment such as rotor-stator high-speed mixing and colloid milling was tested by evaluating changes in hardness of frozen-thawed yolk and particle size distribution of processed yolk. Figures 1A and 1C show that applying the rotor-stator homogenizer to yolk at different speed for 90 seconds did not cause observable changes in the frozen-thawed yolk hardness and particle size distribution. Sirvente at al.,“Structuring and Functionalization of Dispersions Containing Egg Yolk, Plasma and Granules Induced by Mechanical Treatments,” Journal of Agricultural and Food Chemistry 55(23):9537-9544 (2007), which is hereby incorporated by reference in its entirety, also found no change in yolk protein structure following rotor-stator treatment at 20,000 RPM for 90 seconds. Therefore, rotor-stator treatment at 8,000 RPM for 90 seconds was selected to achieve optimal mixing during the preparation of yolk mixtures with additives. [0090] According to Lopez et al.,“Some Factors Affecting Gelation of Frozen Egg

Yolk,” Journal of Milk and Food Technology 17: 334-339 (1954), which is hereby incorporated by reference in its entirety, colloid milling of yolk at 0.003 inch for 3 passes is able to significantly decrease the viscosity of unfrozen egg yolk which also resulted in a lower viscosity gel when the yolk is frozen and thawed. These results showed that colloid milling caused a reduction of hardness from 118.5 to 103.4 g (Figure IB) and a shift in particle size distribution towards an abundance of smaller particles (Figure ID). Protein aggregations occur during freezing, which was reflected through the shift of distribution towards larger particle size. The fresh untreated yolk particles size ranged from 2.5-50 um, and colloid-milled yolk ranged from 0.1-40 um. Freezing caused a shift to larger particles ranging from 2.9-955 um for both untreated and colloid-milled yolk, but the abundance of larger particles in the colloid-milled yolk was less compared to the untreated yolk. This shows that colloid-milling can help to reduce the degree of gelation, although not to the extent of the effectiveness of conventional gelation inhibitors.

Example 11 - Effect of Different Additives and Their Combinations on Yolk Gelation

Effect of Different Additives on Hardness

[0091] At 5% concentration, F1CMC, FLEW, ITEY, proline, sugar, and salt were effective in reducing gelation after short term 45 hour freezing treatment (Figure 2A). Arginine, Tween 80, and PEG were also tested because they have shown the ability to prevent protein aggregation elsewhere (Arakawa et al.,“Suppression of Protein Interactions by Arginine: A Proposed Mechanism of the Arginine Effects,” Biophysical Chemistry l27(l-2):l— 8 (2007); Hillgren et al., “Interaction Between Lactate Dehydrogenase and Tween 80 in Aqueous Solution,” Pharm. Res. 19(4):504-510 (2002), which are hereby incorporated by reference in their entirety). Flowever, it is important to note that egg yolk is a heterogeneous mixture of lipoproteins, and not just pure protein dispersion. These additives are not as effective in preventing lipoprotein aggregations in egg yolk.

[0092] Additives capable of better preventing gelation were further studied to determine the lowest concentration for their optimal gelation-inhibiting effect. Figure 2B shows a negative correlation between the amount of additive and yolk hardness, and 5% addition is the minimum concentration needed to produce good gelation inhibitory effect that is comparable to the industrially practiced 10% salt.

[0093] Tests were conducted to evaluate the effect of different degrees of hydrolysis of

HCMC and hydrolyzed peptides. These results show that different MW of ITCMC did not have a significant impact on gelation reduction (Figures 3A-3B). Increasing the concentration of HCMC resulted in lower hardness, but this trend was not observed when HCMC concentration exceeded 7.5%. The experiments for Figures 3A-3B were conducted separately and a different freezing storage time was used. Yolk with no additive frozen for 5 days (Figure 3A) formed a harder gel than the yolk frozen for 1 day (Figure 3B). This shows that yolk remains dynamic at - 20°C and gelation occurs not only during freezing and thawing, but also during extended freezing storage

[0094] Hydrolyzed egg white and yolk proteins produced under different hydrolysis conditions did not show marked difference in gelation-inhibiting ability. Addition of these peptides at 5% concentration achieved comparable gelation inhibition as 10% salt. Hardness was reduced from 93.02 g to 10.66, 10.11, and 16.53 g by 5% HEW, 5% HEY, and 10% salt, respectively.

