CA2801040A1 - Protein compositions and methods of making and using thereof - Google Patents

Abstract

Described herein are the isolation and use of meat proteins and their applications thereof. In one aspect, meat proteins such as, for example, fish, poultry, bovine, or porcine can be used to make films, meat binders or extenders, and extrudable food articles.

Description

PROTEIN COMPOSITIONS AND METHODS OF MAKING AND USING
THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority upon U.S. provisional application Serial No.
61/181,807, filed May 28, 2009. This application is hereby incorporated by reference in its entirety for all of its teachings.

BACKGROUND
In recent years, consumption of poultry meat and further processed poultry products has greatly increased worldwide. One of the reasons for the increased consumer preference for poultry products is the greater availability of choice poultry cuts, such as wings, thighs and breast. Consequently, in the U.S., the consumption of chicken and turkey from 1950 to 2007 increased from 12 to 52 kg per capita. In Canada, per capita consumption of chicken was 21.5 kg in 1989 and reached 31.8 kg in 2008. As a result, considerable quantity of poultry carcass parts, such as necks, backs, and drumsticks, have become available. This is referred to as "low value"
poultry meat. Utilization of these less desirable parts can be achieved through mechanical deboning to produce mechanically separated poultry meat (MSPM) for the manufacture of variety meats, canned meats and emulsified-type products.

The main problem encountered with MSPM is due to its method of production, which includes grinding meat and bones together and forcing the mixture through a perforated drum with consequent separation into two fractions, such as mechanically separated meat paste and bone residue. This causes the release of a considerable amount of fat and heme components from the bone marrow which becomes incorporated into the meat product, and thorough disruption of muscle cells.
Hence, the fundamental problems with proper utilization of MSPM are the high content of lipids, pigments and connective tissue, which lead to dark meat color, susceptibility to lipid oxidation, undesired textural properties and sometimes unpleasant odor due to the rancidity of fat. These properties may result in problems with further processing and consumer acceptance.

The meat industry often loses vast amounts of revenue each year due to the inability to efficiently recover and use "low value" meat. Even though it may have viable industrial applications, this meat is often discarded as waste post-meat processing and is generally viewed as a useless by-product of meat processing.
Therefore, it would be desirable to find a use for "low value" poultry meat.

SUMMARY
Described herein are the isolation and use of meat proteins and their applications thereof. In one aspect, meat proteins such as, for example, fish, poultry, bovine, or porcine can be used to make films, meat binders or extenders, and extrudable food articles. The advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

Figure 1 shows the solubility (mg/g) profile of mechanically separated turkey meat (MSTM) proteins at pH values from 1.5 to 12Ø Muscle tissue was homogenized in 50 volumes of deionized water and pH was adjusted by using 0.2 M
and 1 M HCl or NaOH. Results are presented as mean (n=4) standard deviation.

Figure 2 shows the extractability of proteins recovered from MSTM by acid-and alkaline-aided extractions. Sarcoplasmic proteins were solubilized in phosphate buffer, while total proteins were solubilized in phosphate buffer (pH 7.4) containing potassium iodide. Results are presented as mean (n=4) standard deviation.
Different letters for respective parameters in the figure represent significant (P <
0.05) difference.

Figure 3 shows the surface hydrophobicity of myofibrillar and sarcoplasmic proteins at different extraction pH values. Hydrophobicity is expressed as initial slopes of relative fluorescence intensity versus protein concentration in the presence of 1-anilino-8-napthalenesulfonate. Results are presented as mean (n=4) standard deviation. Different letters for respective parameters in the figure represent significant (P < 0.05) difference.

Figure 4 shows the total and reactive (free) sulfhydryl content of proteins recovered from MSTM at different extraction pH values. Analyses were performed by using Ellman's reagent. Results are presented as mean (n=4) standard deviation.

Figure 5 shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of different samples from acid and alkaline extraction processes.
Lane 1 is the molecular weight standard. Lanes 2, 3, 4 and 5 refer to the protein after isoelectric precipitation for extraction at pH values of 2.5; 3.5; 10.5 and 11.5, respectively. Lane 6 represents MSTM. Lanes 7, 8, 9 and 10 refer to the final protein isolate for extraction at pH values of 2.5; 3.5; 10.5 and 11.5, respectively.

*MHC - myosin heavy chains.
**MLC - myosin light chains.

Figure 6 shows the effect of time and extraction pH on oxidative stability of protein recovered from MSTM as determined by induced thiobarbituric acid relative substances (TBARs). Results are presented as mean (n=4) standard deviation.

Figure 7 shows cooking loss of proteins recovered from MSTM at different extraction pH. No statistical differences were observed. Data were statistically analyzed by one-way ANOVA. Results are presented as mean (n=4) standard deviation.

Figure 8 shows expressible moisture (expressed as a water loss) of proteins recovered from MSTM at different extraction pH. Results are presented as mean (n=6) standard deviation. Different alphabetical letters in the figure represent significant (P < 0.05) difference between means.

Figure 9 shows the total heme pigments content of proteins recovered from MSTM as different extraction pH. The total original material (raw MSTM) contained 3.77 mg of total heme pigments per lg of meat. Results are presented as mean (n=3) standard deviation. Different alphabetical letters in the figure represent significant (P
< 0.05) difference between means.

Figure 10 shows the hardness, chewiness, springiness and cohesiveness of proteins recovered from MSTM at different extraction pH. No statistical differences were observed. Data were statistically analyzed by one-way ANOVA. Results are presented as mean (n=3) standard deviation.

Figure 11 shows the changes in dynamic viscoelastic behaviour (DVB) of proteins recovered from MSTM at different extraction pH. The samples were prepared with 2.5% of NACI additon. The rheograms show storage modulus (G), loss modulus (G") and tan delta (8) development during heating from 4 to 80 C at 2 C/min.

Figure 12 shows the average storage modulus (G', kPa) at 5, 56.6 and 80 C
for proteins recovered from MSTM at different extraction pH. Results are presented as mean (n=4) standard deviation. Different alphabetical letters in the figure represent significant (P < 0.05) difference between means.

Figure 13 shows the changes in dynamic viscoelastic behaviour (DVB) of proteins recovered from MSTM at different extraction pH. The samples were prepared with 2.5% of NACI addition. The rheograms show storage modulus (G), loss modulus (G") and tan delta (8) development during cooling from 80 to 4 C
at 2 C/min.

Figure 14 shows an exemplary method for solubilizing and isolating a poultry protein.

Figure 15 shows a poultry meat slurry mixed with an acidic or a basic solution prior to centrifugation.

Figure 16 shows a solubilized protein layer after contacting the poultry meat slurry with an acidic or a basic solution followed by centrifugation.
Figure 17 shows a biopolymer film made by the methods described herein.

DETAILED DESCRIPTION

Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific compounds, synthetic methods, or uses as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes mixtures of two or more such proteins, and the like.
"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "optionally a flaxseed oil" means that the flaxseed oil can or can not be included.
Described herein are the isolation and use of meat proteins and their applications thereof. Using the techniques described herein, meat such as, for example, fish, poultry, bovine, or porcine can be used to make films, meat binders or extenders, and extrudable food articles.

Proteins as defined herein may include either a non-post-translationally modified protein, a post-translationally modified protein, or a combination thereof. In one aspect, the protein may include a full-length protein, full-length proteins, or peptide fragments of a full-length protein(s). In one aspect, the protein can be derived from an animal such as a fish, bird, cow, or pig, and in another aspect, the protein can be derived utilizing recombinant DNA technology. If derived from an animal, the protein can be obtained, for example, during meat processing where meat is separated from the bone of the animal. During meat processing, what is termed "low value"
meat may adhere to the bone after processing. In one aspect, this meat is skeletal muscle, which is composed of myofibrillar protein.
In certain aspects, the protein may include, for example, a poultry protein.
Although poultry protein can be derived from "low value" meat, poultry protein can be isolated from any poultry meat. Examples of poultry meat include, but are not limited to, chicken, turkey, duck, ostrich, quails, pigeons, geese, guinea fowls, and swans. In one aspect, poultry protein includes, but is not limited to, poultry myofibrillar protein. In one aspect, the poultry myofibrillar protein includes a chicken myofibrillar protein, a turkey myofibrillar protein, a duck myofibrillar protein, an ostrich myofibrillar protein, quail myofibrillar protein, pigeons myofibrillar protein, geese myofibrillar protein, guinea fowl myofibrillar protein, swan myofibrillar protein, or any combination thereof. In another aspect, the poultry meat is mechanically separated poultry meat (MSPM) having a 20-30% fat content and poor shelf stability. Methods for isolating proteins such as poultry protein are discussed in detail below.
The amount of protein present in the article can vary depending upon the article or application selected and the source of the protein and isolation techniques.
In this aspect, the protein can include 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, and 70 wt% of the total weight of the article or composition, wherein any one wt% can serve either as an upper or lower end point of a wt%
range.
In one aspect, the protein compositions described herein can be used to make films. When making films, the protein composition also includes a plasticizer.
Plasticizers increase the plasticity, elasticity, or fluidity of films described herein. In one aspect, the protein compositions produced herein can be used to make edible films and coatings. Edible plasticizers include glycerol, acetylated monoglycerides, trioctyl citrate, trihexyl citrate, sorbitol, and polyethylene glycol 400. In one aspect, the plasticizer can include 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35 wt%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45 wt%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, 60 wt%, 61 wt%, 62 wt%, 63 wt%, 64 wt%, 65 wt%, 66 wt%, 67 wt%, 68 wt%, 69 wt%, and 70 wt% of the total weight of the edible films and coatings, wherein any one wt% can serve either as an upper or lower end point of a wt% range.
After formation of the protein/ plasticizer composition, the composition is cast to produce a film. In one aspect, the casting step involves applying the protein/
plasticizer composition onto a substrate (e.g. Teflon coated trays or silicone resin plates) set on a level surface followed by drying the protein/ plasticizer composition.
In one aspect, the cast composition can be dried at from 20 C to 50 C for 5 to 15 hours. In another aspect, the cast composition can be dried at room temperature by air blowing for 2 to 24 hours followed by further drying in an environmental chamber from 25 C to 80 C at a relative humidity of 10 % to 30 % for a period of 24 hours to 48 hours. The thickness of the film can vary depending on the amount of protein/
plasticizer mixture that is cast. In one aspect the film can be 0.03 mm, 0.035 mm, 0.04 mm, 0.045 mm, 0.05 mm, 0.055 mm, 0.06 mm, 0.065 mm, 0.07 mm, 0.075 mm, 0.08 mm wherein any one mm can serve either as an upper of lower end point of a mm range.