[0095] Proline also proved to be a very effective gelation inhibitor. The hardness of proline-treated yolk was reduced by 87% compared to the yolk without additive. When added at 10% concentration, it inhibited gelation almost completely and the yolk maintained its fresh texture. However, proline is relatively expensive compared to salt and sugar, and the amount of proline as an additive is not allowed to exceed 4.2% of the total protein content in the food (Food Additives Permitted for Direct Addition to Food for Human Consumption, § 172.320 (2017), which is hereby incorporated by reference in its entirety).

[0096] Sugar works similarly to proline. At 5% addition, gelation was reduced; but at

10% addition, gelation was completely inhibited and the yolk fluidity was preserved. Because of this, the hardness of 10% sugared yolk was not measurable, and 10% salted yolk was used for target comparison among the other additives.

[0097] No significant difference was observed in yolk gelation treated with 5% and 10% salt. Unlike other treatments, the yolk viscosity was markedly increased upon the addition of salt, the yolk mixture was darker, more sticky and transparent. Based on this observation, it is apparent that each additive has a different mechanism in reducing the degree of gelation.

Combination Treatment and Identification of Synergistic Effect

[0098] Based on visual observations, HEW, HEY, proline, and sugar seem to follow similar mechanisms in inhibiting gelation. Although these additives were found effective in lowering the degree of gelation, HEW, HEY, and proline are relatively more expensive than salt or sugar. Preliminary test showed that unlike the other additives, HCMC at high concentration had negative effect on hardness, and it was hypothesized that HCMC can prevent aggregation by forming electrostatic interactions with protein. Therefore, it was in our interest to determine if these additives could work synergistically with each other to inhibit gelation at lower

concentrations, based on the unique gelation-inhibiting mechanism that each of these additives has.

[0099] Figures 4A-G show the hardness of frozen-thawed yolk treated with different combinations of additives. An apparent linear increase in hardness was observed as proline concentration is reduced and HCMC concentration is increased, indicating that proline and FICMC do not prevent gelation synergistically (Figure 4A). Similar trends were observed in treatments involving HCMC-HEY and HCMC-HEW (Figures 4B-4C).

[0100] Interestingly, a synergistic effect was observed between HCMC and sugar. The hardness of yolk containing 2.5% HCMC 2.5% sugar is significantly lower than those containing only 5% HCMC or 5% sugar (Figure 4D). This finding shows that HCMC has the potential to reduce the amount of sugar currently used to prevent gelation in commercial frozen yolk product.

[0101] Conversely, HCMC-salt showed counter-synergistic effect when used in combination (Figure 4E). The yolk gel was harder when the combined additive contained a higher proportion of HCMC to salt. According to Pawlik et al.,“Effect of Ionic Strength on Stabilization of Mineral Suspensions by Carboxymethyl Cellulose and Guar Gum,” 2004 Society for Mining Metallurgy and Exploration (SME) Annual Meeting & Exhibit, Feb. 23-25, Denver, Preprint # 04-059 (2004), which is hereby incorporated by reference in its entirety, CMC molecules coil with increasing ionic strength, making it less soluble in brine solutions.

Therefore, HCMC might have been inactivated and the gelation inhibition was mostly due to salt, as shown through the decreasing hardness with increasing salt content.

[0102] Salt at 5% was more effective in reducing gelation than 5% hydrolyzed proteins, but their combinations had similar effectiveness as the 5% salt (Figure 4F). Similarly, no synergistic effect was formed between sugar and hydrolyzed proteins (Figure 4G). The different trends observed from the effects of adding different additives confirmed that these additives have different gelation-inhibiting mechanisms, which are discussed in more depth in the next section.

Combination of Additives with Colloid Milling

[0103] Figures 5A-5C show how colloid milling affected gelation and particle size distribution. The milling was able to significantly reduce gelation, and the addition of 2.5% proline and 5% HCMC to the milled sample was effective to inhibit gelation comparable to the performance of 10% salt (Figures 5A-5C). Colloid milling was reported to decrease the degree of gelation of frozen yolk; the smaller the clearance of the mill, the less the gelation. The flavor, color, and texture of the colloid milled frozen-thawed yolk was very similar to those of fresh yolk (Lopez et al.,“Some Factors Affecting Gelation of Frozen Egg Yolk,” Journal of Milk and Food Technology 17: 334-339 (1954), which is hereby incorporated by reference in its entirety). The same authors reported that salted colloid milled yolk had a higher degree of gelation compared to the non-milled sample containing the same amount of sodium chloride. This, however, is not supported by the present results, where the salted colloid-milled yolk has a significantly lower hardness than the non-milled salted yolk. The addition of 10% salt caused a shift in distribution towards the smaller particle size. Freezing of the same sample resulted in a distribution profile similar to the non-frozen control

Example 12 - Mechanism of Gelation Inhibition by Selected Additives

[0104] Gelation in frozen-thawed egg yolk is known to be caused by aggregations of surface proteins of LDL particles (Au et al.,“Determination of the Gelation Mechanism of Freeze-Thawed Hen Egg Yolk,” Journal of Agricultural and Food Chemistry 63(46): 10170- 10180 (2015); Chang et al.,“Studies on the Gelation of Egg Yolk and Plasma Upon Freezing and Thawing,” Journal of Food Science 42(6): l658-1665 (1977); Primacella et al.,“Use of

Reconstituted Yolk Systems to Study the Gelation Mechanism of Frozen-Thawed Hen Egg Yolk,” Journal of Agricultural and Food Chemistry (2017), which are hereby incorporated by reference in their entirety). Many factors can lead to protein aggregation, including

concentration of yolk components and dehydration of LDL micelles due to formation of ice crystals, exposure of previously inaccessible hydrophobic regions due to change in protein structure when pH or ionic strength were altered, etc. While most researchers suggested plasma LDL as the main contributor to gelation, recent studies have shown that gelation involves a heterogeneous mix of components including the major granule component HDL (Au et al., “Determination of the Gelation Mechanism of Freeze-Thawed Hen Egg Yolk,” Journal of Agricultural andFood Chemistry 63(46): 10170 10180 (2015); Primacella et al.,“Use of Reconstituted Yolk Systems to Study the Gelation Mechanism of Frozen-Thawed Hen Egg Yolk,” Journal of Agricultural and Food Chemistry (2017), which are hereby incorporated by reference in their entirety). It is postulated that food additives that can interfere with ice crystal formation and growth or prevent protein-protein interactions will inhibit gelation. Gel strength measurement by itself does not provide sufficient information on how the additive prevents gelation. To better understand how each additive was able to successfully inhibit gelation, the changes in protein structure were monitored through the measurement of particle size distribution, protein surface hydrophobicity, and the amount of freezable water was also quantified. The overall gelation-inhibition mechanisms of HCMC, HEW, HEY, and proline are illustrated in Figure 6. Detailed explanation of each mechanism is discussed in the following sections.

Example 13 - Effect of Salt

[0105] The results here show that the additives tested inhibited gelation differently. The addition of salt caused a remarkable increase in the viscosity of yolk mixture even before freezing, but the frozen-thawed yolk did not gel. Figure 7A shows a significant reduction in the particle size of both fresh and frozen-thawed salted yolk. Increasing the salt concentration from 5% to 10% resulted in the growth of the abundance of the smaller yolk particles. The surface hydrophobicity of the system was also shown to increase significantly, especially with the addition of 10% salt (Figure 7B). According to Wang et al,“Relationship between Secondary Structure and Surface Hydrophobicity of Soybean Protein Isolate Subjected to Heat Treatment,” Journal of Chemistry 20l4:Article ID 475389, 10 pages (2014), which is hereby incorporated by reference in its entirety, an increase in surface hydrophobicity can be caused by protein denaturation, dissociation, or expansion of peptide chains, while formation of aggregates causes a decrease in surface hydrophobicity. This finding shows that salt caused dissociation of proteins, altering the protein conformation and exposing more hydrophobic surface for ANS to bind. This is in agreement with previous findings where high level of salt can cause disruption of lipoproteins (Chang et al,“Microstructure of Egg Yolk,” Journal of Food Science