In certain aspects, additional components can be added to the protein/plasticizer composition to enhance the mechanical properties of the edible film. For example, a lipid can be added to the composition in order to increase water barrier capacity. Suitable lipids include, but are not limited to, beeswax, stearic acid, palmitic acid, myristic acid, lauric acid, stearyl alcohol, hexadecanol, tetradecanol, or any combination thereof. In one aspect, the amount of lipid used is 0.5 % by weight.
In certain aspects, the protein compositions described herein can be used to produce an edible film. The edible film may be used as either a coating, a casing, or packaging. For example, the film can be applied as a casing or coating to edible foods including, but not limited to, meat products such as poultry, beef, bison, pork, lamb, goat, fruits, eggs, cheese, or any combination thereof. The edible films and coatings provide numerous benefits in the meat industry including, but not limited to:
1. Application of edible coatings prior to vacuum-packaging of meat may prevent moisture loss, thereby maintaining saleable weight and alleviating texture, flavor, and color changes.
2. Edible coatings on meat cuts may hold in juices, prevent dripping, enhance product presentation, and eliminate the need for placing absorbent pads at the bottom of plastic retail trays.
3. Lipid and myoglobin oxidation in meats may be reduced by using edible coatings of low oxygen permeability.
4. Edible coating solutions that have been heated just prior to application may reduce the loads of spoilage and pathogenic microorganisms and partially inactivate proteolytic enzymes at the surface of coated meat cuts.

5. Volatile flavor loss from and foreign odor pick-up by meat may be restricted with edible coatings.

6. Seasonings and/or browning agents may be imbedded into coatings and applied to meat products prior to cooking.

7. Used as an active packaging, edible coatings carrying antioxidants (e.g., tocopherols) and/or antimicrobials (e.g., organic acids) may be used for direct treatment of meat surfaces, thereby delaying meat rancidity and discoloration and reducing microbial loads.

8. Oil uptake by meat products during deep-fat frying may be reduced through application of coatings prior to battering and breading.

9. Coatings may reduce meat charring and stickiness to the cooking surface during broiling/grilling In other aspects, the protein compositions described herein can be used to produce biodegradable packaging films. With the increasing concern over environmental safety of non-degradable synthetic products from traditional petroleum resource, there is interest in natural degradable products from renewable sources as alternatives to synthetic polymers. In one aspect, poultry protein such as chicken myofibrillar protein is a viable renewable resource for producing environmentally safe industrial products. Modification of the myofibrillar protein can be performed in order enhance the mechanical properties of the film. For example, the use of crosslinkers that possess two or more groups capable of reacting with amino groups present on the protein can be used herein. In one aspect, compounds possessing two or more aldehyde groups such as, for example, glyoxal, can form a strong gel network suitable for use in biodegradable packaging films. In one aspect, the amount of crosslinker that can be used is from 0.25% to 2.0 % by weight of the composition.
In other aspects, the protein compositions described herein can be used to produce surimi. Surimi is a much-enjoyed food product in many Asian cultures and is available in many shapes, forms, and textures. Surimi is a useful ingredient for producing various kinds of processed foods. It allows a manufacturer to imitate the texture and taste of a more expensive product such as, for example, a lobster tail, using a relatively low-cost material. In one aspect, the surimi is poultry surimi.
In another aspect, the protein compositions described herein can be used to make extruded food articles. For example, the extruded article can be a noodle. In order to address the growing obesity and overweight, as well as diabetes problems, low calorie and fiber enriched foods are getting momentum in their development. In many parts of the world, consumption of pasta or noodles is significant. Fiber enriched pasta or noodle with whole grain; hydrocolloids and other fiber source have not made much progress. In one aspect, poultry proteins mixed with a plasticizer (e.g., glycerol) and an alginate (e.g., sodium alginate) can be used to make noodles.
Other materials suitable for making noodles besides alginates include, but are not limited to, (3-glucans, konjac glucomannan, and pectins. An exemplary procedure for making noodles is provided in the Examples. In the preparation of the noodle forming solution, various flavors and colorants can be introduced as needed.
In yet another aspect, the protein compositions described herein can be used as meat binders or extenders used in processed meat products. In one aspect, the compositions described herein can be used in processed poultry meat products such as, for example, white meat chicken nuggets. In other aspects, the meat binder can be added to restructured meat products, comminuted meat products, emulsified meat products, marinated meat products, or any combination thereof. In one aspect, the restructured meat product includes chicken nuggets, cordon blue, or a combination thereof; the comminuted meat product includes sausages, hamburgers, or a combination thereof; and the emulsified meat products includes bologna, hot dogs, or a combination thereof. In one aspect, when the meat binder is added to marinated meat products, the meat binder is mixed with water and added to a meat products by, for example, injection of the meat binder into the meat products. In another aspect, the protein composition is a dry powder that is mixed with one or more spices to produce protein-fortified spice composition.
The use of the protein compositions described herein as meat extenders or binders have numerous benefits including:
1. Increase raw and cooked yields dramatically with cleaner label.
2. Restore succulence to frozen meats.
3. Reduce or eliminate phosphates and soybean protein isolates.
4. Pre-cook yields equal to or better than phosphates and soybean proteins isolates.
5. Cook yields superior to phosphates and soybean protein isolate.
6. Recover meat from trimmings as a soluble protein marinade.
7. Establish a new control of quality/consistency in intact meat cuts.
In other aspects, the protein composition can be used as a nutritional supplement. For example, the protein composition can be a dry powder that can be added to sports drinks. Alternatively, the protein composition can be added to foods such as energy bars and gels.
The protein compositions described herein can be optionally supplemented with additional ingredients depending upon the application and nature of the proteins.
In one aspect, these additional ingredients optionally include fish oil, flaxseed oil, an essential oil including citrus or various spices, or any combination thereof;
these spices can include, but are not limited to, black pepper, white pepper, nutmeg, celery seed, glow, fennel, coriander, ginger, and tumeric. Fish oil and flaxseed oil both contain essential fatty acids. Essential fatty acids include the omega-3 and omega-6 families. Essential fatty acids are fatty acids that cannot be constructed within an organism from other components by any known chemical pathways and must therefore be obtained directly via ones diet. Essential fatty acids are crucial to make eicosanoids, endocannabinoids, lipoxins, resolvins, isofurans, neurofurans, isoprostanes, hepoxilins, epoxyeicosatrienoic acids (EETs) and neuroprotectin D.
When present in the biopolymer film, the fish oil, flaxseed oil, an essential oil including citrus or various spices, or any combination thereof can include 0 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, and 10 wt% of the total weight of the biopolymer film wherein any one wt% can serve either as an upper of lower end point of a wt% range.
In other aspects, the protein compositions can be optionally supplemented with anti-microbial agents, anti-viral agents, anti-oxidants, release agents, time-release agents, colorants, flavors, cross-linking agents, or any combination thereof.
In one aspect, anti-microbial agents prevent the growth of bacteria and further aid in preserving a film. If the film is used as a casing or coating for edible food, anti-microbial agents in the edible film may further help to preserve the shelf-life of that edible food. Anti-microbial agents include, for example, bactriocines, (i.e.
Pediocin PA-1, nisin, etc.) and enzymes including, but not limited to, lysozyme, butyric acid, and lauric acid.