42(5): 1193-1200 (1977); Kaewmanee et al.,“Changes in Chemical Composition, Physical Properties and Microstructure of Duck Egg as Influenced by Salting,” Journal of Food

Chemistry 112:560-569 (2009)), which are hereby incorporated by reference in their entirety). According to Telis et al.,“Viscoelasticity of Frozen/Thawed Egg Yolk as Affected by Salts, Sucrose and Glycerol,” Journal Of Food Science 63:20 24 (1998), which is hereby incorporated by reference in its entirety, salt dissociates into ions when in solution, and these ions electrostatically shield proteins, increasing repulsion, which explains the increase in yolk viscosity when salt is added.

[0106] The melting and crystallization transition temperatures and amount of freezable water were also significantly reduced with salt addition (Figure 7C, melting transition Tm). Wakamatu et al.,“On Sodium Chloride Action in the Gelation Process Of Low Density Lipoprotein (LDL) from Hen Egg Yolk,” Journal of Food Science 48(2):507-5l2 (1983), which is hereby incorporated by reference in its entirety, suggested that as an inhibitor of gelation in LDL solutions, NaCl increased the unfreezable water through formation of LDL-water-NaCl complex where water did not freeze. Chang et al.,“Studies on the Gelation of Egg Yolk and Plasma Upon Freezing and Thawing,” Journal of Food Science 42(6): 1658-1665 (1977), which is hereby incorporated by reference in its entirety, found that salt only inhibited gelation when the yolk is frozen at a temperature higher than the eutectic temperature of coexisting salt. This suggested that LDL aggregation is caused by the progressive removal of water from LDL due to ice formation, and salt prevents this by forming complexes with water and LDL.

Example 14 - Effect of Sugar

[0107] Sucrose showed a completely different mechanism in inhibiting gelation. Its addition lowered the viscosity of the unfrozen yolk and 10% sucrose yolk was completely fluid. Sugars have been commonly used as stabilizers to protect proteins from degradation during lyophilization and frozen storage. Two main hypotheses have been proposed to explain the stabilization mechanism of sugar: the“water substitution” hypothesis and the“glass dynamics” hypothesis. In the“water substitution” hypothesis, stabilizers form hydrogen bonds at specific sites on protein surface and thus substitute for the stabilization function of water that is lost during freezing induced dehydration. The glass dynamic hypothesis suggests that sugar forms a rigid, inert matrix in which the protein is molecularly dispersed, limiting the mobility necessary for protein aggregation (Wang et al.,“Impact of Sucrose Level on Storage Stability of Proteins in Freeze-Dried Solids: I. Correlation of Protein-Sugar Interaction With Native Structure

Preservation,” Journal of Pharmaceutical Sciences 98(9):3131-3144 (2009), which is hereby incorporated by reference in its entirety). Lee et al.,“The Stabilization of Proteins by Sucrose,” Journal of Biological Chemistry 256(14):7193-7201 (1981), which is hereby incorporated by reference in its entirety, also agreed that sucrose does not affect protein conformation. Its stabilizing effect is by increasing the free energy of the system while being preferentially excluded from the protein domain.

[0108] The results here are in agreement with these hypotheses. There were no significant changes in lipoprotein size and hydrophobicity caused by the addition of sugar (Figures 7A-7B), and the melting transition (Tm) and freezable water were reduced (Figure 7C), but not as significantly as salt did. The changes in lipoprotein particle size and surface hydrophobicity at 10% addition are relatively small in the frozen-thawed sample compared to the unfrozen sample, showing that sugar is a very effective gelation inhibitor.

Example 15 - Effect of HCMC

[0109] CMC, an anionic water soluble polymer derived from cellulose, is widely used as food additive and is known to form charge-charge electrostatic complexes with proteins (Imeson et al.,“Protein Recovery From Blood Plasma by Precipitation With Polyuronates,” International Journal of Food Science & Technology l3(4):329-338 (1978), which is hereby incorporated by reference in its entirety). In egg yolk plasma, an interaction between the hydrocolloid CMC and the LDL particles is most likely caused by the negatively charged carboxyl groups of the CMC and the positively charged side chains of the amino acids of the LDL surface proteins, leading to an agglomeration of the LDL micelles (Ulrichs et al.,“A Commercially Viable Procedure For Separating Pasteurised Egg Yolk Into Three Technologically and Functionally Valuable Fractions,” Arch.Gefliigelk 74(4):279-284 (2010), which is hereby incorporated by reference in its entirety). For this study, CMC is partially hydrolyzed for the production of low viscosity, low molecular weight material that will not significantly agglomerate the LDL, but rather to prevent protein aggregation.

[0110] Figure 7A shows how HCMC affected particle size distribution in fresh and frozen-thawed yolk. The measured yolk particles were relatively smaller compared to fresh yolk, and this supports the statement that HCMC forms electrostatic interaction with proteins, thus increasing the net negative charge and repulsive forces between proteins (Huan et al., “Influence of the Molecular Weight of Carboxymethylcellulose On Properties and Stability of Whey Protein-Stabilized Oil-In-Water Emulsions,” Journal of Dairy Science 99(5):3305-33 l5 (2016), which is hereby incorporated by reference in its entirety). Gelation still caused the distribution to shift towards larger particles, but not to the extent of the untreated frozen-thawed yolk. Protein surface hydrophobicity test also confirmed this mechanism. HCMC -treated yolk had a significantly higher surface hydrophobicity than untreated yolk before freezing (Figure 7B). The negative net charges kept proteins apart in aqueous dispersion that made it easier for the hydrophobic ANS probe to access the hydrophobic region on the yolk proteins. The melting transition and freezable water were also significantly reduced due to the high solubility of HCMC.

Example 16 - Effect of Proline

[0111] Amino acid proline has been reported to suppress protein aggregation during refolding of bovine carbonic anhydrase and egg white lysozyme (Kumat et al.,“The Role of Proline in the Prevention of Aggregation During Protein Folding in vitro,” IUBMB Life , 46(3):509-5l7 (1998); Samuel et al.,“Proline Inhibits Aggregation During Protein Refolding,” Protein Science 9(2):344-352 (2000), which are hereby incorporated by reference in their entirety). Figure 7B shows how surface hydrophobicity was significantly reduced following the addition of proline. It is possible that the previously available hydrophobic region had been bound to proline and no longer accessible to ANS. No significant change in protein size distribution was observed except that the smaller size population present in fresh untreated yolk is no longer present, and the size distribution became more uniform (Figure 7A). There was a slight shift towards larger particle size in the frozen-thawed sample, meaning that some aggregations still occurred. The melting transition temperature and freezable water also decreased (Figure 7C).

[0112] According to Rudolph et al.,“A Calorimetric and Infrared Spectroscopic Study of the Stabilizing Solute Proline,” Biophysical Journal 50(3):423-430 (1986), which is hereby incorporated by reference in its entirety, proline forms hydrophobic stacking in aqueous solution through the formation of hydrogen bonding between the imino group of proline with the negatively charged carboxyl group of the adjacent proline molecule. The carboxyl groups can also form hydrogen bonding with the solvent water molecules (Samuel et al.,“Proline Inhibits Aggregation During Protein Refolding,” Protein Science 9(2):344-352 (2000), which is hereby incorporated by reference in its entirety). This amphiphilic proline assembly suppresses aggregation by shielding the hydrophobic, aggregation-prone region of the proteins (Kumat et al.,“The Role of Proline in the Prevention of Aggregation During Protein Folding in vitro,” IUBMB Life 46(3):509-5 l7 (1998), which is hereby incorporated by reference in its entirety).