Anti-viral agents can include, for example lauric acid.
Anti-oxidants can be added to the protein compositions to further enhance the nutritional value of the article (e.g., film) and to further preserve the article. Anti-oxidants include, for example, vitamins such as vitamin E, alpha-lipoic acid, rosemary extract, oregano extract, green tea extract, blueberry extract, BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), beta-carotenes, licopenes, or any combination thereof.
In one aspect, time-release agents can be incorporated in the protein composition. Time-release agents include, for example, gelatin, collagen, alginate, beta-glucans, guar gum, or any combination thereof.
In one aspect, colorants can be added to the protein composition. For example, ff the film is used as a casing or coating for edible food, these colorants may enhance the edible food's appearance thus making the edible food appear fresh.
Colorants include, for example, beet root extract, carminic acid, beta-carotenes, or any combination thereof.
In certain aspects, the protein composition includes a flavor or flavoring agent.
Flavors include, for example, chicken, beef, turkey, ostrich, bison, goat, protein hydrosilate, or any combination thereof.
In one aspect, cross-linking agents can be used in the protein compositions described herein to further aid in strengthening or solidifying the biopolymer films.
Crosslinking agents include, for example, transglutaminases, ferulic acid, glyoxal, glutaraldehyde, or any combination thereof. As discussed above, crosslinking agents can enhance the mechanical properties of a biodegradable film produced by the protein compositions.
When present in the protein composition, the anti-microbial agents, the anti-viral agents, the anti-oxidants, the release agents, the time-release agents, the colorants, the flavors, cross-linking agents, or any combination thereof can include 0 wt %, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, and 30 wt% of the total weight of the composition wherein any one wt%
can serve either as an upper of lower end point of a wt% range.
In one aspect, the protein compositions can be produced by first isolating a poultry protein. In one aspect, the poultry protein can be isolated by the following steps:
(a) contacting a low value poultry meat with an acidic solution or basic solution;
(b) centrifuging the low value poultry meat produced in step (a);
(c) isolating one or more proteins from the low value poultry meat produced in step (b).
In one aspect, if the protein composition is used to make a meat binder or extender, after step (c), the isolated protein may be dried by air drying or lyophilization to produce a dry powder. Lyophilization can include freeze drying the isolated protein at 50 C to 55 C for 8 to 12 hours.
In certain aspects, the compositions, including the biopolymer films are desired. In one aspect, when the protein composition is used to make a film, the method involves:
(a) isolating one or more proteins from a poultry meat to produce a first composition;
(b) admixing a plasticizer with the first composition to produce a second composition; and (c) casting the second composition to produce the film.
In one aspect, the one or more proteins are derived from a poultry meat. In certain aspects, the poultry meat is prepared as a meat slurry. The meat slurry may include poultry meat that is tempered from 0 C to 4 C overnight and then minced in a commercial-grade food processor with ice water. Next, the meat slurry containing the one or more proteins is contacted with either an acidic solution or a basic solution for a sufficient time and concentration to remove or isolate the desired proteins from the poultry meat. Not wishing to be bound by theory, the acidic and the basic solutions solubilize the poultry protein present in the poultry meat (i.e., meat slurry).
In one aspect, the poultry meat is contacted with an acidic or basic solution from 30 minutes to 1 hour. The acidic and basic solutions used herein can have various molarities ranging from 0.05 M to 2 M. In one aspect, the one or more proteins derived from a poultry meat are contacted with an acidic solution at a pH
from 2.0 to 4Ø In another aspect, the one or more proteins derived from a poultry meat are contacted with a basic solution at a pH from 10.5 to 12Ø In each of these aspects, a protein layer can be formed after contacting with either the acidic or basic solution. In one aspect, the acidic solution includes HCl, citric acid, acetic acid, or any combination thereof. In another aspect, the basic solution includes NaOH, NaHCO3 and phosphate buffers, or any combination thereof. Although it is desirable to use an aqueous solution of acid or base, it is also possible to use other organic solvents as well.
In one aspect, before contacting the meat slurry with either an acidic or basic solution, 2 to 10 mmole/L of citric acid can be added to promote the removal of polar lipids (i.e. phospholipids and cholesterol). In a further aspect, before contacting the meat slurry with either an acidic or basic solution, 8 to 16 mmol/L of calcium chloride (CaC12) can be added to promote the removal of polar lipids (i.e.
phospholipids and cholesterol). In yet another aspect, before contacting the meat slurry with either an acidic or basic solution, 8 to 16 mmol/L of calcium chloride (CaC12) and 2 to mmol/L of citric acid can be added to promote the removal of polar lipids (i.e.
phospholipids and cholesterol). By removing the polar lipids such as phospholipids, lipid oxidation can be reduced or prevented.
After solubilizing the protein with an acidic solution or a basic solution, a protein layer is generally produced. The protein layer is next separated or isolated from the fat and unwanted sediment, which is also present within the meat slurry. In one aspect, in order to further enhance separation and isolation of the protein layer, the mixture can be centrifuged. In one aspect, the meat slurry that has been contacted with either an acidic or basic solution is centrifuged between 6,000 rpm to 20,000 rpm for a period of 5 minutes to 1 hour. In certain aspects, the meat slurry that has been contacted with either an acidic or basic solution is centrifuged between 6,000 rpm to 20,000 rpm for a period of 5 minutes to 20 minutes. In this aspect and upon completing the centrifugation step, three layers are formed - an upper layer, a middle layer, and a bottom layer. The upper layer is a fat layer. The middle layer contains the solubilized protein (i.e. the protein layer), and the bottom layer contains unwanted sediment. In this aspect, the solubilized protein is carefully removed from the fat and sediment layers. In one aspect, the protein layer is an isolated protein layer that includes, for example, an aqueous layer of protein, a precipitated layer of protein, or a combination thereof. As defined herein, "isolate" means removing a desired protein or proteins from a mixture of proteins, fats, and carbohydrates or sugars. In this aspect, the isolated protein layer is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% poultry myofibrillar protein. In another aspect, the isolated protein layer is essentially free of connective tissue or connective tissue protein.
In certain aspects, when the protein composition is used to make a meat binder or extender, the protein layer can be isolated and the pH can be adjusted by contacting the protein layer with a second acidic or basic solution. In this aspect, the pH can be adjusted to the isoelectric point of the desired protein and subsequently precipitated and recovered. For example, the pH may be precipitated at a pH 5.2, which can be desired protein's isoelectric point. Next, the precipitated protein can be adjusted to pH values ranging from 6.2 to 7.0 and subjected to a second centrifugation step to further isolate the protein of choice. After the second centrifugation step, the isolated protein may be dried by air drying at 50 C to 55 C for 12 to 20 hours or by lyophilization to produce a meat binder.
In a further aspect, the protein may be separated using a chromatography step.
This chromatography step can include, for example, high-performance liquid chromatography (HPLC), affinity chromatography, size exclusion chromatography, ion exchange chromatography, or any combination thereof.
In one aspect, the isolated protein layer can be mixed with 2 to 10 wt%

sucrose, 2 to 10 wt% sorbitol, and 0 to 1 wt% sodium tripolyphosphate. In certain aspects, the isolated protein layer mixed with 2 to 10 wt% sucrose, 2 to 10 wt%
sorbitol, and 0 to 1 wt% sodium tripolyphosphate can be dried and subsequently used as a meat binder.
In another aspect, the protein solution is contacted and admixed with a plasticizer to produce a film. In one aspect, the protein is 1 wt% to 3 wt%
and the plasticizer is added at a range from 0.5 wt% to 1.5 wt%. In another aspect, fish oil, flaxseed oil, anti-microbial agents, anti-viral agents, anti-oxidants, release agents, time-release agents, colorants, flavors, cross-linking agents, or any combination thereof can be optionally added to the protein/ plasticizer mixture. In one aspect, the protein and plasticizer are mixed together for 1 to 3 hours by stirring, and then allowed to dry.

EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

1. Chemical and Functional Properties of Recovered Proteins from Acid-and Alkaline-Aided Extractions Material and Methods 1. Materials Mechanically separated turkey meat (MSTM) obtained from Lilydale Inc.
(Edmonton, AB, Canada). MSTM (250 g) was filled into polyethylene bags and kept at -20 C until use. Before extraction, samples were thawed overnight at 4 C.
All the reagents and chemicals used in the study were of analytical grade.

2. Methods Protein solubility In order to find the effect of different pH on the solubility of proteins in raw MSTM, a solubility curve was created, as described by Kim YS, Park JW, Choi YJ.
New approaches for the effective recovery of fish proteins and their physicochemical characteristics. Fish Sci 2003;69(6):1231-1239. Six grams of raw MSTM was mixed with 300 mL of refrigerated, deionized water in a homogenizer (Fisher Scientific, Power Gen 1000 S1, Schwerte, Germany) at a setting of 3 for 1 minute. The pH
of the homogenate (30 mL) was adjusted from pH 1.5 to 12.0 in 0.5 intervals, using 0.2 M
and 1 M HCl or NaOH, with the aid of a pH meter (Denver Instrument, Ultra Basic, UP-10, Colorado, USA). The homogenate was centrifuged at 25,900 x g at 4 C for min. The protein concentration of the supernatant was determined by Biuret method.
The protein solubility of the middle layer was expressed as milligram per gram of meat. Four replications were carried out for each measurement.

Extraction procedures Preparation of protein isolate by acid-aided process The acid-aided protein recovery from MSTM was done as per the methods of Liang and Hultin (Liang Y, Hultin HO. Functional protein isolates from mechanically deboned turkey by alkaline solubilization with isoelectric precipitation. J
Muscle Food 2003;14(3):195-205) and Betti and Fletcher (Betti M, Fletcher DL. The influence of extraction and precipitation pH on the dry matter yield of broiler dark meat. Poult Sci 2005;84(8):1303-1307) with some modifications. Protein extractions were carried out under low temperature conditions (4 C) in order to maintain the functionality of the final product. For each test, 200 g of MSTM were homogenized with cold (1-3 C) distilled water/ice mixture at 1:5 ratio (meat: water/ice, wt/vol) using a 900-Watt Food Processor (Wolfgang Puck WPMFP15, W.P. Appliances Inc., Hollywood, FL, USA) for 15 min. After homogenization, 1200 mL of the meat slurry was transferred to a beaker and placed at 4 C for 30 min. Further, the proteins in the homogenate were solubilized by drop-wise addition of 2 M HC1 to reach the maximum solubility points at acid conditions with pH values of 2.5 and 3.5, as determined from the solubility profile. The protein suspension was centrifuged using an Avanti J-E refrigerated centrifuge (Beckman Coulter Inc., Palo Alto, CA, USA) at 25,900 x g for 20 min at 4 C. After centrifugation, three layers were formed: an upper layer of MSTM neutral lipids; a middle layer of water-soluble proteins and a bottom layer of water-insoluble proteins and membrane lipids (this layer is termed the sediment fraction in the text). The middle layer of soluble proteins was collected and pH was adjusted to 5.2 by 2 M NaOH in order to isoelectrically precipitate proteins.
The precipitated proteins were thereafter centrifuged at 25,900 x g for 20 min at 4 C.
The precipitate was re-suspended in water/ice mixture (water/ice, 350mL/350g) by homogenization for 10 min and pH of the homogenate was then adjusted to 6.2.
The proteins were finally collected via centrifugation at 25,900 x g for 20 min at 4 C.
After complete extraction the moisture content of the resulting protein isolates was adjusted to 80%. Cryoprotectants (5% sorbitol, 4% sucrose, 0.3%
tripolyphosphate, 0.4% sodium bicarbonate and 0.03% sodium nitrite) were mixed with protein isolates in a pre-chilled Wolfgang Puck WPMFP15 900-Watt Food Processor (W.P.
Appliances Inc., Hollywood, FL, USA). The isolated proteins were stored in the freezer at -20 C until analysis.

Preparation of protein isolate by alkaline-aided process The extraction process was carried out in the same sequence as acid-aided extractions, and differs just in the first solubilization step. Here, the MSTM
proteins were initially solubilized at pH values of 10.5 or 11.5.