Example 17 - Effect of Hydrolyzed Proteins

[0113] Hen egg white and egg yolk proteins were enzymatically hydrolyzed using pepsin for production of short-chain peptides. Enzymatic hydrolysis is known to increase the value of food proteins by modifying their physical and nutritional properties. Other than reducing molecular weight, increasing the number of ionizable groups, and exposing the initially buried hydrophobic groups, enzymatic hydrolysis improves the solubility of proteins and modulates their surface or interfacial properties such as stabilization of emulsions and foams (Foegeding et al.,“Food Protein Functionality: A Comprehensive Approach,” Food Hydrocolloids, 25: 1853- 1864 (2011), which is hereby incorporated by reference in its entirety). Hydrolysis of egg protein using pepsin has been shown to produce peptides with strong antioxidant activity and angiotensin I-converting enzyme (ACE) inhibitory activity (Davalos, et al.,“Antioxidant Activity of Peptides Derived From Egg White Proteins by Enzymatic Hydrolysis,” Journal of Food Protection 67(9): 1939- 1944 (2004), which is hereby incorporated by reference in its entirety).

[0114] Based on these results, it is believed that peptides were able to inhibit gelation not only by preventing the growth of ice crystals, but also by hydrophobically shielding the yolk proteins. The significant reduction in the surface hydrophobicity of yolk treated with both HEW and HEY (Figure 7B) suggests that less hydrophobic region was available on the protein surface, similar to that of proline-treated yolk. The DSC results for both HEW and HEY (Figure 7C) also show similar melting transition temperature and amount of freezable water as proline, which suggest that these additives functioned following the same mechanism as proline. While proline as a food additive has a limit of 4% for safe consumption, HEW and HEY do not have a set limit. In fact, these additives can provide added value due to their high-protein and additional health benefits.

[0115] Previous studies showed that peptide effectiveness in inhibiting crystal formation and growth is dependent on the size range and peptide source. Peptides in the range of molecular weight of about 2-5 kDa from hydrolyzed gelatin were shown to inhibit recrystallization of ice in ice cream mix (Damodaran,“Inhibition of Ice Crystal Growth in Ice Cream Mix by Gelatin Hydrolysate,” Journal of Agricultural and Food Chemistry 55: 10918-10923 (2007), which is hereby incorporated by reference in its entirety). Peptides from collagen source inhibited ice recrystallization at molecular weight range of 0.6-2.7 kDa (Wang et al,“Ice-Structuring Peptides Derived From Bovine Collagen,” Journal of Agricultural and Food Chemistry 57(l2):550l- 5509 (2009), which is hereby incorporated by reference in its entirety). It is accepted that the inhibition mechanism involves binding of these peptides to the ice-liquid interface, which primarily involves hydrogen bonding. As measured by SDS-PAGE, the peptides produced from egg white and yolk hydrolysis were no larger than 15 kDa. Future work will further identify the optimal size of the egg peptides for yolk gelation inhibition.

[0116] Novel yolk gelation inhibitors that can potentially replace salt or sugar were identified and assessed. HEW, HEY, HCMC, and proline have been proven to effectively inhibit gelation of frozen-thawed yolk through different mechanisms. These additives can be used in combination, or with sugar and colloid milling for further reduction in gelation. Particularly, hydrolyzed egg proteins prevented gelation as the typically used 10% salt or sugar, and they were effective at an addition level of 5%. The different mechanisms of gelation inhibition are discussed according to this observation. There is great potential for using such egg derived ingredients to replace salt and sugar to effectively prevent yolk gelation in commercial yolk freezing storage operations.

[0117] Although the invention has been described in detail, for the purpose of illustration, it is understood that such detail is for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.