Total protein content and recovery yield The total protein contents of both the raw material and final protein isolates from different solubilization methods was estimated by the Biuret procedure.
Meat sample (1 g) was dispersed in 20 mL of 0.5 M NaOH, heated in the boiling water for min and cooling, in an ice-water bath. After cooling the solution was filtered through Whatman No. 1 filter paper. Then, 15 mL of the filtrate was centrifuged with mL of anhydrous ether (J-6B/P Beckman, Beckman Instruments, Inc, CA, USA) at 2278 x g for 10 min. After centrifugation, 1 mL of the lower phase was taken and mixed with 4 mL of Biuret reagent and the absorbance was measured at 540 nm (V-530, Jasco Corporation, Tokyo, Japan). Bovine serum albumin (Hy-Clone, UT, USA) was used as a standard.

Protein recovery of acid- and alkaline-aided treatments was determined according to the method described by Omana et al. (Omana D, Xu Y, Moayedi V, Betti M. Alkali aided protein extraction from chicken dark meat: chemical and functional properties of recovered proteins. Process Biochem 2010;45(3):375-381).
The recovery yield was expressed as a difference in total protein content of isolates (after isoelectric precipitation or final) and raw MSTM.

Protein extractability Frozen MSTM protein isolates were thawed overnight at 4 C. Sarcoplasmic and total protein extractability was determined by homogenizing (Fisher Scientific, Power Gen 100051) 2 g of sample at speed setting of 1 for 45 sec in 20 mL of 30 mM
phosphate buffer (pH 7.4) and 50 mM phosphate buffer containing 0.55 M
potassium iodide (pH 7.4), respectively. The homogenate was centrifuged in Avanti J-E
refrigerated centrifuge (Beckman Coulter Inc., Palo Alto, CA, USA) at 15,300 x g for 15 min at 4 C. The supernatant was filtered through Whatman No. 1 filter paper and the protein content of the clear filtrate was determined by Biuret method.

Surface hydrophobicity Sarcoplasmic and myofibrillar protein surface hydrophobicity were determined using 1-anilino-8-naphthalenesulfonate fluorescent probes (ANS; 8 mM in 0.1 M phosphate buffer, pH 7.0) according to the method described by Kim et al.
Sarcoplasmic proteins were extracted by homogenizing 2 grams of meat sample in mL of 30 mM phosphate buffer (pH 7.4) for 45 sec, followed by centrifugation at 15,300 x g for 15 min at 4 C. The supernatant was used as sarcoplasmic protein solution. The pellet obtained after centrifugation was re-suspended in 50 mM
phosphate buffer containing 0.55 M potassium iodide (pH 7.4), homogenized and centrifuged as described above. The supernatant was filtered through Whatman No. 1 filter paper and protein concentration was determined using Biuret method. The protein solutions were serially diluted with the same buffer to the final volume of 4 mL to obtain concentrations ranging from 0.008% to 0.03%. After mixing with 20 tl of ANS solution, fluorescence was measured using a fluorescence plate reader (Fluoroscan Ascent FL; Thermo Electron Corp., Vantaa, Finland) at an excitation wavelength of 355 and emission wavelength of 460 nm. The net relative fluorescence intensity (RFI) was obtained by subtracting the RFI of each sample measured without ANS from that with ANS. The initial slope of the RFI versus protein concentration (expressed in percents) was calculated by linear regression analysis and used as an index of the protein surface hydrophobicity.

Total and reactive suljhydryl content The estimation of total (T-SH) and reactive (R-SH) sulfhydryl groups were performed using protocols of Choi and Park (Choi YJ, Park JW. Acid-aided protein recovery from enzyme-rich Pacific whiting. J Food Sci 2002;67(8):2962-2967) and Kim et al., respectively. Protein extracts were prepared by homogenizing (setting 3 for 1 min) 2.5 gram of recovered protein in 25 mL of tris-glycine buffer (pH
8.0) containing 5 mM of EDTA. The homogenate was filtered before use. For T-SH

estimation, to 1 mL of the filtrate, 4 mL of 10 M Urea and 50 l of Ellman's reagent ((10 mM 5,5'-dithiobis (2-nitrobenzoic acid)) were added and mixed well by vortex mixer (Fisher, Scientific, On, Canada). In case of R-SH, 1 mL of filtrate was mixed with 4 mL of tris-glycine buffer (pH 8.0) and 50 l of Ellman's reagent. The mixture was kept for 1 h at 4 C with intermittent stirring. The absorbance of the solutions was measured at 412 nm against a blank of Ellman's reagent at the same concentration without proteins using a spectrophotometer (V-530, Jasco Corporation, Tokyo, Japan). The SH content was calculated by using molar extinction coefficient of 13, 600 M-1 cm -1 and was expressed as mol/g of protein. The protein content of the filtrate was determined by Biuret method.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Proteins were separated according to the procedure described by Laemmli (Laemmli UK. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 1970;277:680-685). Precast 10-20% ready gels (Bio-Rad Laboratories Inc., Hercules, CA) were used to separate proteins in a Mini-PROTEAN
tetra cell attached to a PowerPack Basic electrophoresis apparatus (Bio-Rad Laboratories Inc., 1000 Alfred Nobel Drive, Hercules, CA, USA). For each sample, 20 g of protein was loaded and the run was carried out at constant voltage of 200 V.
After staining and destaining, gels were scanned using an Alpha Innotech gel scanner (Alpha Innotech Corp., San Leandro, CA) with FluorChem SP software. Standard protein marker from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA) was loaded into a separate well.

Amino acid analysis Amino acid analysis was carried out on a Beckman System 6300 High Performance Analyzer by post-column ninhydrin methodology after hydrolysis of proteins in 6 N HCl and 0.1% phenol for 1 h at 160 C. Pickering Laboratories 15 cm sodium column and Pickering's sodium eluent buffers were used in the study.
Data was collected and analyzed using Beckman System Gold software.

Total lipid extraction Total lipid content was determined using the method of Folch et al. (Folch J, Lees M, Stanley GHS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226(l):467-509). Accordingly, 10.0 g of processed meat and 5.0 grams of raw meat were separately extracted with 120 mL
of Folch solution (chloroform: methanol solution, 2: 1, vol/vol) by homogenization for 10 min. After 30 min, the homogenates were filtered through Whatman No. 1 filter paper. To allow clear phase separation, 40 mL of 0.88% (vol/vol) sodium chloride solution was added and the mixture was carefully transferred to a separating funnel. After separation, the chloroform phase was filtered through anhydrous sodium sulphate (Fisher Scientific, NJ, USA) and transferred into a pre-weighed round-bottom flask, while the upper phase was discarded as it was rich in non-lipid components. Thereafter, the chloroform was evaporated at 40 C using a rotary evaporator (Rotavapor, RE 121, Buchi, Switzerland). The flasks were then placed in a hot air oven for drying at 60 C for 30 min and weighed accurately after desiccation for 30 min. For further analysis of lipid classes, the total lipid extract was washed with 10 mL of chloroform and dissolved lipids were transferred into pre-weighed vials and frozen at -20 C. Lipid reduction was calculated from the difference in lipid content between raw and treated materials and expressed as percentage.

Fractionation of the main lipid classes The method of Ramadan and Morsel (Ramadan MF, Morsel JT. Determination of the lipid classes and fatty acid profile of Niger (Guizotia abyssinica Cass.) seed oil.
Phytochem Anal 2003; 14(6):3 66-3 70) was used to separate the triglycerides (neutral lipids) and phospholipid (polar lipids) fractions in total lipid extracts. The separation of two lipid classes was accomplished using a glass column (30 cm x 2 cm;
height x diameter) (Chemiglass Life Sciences, NJ, USA) packed with silica gel (70-230 mesh;
Whatman, NJ, USA) by applying the slurry of the adsorbent in chloroform (1:5, wt/vol). Lipid solution (9 mL) obtained from the total fat extraction was applied to the column. Neutral lipids were eluted first using 60 mL of chloroform. After the triglycerides were removed, 60 mL of methanol was applied to the column, which resulted in elution of polar lipids. The obtained fractions were completely evaporated to dryness and kept in a hot air oven at 60 C for 30 min. The final weight of the flasks was taken after desiccating for 30 min. Both neutral and polar lipid parts were determined gravimetrically and expressed as percentage.

TBARs measurement Lipid susceptibility to oxidation was measured by the induced thiobarbituric acid reactive substances (TBARs) method as a modification of the procedure of Kornburst and Mavis (Kornburst DJ, Mavis RD. Relative susceptibility of microsomes from lung, heart, liver, kidney, brain and testes to lipid peroxidation:
correction with vitamin E content. Lipids 1980;15:315-322). Briefly, 3 g of sample was homogenized in 27 mL of 1.15% KC1 with a Power Gen 1000 Si homogenizer (Schwerte, Germany) for 1 min at setting 3. A 200 l aliquot of the homogenate was mixed with 1000 l of 80 mM Tris-maleate buffer (pH 7.4), 400 l of 2.5 mM
ascorbic acid and 400 l of 50 mM ferrous sulphate and incubated for 0, 30, 60, 100 and 150 min in a 37 C water bath. After incubation, 4 mL of TBA-TCA-HCl mixture (26 mM TBA (thiobarbituric acid), 0.92 M TCA (trichloroacetic acid) and 0.8 mM
HC1) was added to the sample and further the test tubes were placed in boiling water for 15 min. After cooling to room temperature, the absorbance was recorded at nm against the blank containing all the reagents except homogenate. TBARs concentration was calculated using the extinction coefficient of F532 = 1.56 x 105 M-i cm i. The extent of lipid oxidation was expressed as nanomoles of malonaldehyde (MDA) per gram of meat.

Analysis of connective tissue components Collagen and glycosaminoglycan concentration in raw meat, sediment after first centrifugation, and final isolates were estimated by analyzing hydroxyproline and uronic acid content, respectively. Samples were dehydrated and defatted with several changes of acetone and then with chloroform: methanol (2:1, vol/vol) solution.
For hydroxyproline analysis, dry-defatted samples (50-100 mg) were hydrolyzed in 6 N

HCl in the presence of nitrogen at 110 C for 20 h. Then, hydrochloric acid was removed by evaporation in a hot water bath (80 C) with nitrogen flushing. The dried preparation was cooled to room temperature, dissolved in water and filtered (Whatman No. 1). The clear filtrate was subjected to the colorimetric method of hydroxyproline analysis as reported by Stegemann and Stalder (Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta 1967;18:267-273).

For uronic acid determination, dry-defatted samples (50-200 mg) were digested with twice crystallized papain (4 g/mg of tissue) in 20 volumes of 0.1 M
sodium acetate buffer (pH 5.5) containing 0.005 M EDTA and 0.005 M cysteine hydrochloride at 65 C overnight. After proteolysis, trichloroacetic acid was added to each digest to a final concentration of 7% (wt/vol) and the mixture was held at 4 C
overnight. After the removal of precipitated proteins by filtration (Whatman No. 1), the filtrate was dialyzed in running tap water for 24 h and then for another 24 h in double deionized water at 4 C. The uronic acid content in glycosaminoglycan containing fraction retained in the dialysis tube was determined by the carbazole reaction with glucuronolactone as a standard. The reaction mixture consisted of 0.5 mL of solution containing glycosaminoglycan or glucuronolactone standard, 3.0 mL
of sulfuric acid reagent (0.2 M sodium tetraborate decahydrate in sulfuric acid) and 0.1 mL of 0.5% (wt/vol) carbazole in methanol.

Statistical analysis The entire experiment, from MSTM through final protein isolate was replicated at least three times. The results were expressed as mean value standard deviation. Data were subjected to one-way-analysis of variance (ANOVA) using the General Linear Model procedure of the Statistical System Software of SAS
institute (SAS user's guide. Statistics, Version 9.0, SAS Institute. Inc., Cary, NC. USA
2006).
To identify significant differences among mean values within the evaluated parameters at various pH treatments, HSD Tukey's adjustment with a 95%
confidence level (P <w 0.05) was performed.

Results and Discussion Protein solubility The basis for using pH-shifting processing on MSTM utilization is the fact that solubilization of muscle proteins is maximum at low and high pH values.
Solubility is not only significant for the determination of the optimum conditions for protein extraction, but also of great importance in food industry applications. The high solubility at certain pH values is required for efficient separation of the soluble proteins from undesirable meat constituents (lipids, connective tissue, impurities, etc.). However, low solubility is needed to precipitate the solubilized proteins at isoelectric point for better recovery. In order to investigate the effect of different pHs on MSTM proteins, a solubility curve was constructed with pH range from 1.5 to 12.0 in 0.5 increments (Figure 1). The lowest solubility (or highest precipitation) in deionized water occurred at pH 5.5, which is in the range of isoelectric points for the majority of muscle proteins. At the isoelectric point, the negative and positive charges are equal, thus association among protein molecules is strong due to the ionic linkages. As a consequence, protein-water interactions are replaced by protein-protein interactions and precipitation occurs. An increase in solubility was observed with either acidification or alkalization, when the proteins become positively or negatively charged, respectively. These net charges provide more binding sites for water, resulting in electrostatic repulsion among molecules, hydration of charged residues and increased protein-solvent interactions contributing to the increased solubility. The highest protein solubility in acidic conditions, (186.2 mg/g) was attained at pH 2.5, while for alkaline conditions a maximum value of 245.3 mg/g was found with pH

11.5. The rapid increase in solubility on the acidic side compared to the alkaline might be attributed to more ionizable groups with pKa values between 2.5 and 7.0 than between 7.0 and 11Ø The protein solubility profile showed a U-shaped pattern;
however, unlike the typical solubility curve for fish muscle protein homogenates, the solubility was found to be maximum at pH 11.5 and decreased at pH 12Ø
Therefore, additional pH points 11.25 and 11.75 were added to the MSTM protein solubility analysis. The results confirmed the decreasing solubility with increasing pH
from 11.5 to 12Ø This finding indicated that poultry meat proteins are likely to behave differently when exposed to the extreme alkaline conditions compared to fish muscle proteins.

The pH-shifting process, which is widely used for extraction of proteins from fish sources, was found to be possible to apply for the recovery of poultry meat proteins, MSTM in particular. Based on the solubility study, four pH values (2.5; 3.5;
10.5 and 11.5) were selected as solubilization pHs for the protein extraction from MSTM.

Protein content and recovery yield A high recovery yield is important for economic reasons. The yield of protein achieved by acid or alkaline treatments is predominantly driven by three major factors: the solubility of the protein during exposure to low or high pH, the size of the sediments after centrifugation and the solubility at precipitation pH. The results obtained for different extraction stages are shown in Table 1.

Table 1. Protein content (%) and recovery yield (%) during different stages of protein extraction from MSTM' Extraction Protein yield Final protein Final protein pH after pI, % content, % yield, %
pH 2.5 70.6 1.7 18.5b 0.6 66.4a 5.4 pH 3.5 69.1 2.2 18.2b 1.3 57.1b 4.7 pH 10.5 67.3 6.9 19.6a 0.2 63.6ab 6.3 pH 11.5 68.7 1.4 19.0ab0.2 64.8a 2.5 'Results are presented as mean (n = 4) standard deviation.
Different letters within a column indicate significant differences; P < 0.05.
pI refers to the isoelectric precipitation.

The yield of the proteins recovered by isoelectric precipitation indicated no significant difference (P = 0.7972) due to the extraction pH. The final protein content was found to be different between acid and alkaline treatments, with a tendency to increase from low to high pH values. Final protein content was found to be maximum (19.6%) when MSTM was solubilized at pH 10.5, and minimum when solubilized pH
2.5 and 3.5. The highest final recovery yields were found at extraction pH of 2.5 and 11.5 (66.4% and 64.8% respectively), while the lowest was observed at pH 3.5 (57.1%) (P = 0.0097). The increase in recovery yield for pH of 2.5 and 11.5 is highly associated with the solubility profile (Figure 1), which showed the highest solubility at these pH values. Slight decrease of recovery yield at pH 10.5 resulted mainly from decreased amount of solubilized proteins as indicated by the MSTM solubility profile.
In general, the per cent of loss in recovery yield between precipitation (pH
5.2) and re-adjusting to pH 6.2 was found to be around 6%. The results indicated that optimizing pH during solubilization is the prerequisite step to achieve the maximum protein recovery from MSTM.

Extractability of recovered proteins Extractability is an important property since the amount of protein available in the solution affects the functional properties expected from proteins. The conformation of proteins, which is related to the environment, plays a significant role in determination of protein functionality. Also protein extractability relates to the surface hydrophobic (protein-protein) and hydrophilic (protein-solvent) interactions.

The highest total protein extractability (Figure 2) was observed at pH 10.5, with a value of 73.7 mg/g. The difference in extractability between solubilization pHs can be explained by the different degrees of denaturation and the consequences of different degree of protein refolding after pH readjustment to 6.2. The results indicated that protein isolates prepared at pH 10.5 were less denatured compared to those prepared at pH 2.5, 3.5 and 11.5. SDS-PAGE profiles of protein after isoelectric precipitation also revealed that protein hydrolysis was comparatively less at pH 10.5, which may be the reason for less denaturation (Figure 5). The lowest amount of solubilized total proteins (62.3 mg/g) was found at extraction pH of 2.5.
Sarcoplasmic protein extractability from recovered proteins as a function of pH was not significantly (P = 0.0563) different among treatments (Figure 2). The sarcoplasmic protein fraction comprised around 58% of total soluble proteins, which confirms the fundamental theory of the pH-shifting method, that a sizeable amount of sarcoplasmic proteins are recovered during acid- and alkali-aided processes.

Protein surface hydrophobicity Hydrophobic interactions play a major role in defining the conformation and interactions of protein molecules in solution, thereby affecting the stability of native protein structures. Surface hydrophobicity of proteins helps to determine the rate of protein unfolding due to different processing methods.

Myofibrillar protein hydrophobicity (Figure 3) was shown to be significantly different (P < 0.0001) between treatments and the trend was similar to that observed in protein extractability (Figure 2). Extraction at pH 10.5 showed highest myofibrillar hydrophobicity (Ho = 465). Similar values were observed for extractions conducted at pH values of 3.5 and 11.5 while at pH 2.5 extracted samples represented the lowest value. The myofibrillar hydrophobicity was found to increase with an increase in total protein extractability (Figures 2 and 3). Even though the observed results appear to be in contradiction, it is important to point out that protein extractability depends not only on the amount of hydrophobic groups exposed to the protein surface, but also on the intrinsic factors such as protein conformation and surface polarity/hydrophobicity ratio. In these circumstances, although proteins isolated at pH 10.5 showed the highest surface hydrophobicity and protein extractability, it might be possible that after the readjustment to pH 6.2, the amount of polar and ionic groups were still predominant over the non-polar groups even if these latter were exposed to the surface.
Therefore, exploring the surface polarity/hydrophobicity ratio could be a better indicator of protein denaturation than surface hydrophobicity by itself.

Sarcoplasmic protein hydrophobicity of the extracted proteins was significantly higher (P < 0.0001) for the alkali processed samples compared to acidic treatments (Figure 3). The cause of increased hydrophobicity might be due to the change in protein conformation, particularly due to partial protein unfolding.
As a result, the intramolecular bonds which stabilize protein structure are ruptured, thus facilitating the exposure of hydrophobic groups to the surface.

Sulfhydryl content Sulfhydryl groups are considered to be the most reactive functional group in proteins. The total and reactive sulfhydryl content of proteins extracted at different pH
values indicated no significant difference between treatments (P = 0.5825 and P =
0.9841, respectively), even though hydrophobicity was higher at pH 10.5.
However, an increase in total and reactive sulfhydryl group content was found for pH
treated samples compared to raw MSTM (data not shown), which is probably related to the protein unfolding, resulting in exposure of sulfhydryl groups to the protein surface.
The ratio T-SH/R-SH for raw and processed meat was also characterized. For raw MSTM, the ratio was equal to 1.42. A slight decrease of T-SH/R-SH ratio for protein isolates (1.32 and 1.36 for acid and alkaline extractions, respectively) was observed, which may be the result of increasing the amount of disulfide bond formation.
SDS-PAGE profile Protein bands corresponding to myosin heavy chains (MHC), actin, tropomyosin a and (3 were most abundant after isoelectric precipitation fractions and in the final protein isolate (Figure 5). The electrophoretic profile of the pI
precipitated proteins showed less hydrolysis of myosin heavy chains (MHC) for pH 10.5 treated samples, suggesting lower level of denaturation extension. No difference in protein profile was observed among different pH treatments for the final protein isolates. The presence of myosin light chains of low molecular weight showed the degradation of myosin into its subunits. The intensity of bands corresponding to myosin heavy chain, actin, tropomyosin a and (3 were increased in the extracted samples suggesting that the concentration of these proteins increased in the final protein isolates.
Hence, this may have effects on the improved functionality of proteins in the final isolates compared to that of raw material. Unpublished results showed appreciable gelation, emulsion and foaming capacities of the MSTM protein isolates. With improved functionalities of protein isolates, MSTM can be better utilized for value-added processing.

Amino acid composition The amino acids composition of raw MSTM and protein isolates obtained by extractions at different pH are shown in Table 2. Glutamic acid was found to be the predominant amino acid and was significantly (P = 0.0143) higher in acid treated samples compared to raw meat. Lysine, an essential dietary amino acid, was found to be significantly (P = 0.0023) increased for the acid treated samples compared to the raw meat. No significant difference (P > 0.05) was found between raw and processed meat for alanine, glycine, isoleucine, leucine, phenylalanine, proline, threonine, tyrosine and valine. A significant (P < 0.0001) loss of methionine for the pH
treated samples was observed. The reason for the methionine loss might be due to its oxidation during the extraction process, where the proteins are exposed to acidic or alkali environment. It was reported that methionine can be oxidized to methionine sulfoxide and methionine sulfone during processing. The amino acid histidine was found to decrease 82% on average for all extraction pH values, excluding pH
11.5 where it was not detected. The ratio of total essential amino acids to total amino acids showed no statistically significant (P = 0.1575) difference, suggesting no effect of acid and alkaline extractions on amino acid concentrations.

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Lipid reduction and TBARs Lipid reduction is the principal factor for producing functional protein isolates from MSTM since the raw material is highly rich in triacyglycerols and membrane phospholipids. The latter contribute greatly to the oxidative reactions due to the high content of unsaturated fatty acids. The amount of lipids that can be removed is linked to the fat content of the starting material. The total, neutral and polar lipids content of raw MSTM were 23.5, 14.3 and 7.5%, respectively. Acid and alkaline extractions of MSTM resulted in protein isolates with significantly (P < 0.0001) reduced lipid content compared to the initial material (Table 3). However, no significant (P
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large reduction of lipids from MSTM by the pH-shifting technique was expected, as at extreme pH values, the proteins are solubilized, favoring the release of the storage and membrane lipids. During the centrifugation step the lipids are released to the aqueous environment due to the differences in solubility and particle density. The meat: water ratio (1:5, wt/vol) used in the study also contributed to the high removal of lipids from MSTM. Several studies have showed that a pH-shifting process is effective for lipids removal.

The effect of different extraction pHs on TBARs development in the MSTM
protein isolates is shown in Figure 6. Analysis on lipid oxidation showed no significant difference among different pH treatments (P > 0.05). However, there was a significant (P < 0.001) decrease in the amount of malonaldehyde (MDA) for recovered meat compared to the raw meat (data not shown). The reason for decreased lipid oxidation is probably due to the higher removal of membrane lipids. No difference in the amount of MDA in the samples processed at different pH
refers to the analysis of polar lipids content (Table 3), wherein no differences were found among the various extraction pH conditions.

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Analysis of connective tissue fractions The extracellular matrix of connective tissue is composed of collagen fibers embedded in an amorphous ground substance containing glycosaminoglycans.
Glycosaminoglycans are linear unbranched polymers of repeating disaccharide units of hexosamine and uronic acid. Thus, the amount of uronic acid residues is important for quantitative analysis of glycosaminoglycans. Collagen is the major protein of connective tissue, with relatively small amounts of glycosaminoglycans.
Determination of the amino acid hydroxyproline is an accurate way of measurement of collagen, since there are no other known animal proteins containing any appreciable amounts of this amino acid. The collagen and glycosaminoglycans concentrations were estimated by determining hydroxyproline and uronic acid, respectively, in MSTM sediment obtained after the first centrifugation and in the final protein isolate (Table 4). The hydroxyproline concentration in the final isolates (< 1 g/mg) was on average 23 and 82 times lower compared to MSTM (z 12 g/mg) and the sediment fraction (23.3-59.1 g/mg), respectively, with no significant difference among pH treatments (P = 0.5026). This indicates that the myofibrillar and sarcoplasmic proteins were the major part of the extracted proteins, while most of the connective tissue (collagen) present in the sediment. At acidic extraction conditions, the concentration of hydroxyproline in the sediment was 1.6 times higher at pH
2.5 compared to pH 3.5.

A similar trend between these pH values was observed for the uronic acid concentration. The ratio of uronic acid to hydroxyproline, which represents the estimation of amorphous ground substance to collagen fiber, was similar between pH
2.5 and 3.5 and was almost identical to the value (0.14) corresponding to MSTM.
When the proteins were extracted in alkaline conditions, it was found that hydroxyproline and uronic acid concentrations in the sediment fractions were more than two times higher (P = 0.0005 and P < 0.0001, respectively) at pH 11.5 than pH
10.5. However, the uronic acid values were lower compared to the corresponding values observed at acidic pH, which resulted in the lower (> 3 times) uronic acid to hydroxyproline ratio.

The results give an insight into the possibility of extracting proteoglycans from the sediment fraction during the co-extraction of valuable components from MSTM.
Table 4. Hydroxyproline and uronic acid contents ( g/mg of dry-defatted weight) in mechanically separated turkey meat (MSTM) and its protein fractions obtained during extraction process' Hydroxyproline Hydroxyproline Uronic acid Uronic acid content in the content in the content in the content in the Treatment sediment after 1sr final protein sediment after ls` final protein centrifugation isolate centrifugation isolate Raw MSTM 11.6 0.8 - 1.7 0.1 -pH 2.5 50.6a 4.0 0.5 0.1 7.6a 0.1 0.5' 0.1 pH 3.5 31.7b 5.2 0.6 0.2 4.3b 0.0 0.4b 0.0 pH 10.5 23.36 4.5 0.6 0.3 1.6 0.2 0.9a 0.0 pH 11.5 59.la 5.5 0.3 0.1 3.5b 0.5 0.8a 0.1 'Results are presented as mean (n = 2) standard deviation. Different letters within a column indicate significant differences; P < 0.05.

Conclusions The study demonstrated that at pH 2.5 and 11.5, the proteins from MSTM
were most soluble, leading to the highest protein yields at these pH values.
Among the different extraction pH values, the highest total extractability was achieved at pH
10.5. Acid- and alkaline-aided extractions were equally effective in removing total, neutral and polar lipids from MSTM. Consequently, TBARs analysis showed no difference between acid and alkaline treatments; however the values were significantly lower compared to raw MSTM (P < 0.0001). SDS-PAGE profiles for both acid and alkaline extractions indicated higher concentration of myosin heavy chains, actin and tropomyosin compared to MSTM indicating concentration of myofibrillar proteins. No statistical difference in the ratio of total essential amino acids to total amino acids between MSTM and extracted proteins indicated that there was no substantial effect of acid and alkaline treatments. Analysis of uronic acid content revealed that most of the proteoglycans accumulated in the sediment fractions, hence paving the way for a co-extraction technology of the valuable components from MSTM.

II. Effect of Acid- and Alkaline-Aided Extractions on Functional and rheological properties of proteins recovered Material and Methods Raw material and chemicals Mechanically separated turkey meat (MSTM) was obtained from Lilydale Inc.
(Edmonton, AB, Canada). MSTM (250 g) was filled into polyethylene bags and kept at -20 C until use. Before extraction, samples were thawed overnight at 4 C.
All the reagents and chemicals used in the study were of analytical grade.

Preparation of protein isolate by acid-aided process The acid-aided protein recovery from MSTM was accomplished by the method described by Liang and Hultin and Betti and Fletcher with slight modifications. Protein extractions were conducted under low temperature conditions (4 C) in order to maintain the functional properties of the final product.
To prepare the protein isolate, 200 g of MSTM was homogenized with cold (1-3 C) distilled water/ice mixture at 1:5 ratio (meat:water/ice, wt/vol) using a 900-Watt Food Processor (Wolfgang Puck WPMFP15, W.P. Appliances Inc., Hollywood, FL, USA) for 15 min. Homogenate (1200 mL) was transferred to a beaker and allowed to stand for 30 min at 4 C. The proteins in the homogenate were then solubilized by drop wise addition of 2 M HCl to reach the maximum solubility at pH values of 2.5 and 3.5, as determined from the solubility profile, as reported by Hrynets and others (2010). Acidic or alkaline homogenates were centrifuged in Avanti R J-E
refrigerated centrifuge (Beckman Coulter Inc., Palo Alto, CA, USA) at 25,900 x g for 20 min at 4 C. Three phases were formed after centrifugation: a top phase of MSTM neutral lipids; a middle aqueous phase of water-soluble proteins and a sediment phase, containing a water-insoluble protein fraction and membrane lipids. The middle layer of soluble proteins was collected and pH was adjusted to isoelectric point (pH
5.2) with 2 M NaOH. The precipitated proteins were recovered by centrifugation at 25,900 x g for 20 min at 4 C. The precipitate was re-suspended in water/ice mixture (water/ice, 350mL/350g) by homogenization for 10 min. The pH of the resultant homogenate was adjusted to 6.2 by drop wise addition of 2 M NaOH. Proteins were finally collected by centrifugation at 25,900 x g for 20 min at 4 C. The moisture content of the recovered protein isolates was adjusted to 80%. Cryoprotectants (5%
sorbitol, 4% sucrose, 0.3% tripolyphosphate, 0.4% sodium bicarbonate and 0.03%
sodium nitrite) were mixed with protein isolates in a pre-chilled Wolfgang Puck WPMFP15 900-Watt Food Processor (W.P. Appliances Inc., Hollywood, FL, USA).
The isolated proteins were stored in the freezer at -20 C until analysis.

Preparation of protein isolate by alkaline-aided process For the alkali extraction process the procedures described above were followed except for the first solubilization step. Here the MSTM proteins were initially solubilized at pH values of 10.5 or 11.5.

Cooking loss Raw samples were prepared by manually grinding protein isolate samples (12 g) with 2.5% NaCl in a pestle and mortar for 10 min. The paste was packed in polypropylene capped tubes (1.7 cm x 10 cm, Simport, QC, Canada) without air pockets. The stuffed tubes were then heated in the water bath at 95 C until the internal temperature reached 75 O C. The internal temperature was measured using thermocouples, inserted in the centre of the sample. After cooking, the gel was removed from the tubes and accurately weighed individually. The samples were then stored in polyethylene bags at 4 C overnight prior to texture profile analysis. The cooking loss was calculated as follows:

Cooking loss (Original sample weight - weight after cooking) x 100 Original weight Determination of expressible moisture (EM) The expressible moisture of the protein isolates, as an estimation of water loss, was evaluated by using a texture profile analyzer (TA-XT Express, Stable micro systems, Ltd., Surrey, England), which was set to the adhesive test mode prior to the measurements. Three grams of meat sample were placed on the pre-weighed filter paper (Whatman No. 1), and pressed between two glass plates, with a target force of 1000 g for 2 min. After squeezing, the filter paper along with the absorbed water was immediately weighed. Expressible moisture was expressed as percentage. Six press tests were performed for each treatment. The following formula was used to calculate the expressible moisture:

Expressible moisture (Wet paper - Dry paper) x 100 Meat weight Emulsifying activity index (EAI) and emulsion stability index (ESI) The measurements of emulsifying activity index and emulsion stability index were conducted according to the method described by Moure and others (2002) with slight modifications. Oil-in-water emulsion was prepared by mixing corn oil with protein solution (myofibrillar or sarcoplasmic at the concentration of 0.4 mg/ml) at 1:3 ratio (vol/vol) in an homogenizer (Fisher Scientific, Power Gen 1000 S1, Schwerte, Germany) operated for 1 min at setting 3. Immediately after homogenization, 0.05 mL of emulsion was diluted to 5 mL with 0.1 % sodium dodecyl sulphate (SDS) solution and the absorbance was measured at 500 nm in a 1-cm path cuvette using a spectrophotometer (V-530, Jasco Corporation, Tokyo, Japan).
The EAI was calculated from the following equation:

EAI = 2.33 x AO

where AO is the absorbance estimated just after emulsion preparation. The emulsion stability index was determined by measuring the absorbance of these emulsions after min of standing. The ESI was deduced as follows:

An ESI = 10 x Ao - Aio where A10 is the absorbance determined after 10 min.
Foam expansion (FE) and foam volume stability (FVS) The measurement of foamability was performed as the method described by Wilde and Clark (1996). Known volumes of proteins (myofibrillar and sarcoplasmic) were whipped using a vortex mixer (Fisher Scientific, On, Canada) at speed 10 for 2 min.

Foamability or foam expansion was expressed as percentage volume increase after mixing using the following equation:

Foam expansion (%) = Foam volume (mL) x 100 Initial liquid volume The stability of the foam volume was calculated as percentage of foam remaining after 30 min at 25 C.

Foam volume stability (%) = Volume of foam (mL) retained after 30 minx 100 Volume of foam after whipping Total pigment determination The total pigment content was evaluated by direct spectrophotometric measurement according to the method of Fraqueza and others (Fraqueza MJ, Cardoso AS, Ferreira MC, Barreto AS. 2006 "Incidence of pectoralis major turkey muscles with light and dark color in a Portuguese slaughterhouse" Poult Sci 85 (11):1992-2000), with slight modifications. For each run, 10 g of the sample was weighed into 50 mL capped glass tubes and 40 mL of acetone, 1 mL of HC1, and 1 mL of water, were added. The mixture was vortexed for 3 min and allowed to stand for 1 hour at room temperature. The extract was filtered through Whatman No. 1 filter paper, and the absorbance was read at 640 nm against an acid-acetone blank using a UV/VIS
spectrophotometer (V-530, Jasco Corporation, Tokyo, Japan). The absorbance value was multiplied by a coefficient of 17.18 and the concentration of total heme pigments was expressed in milligrams of myoglobin per gram of meat.

Color characteristics The color characteristics of samples were measured on the surface of raw MSTM and freshly prepared protein isolates using a Minolta CR-400 (Konica Minolta Sensing Americas, Inc, Ramsey, NJ 07446). A white standard plate was used to calibrate the colorimeter. Tristimulus color coordinates L*, a* and b* were recorded.
The L* value on a 0 to 100 scale denotes the color from black (0) to white (100). The a* value denotes redness (+) or greenness (-), and the b* value denotes yellowness (+) or blueness (-). Three readings per treatment sample were taken and the average reading was recorded. The intensity of the red, saturation, Hue and whiteness were calculated as follows:

Intensity of the red = a* / b*
Saturation = (a2 + b2)v2 Hue = arctan b*/a*

Whiteness = 100 - [(100-L* )2 + a*2 + b*2]12 Texture profile analysis Texture profile analysis was carried out on cooked samples by employing a texture profile analyzer (TA-XT Express, Stable micro systems, Ltd., Surrey, England). The samples were cut into cylinders (17 mm diameter, 10 mm height) and subjected to the TPA mode analysis. Three samples per treatment were compressed to 50% of their original height for 2 cycles with the aluminum cylinder probe (d = 5 cm).
The time between two compressions was set as 1 s. Determination of texture attributes were performed at the trigger force of 5 g with the speed of 5 mm/s.
Attributes were calculated as follows. Hardness: the maximum force required for the first compression. Chewiness: the work needed to chew a solid sample to a steady state of swallowing. Springiness: the ability of the sample to recover to its original shape after the first compression. Cohesiveness: represents how well the product withstands a second deformation relative to how it behaved under the first deformation.
Measurements of samples were carried out at room temperature. Data were recorded and analyzed automatically by software provided with the instrument.

Dynamic viscoelastic behavior of isolated proteins The dynamic viscoelastic (DVB) behavior of isolated proteins during heating and cooling was monitored using a Physica MCR Rheometer (Anton Paar GmbH, Virginia, USA) under oscillatory mode, employing a 2.5 cm parallel plate measuring geometry. Four grams of protein isolate were mixed thoroughly with 2.5% of sodium chloride (w/w) in a pestle and mortar to obtain a fine ground paste. The paste was subjected to DVB measurements. The gap between measuring geometry and peltier plates was adjusted to 1.0 mm. Approximately 2 g of paste was placed on the peltier plate at 4 C. Once the sample was pressed by lowering the measuring geometry plate, excess sample was removed with a stainless steel spatula. The samples were heated from 4 to 80 C at a rate of 2 C/min and cooled from 80 to 4 C
at the same rate. To determine the linear viscoelastic region (LVR) an amplitude sweep was carried out in a range of deformation from 0.1 to 10%. After determining LVR, measurements of the samples were conducted by applying a controlled strain (0.5%) with a constant frequency set at 1 Hz. The two sine waves had a phase difference tan = , which gave elastic (storage modulus G') and viscous (loss modulus G' ' ) elements of gel. These two values along with tan = were recorded simultaneously throughout the heating and cooling processes by the instrument. Four replications were performed, each using a fresh paste preparation and the average values were plotted.

Statistical analysis All data were analyzed by one-way-analysis of variance (ANOVA) using General Linear Model procedure of the Statistical System Software of SAS
institute (Version 9.0, SAS Institute. USA. 2006) and reported as means and standard deviation among means. The entire experiment, from MSTM through final protein isolate was replicated at least three times. Comparison of means within the evaluated parameters at various pH treatments was carried out by HSD Tukey's adjustment with a 95% confidence level. Significance of difference was established at P <
0.05.
Results and discussion Cooking loss and expressible moisture The ability of meat proteins to retain water is one of the most important quality attributes influencing product yield and it also has an impact on eating quality of the product. Cooking loss provides an insight into the tenderness of a meat product, which is related to the ability of proteins to bind water and fat. Expressible moisture is a measure of the water holding capacity (WHC) of meat proteins and changes in WHC indicate the changes in the charge and structure of myofibrillar proteins.
In the present study the effect of different pH of extraction on WHC was assessed by estimation of cooking and water loss. No significant (P = 0.5699) difference was found for cooking loss (Figure 7) between different treatments. However, cooking loss of protein isolates was significantly lower (6.23% on average; P <
0.0001) compared to raw MSTM (29.27%; Table 5). Such a significant difference in cooking loss between raw and processed meat is probably due to the difference in composition of those two materials. Total lipid content of raw meat and isolated proteins was 23.50% and 1.81%, respectively. Therefore, while subjected to heat treatment raw meat will be loosing more fat in addition to the water loss resulting in higher cooking loss.

The results obtained from the analysis of expressible moisture, as an evaluation of water loss, are presented in Figure 8. Expressible moisture varied between treatments and the highest (14.26%: P = 0.0249) was obtained for samples processed with pH 2.5. Proteins extracted with pH 10.5 represented the lowest water loss of 12.86%. This decrease in water loss, which refers to the higher ability to retain water, is probably the result of higher protein content of samples extracted with pH
10.5. Extraction of proteins at this pH also resulted in the highest surface hydrophobicity of myofibrillar proteins. The exposure of hydrophobic amino acids to the protein surface may increase the number of hydrophobic interactions, leading to the formation of a gel network with higher ability to entrap water. Water loss was found to be significantly (P < 0.0001) higher for raw MSTM (46.70%) compared to the processed meat. These results suggest that WHC of MSTM could be greatly improved by the extraction treatments.

Table 5. Characteristics of raw mechanically separated turkey meat (MSTM)l Parameter Value Cooking loss (%) 29.27 5.1 Expressible moisture (%) 46.70 0.61 EAI (myofibrillar proteins) 2.37 0.18 EAI (sarcoplasmic proteins) 1.01 0.07 ESI (myofibrillar proteins) 56.67 3.19 ESI (sarcoplasmic proteins) 7.80 0.53 FE (myofibrillar proteins) (%) 93.30 20.82 FE (sarcoplasmic proteins) (%) 10.0 0 FVS (myofibrillar proteins) (%) 61.69 10.85 FVS (sarcoplasmic proteins) (%) 0 Total heme pigments (mg/g of meat) 3.77 0.69 Hardness (gram force) 142.82 20.33 Springiness 0.66 0.03 Chewiness 43.63 11.27 Cohesiveness 0.46 0.02 'Results are presented as mean standard deviation.

Emulsion activity index (EAI) and emulsion stability index (ESI) Emulsion is a heterogeneous system consisting of at least two immiscible liquid phases, one of which is dispersed in the other in the form of droplets.
Emulsion is stabilized through physical entrapment of fat globules within protein matrix followed by formation of an interfacial protein film around the small fat globules. The ability of protein to adsorb at the water-oil interface during the formation of emulsion avoiding flocculation and coalescence is indicated by EAI. On the other hand, ESI
estimates the rate of decrease of the emulsion turbidity due to droplet coalescence and creaming, leading to emulsion destabilization. Therefore, EAI and ESI increase when proteins favor emulsion formation and stabilization, respectively. The emulsification properties of acid and alkaline extracted proteins were evaluated by their ability to form and stabilize emulsion with oil and the results are presented in Table 6.

EAI of myofibrillar proteins was significantly different (P = 0.0184) between treatments, with the highest value obtained at pH 11.5. In general, alkali extracted protein showed slightly higher EAI compared to acid extracted protein. Among the myofibrillar and sarcoplasmic proteins, the latter showed significantly lower emulsification ability (P = 0.0010).

Among treatments, there was a tendency to be significantly higher for (P =
0.0592) ESI of myofibrillar proteins for alkali extracted samples. The latter values were also around 7 times higher compared to the ESI of sarcoplasmic protein fraction.
This effectiveness of myofibrillar proteins is probably due to the ability of myosin to display both hydrophobic affinity for fat and hydrophilic affinity for water.
Myosin provides a distribution of polar and non-polar amino acids thus enhancing the orientation between two unlike phases. High length to diameter ratio of the myosin molecule also contributes to the molecular flexibility and rearrangement at the protein film interface. ESI of sarcoplasmic proteins showed significant (P = 0.0039) difference in stability indexes with alkali extracted samples representing the highest values (6.42 and 6.60%).

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c, con kr) Foam expansion (FE) and foam volume stability (FVS) Food foams are dispersions of gas bubbles in a continuous liquid or semisolid phase. Foaming is responsible for the desirable rheological properties of many foods.
The behavior of the proteins at the liquid/air interface is important since the formation of protein film around air bubbles is essential for foam capacity and stability. The foam capacity and foam volume stability of protein isolates from MSTM prepared at different extraction pH are presented in Table 6. The foaming properties between the pH treatments were found to have no significant differences (P > 0.05), except foam expansion of sarcoplasmic proteins. The latter was significantly (P = 0.0014) lower when proteins were extracted at pH 2.5. Therefore, reduced foam expansion of sarcoplasmic proteins at pH 2.5 might be associated with the low hydrophobicity at this pH. FE of sarcoplasmic proteins were found to be significantly (P =
0.0019) lower compared to that of myofibrillar proteins. However there was no significant (P
= 0.8550) difference between the FVS of these two protein fractions. This suggests that even though myofibrillar proteins have higher ability to form the foam, the stability might be maintained at the same level by both myofibrillar and sarcoplasmic protein fractions. FE (myofibrillar and sarcoplasmic) and FVS of sarcoplasmic proteins were found to be significantly (P < 0.0001) lower for raw MSTM (Table 5) compared to processed samples. This denotes that conformational changes of proteins during acid and alkaline treatments lead to improvement of foaming properties.

Total pigments and color characteristics Color is an important factor for determining consumers' perception of product quality and significantly influences purchasing decisions. Color is also a principal characteristic when different processing treatments are compared, especially considering increased interest of current markets in isolates as white as possible. The two pigments which are mainly responsible for the color of MSTM are myoglobin and hemoglobin, thus their effective removal could greatly improve color characteristics of recovered meat. The total pigment analysis showed significantly (P
< 0.0079) higher content in alkali extracted samples (0.56 mg/g of meat) compared to that of acid treatments (0.44 mg/g of meat) (Figure 9). The highest total pigment removal was found when MSTM proteins were extracted at pH 2.5 (88.96%), while the lowest was for extractions at pH 10.5 and 11.5. Stronger protein-protein interactions at alkaline pH probably result in higher aggregation of sarcoplasmic proteins leading to precipitation into sediments after isoelectric precipitation. In general, extractions showed 86.72% removal of total pigments, resulting in around 0.5 mg of heme pigments per 1.0 g of meat.

Color characteristics (L*, a*, b*, a*/b*, saturation, Hue and whiteness) of the recovered proteins by the pH-shifting process are presented in Table 7. The results are shown in comparison to the initial material (raw MSTM). In general acidic and alkaline isolates greatly (P < 0.0001) decreased in the redness (a*), with no difference being found within pH treatments. The decrease in redness is due to the removal of pigments during extraction (Figure 9). Yellowness (b*) values remained constant (P =
0.0984) for raw MSTM and different extraction treatments. Lightness (L*) was significantly increased (P = 0.0035) for the samples processed with extraction pH of 2.5. The concentration of total pigments is the influential factor for the L*
values.
Thus, the extension of total pigment removal, which was observed to be higher for acid treated samples contributed to the increased lightness. Whiteness increased significantly (P = 0.021) compared to the raw meat, with the highest value (64.82) observed at pH 2.5. This is expected, because the whiteness values are mainly influenced by the lightness, which was the highest for samples extracted with pH 2.5.
Both lightness and whiteness values are in agreement with the results obtained from the analysis of total pigment content, which indicated the highest removal at extraction pH of 2.5. A significant decrease (P = 0.0036) was observed for a*/b*, which indicates a decrease in intensity of the redness value. The ratio decreased from the original value of 0.45 found for raw MSTM to 0.24 in general for processed meat.
High a*/b* obtained for the raw meat is primarily because of the high total pigment content. Saturation values determine how different the color is from gray and expressed as depth, vividness and purity. There was a significant decrease in saturation (P = 0.0036) observed between raw MSTM and acid extracted meat. The samples having a dominant red color would give a higher saturation value than the samples with a more homogenous structure. In this study the lower purity of alkali extracted meat compared to acid treatments might be the result of higher total pigment content. Hue angle shows the degree of departure from the true red axis to the CIE
color space. The Hue values were found to be significantly (P = 0.0046) higher for proteins recovered at pH 2.5; 3.5 and 11.5 compared to raw MSTM. This is expected, since increased Hue angle indicates a decrease in perceived redness. As the result of extraction procedures, the red color was decreased due to the removal of heme pigments. Consequently samples decreased in darkness with the dominance of Hue, which is an indication that the color shifted slightly to the yellowish spectrum.

Table 7. Color characteristics of proteins recovered from MSTM at different extraction pH' Parameter Raw MSTM 2.5 3.5 10.5 11.5 a* 7.45 0.24a 3.66 1.12b 2.84 0.76b 4.51 0.21b 3.58 1.19b b* 16.49 0.36 14.43 2.25 14.44 0.98 16.05 0.25 15.82 0.39 L* 58.94 1.55b 69.84 1.75a 61.59 b b 4.74ab 59.90 2.75 57.40 4.08 a*/b * 0.45 0.02a 0.27 0.13b 0.20 0.07b 0.28 0.02ab 0.22 0.07b Saturation 18.10 0.31a 14.95 1.88b 14.74 0.81b 16.67 0.20ab 16.24 0.64ab Hue 65.67 0.95b 75.23 6.67a 78.75 3.68a 74.30 0.89ab 77.36 3.81a Whiteness 52.46 1.27b 64.82 1.99a 57.85 4.38ab 54.93 2.47b 53.19 4.36b 'Results are presented as mean (n=3) standard deviation.
Least square means in each row corresponding to respective parameters with different superscripts are significantly different (P < 0.05) from one another.

Texture profile analysis and dynamic viscoelastic behavior of isolated proteins Complimentary information on textural properties of protein isolates was obtained using small and large deformation tests. A small deformation test was applied to investigate elastic and viscoelastic properties of gels, which is related to el quality and strength. Uniaxial compression of a gel sample between two flat parallel plates (large deformation test) was used to determine textural properties, such as hardness, chewiness, springiness and cohesiveness.

Texture profile analysis (TPA) of the MSTM protein gels is summarized in Figure 10. No significant differences (P > 0.05) were found for any of the parameters.
Generally, the higher hardness of the gels developed from MSTM protein was observed at pH 10.5, with the value of 1773 gram force. The lowest value for chewiness (934) was observed at pH 3.5. The lower value for this parameter is associated with the higher ability to form a viscoelastic network (Figure 11 A, B), as chewiness represent the ability of the sample to regain its shape after compression.
Chewiness is also one of the important characteristics, which associates with meat tenderness. No significant difference found for springiness value is probably due to the same water content between samples, as the extraction process was followed by adjustment of water content to 80%. While no difference among treatments was found for cohesiveness, the samples extracted at pH 11.5 appeared to be higher.

Gelation of muscle protein is a multi-step thermodynamic process which involves protein unfolding and aggregation prior to the formation of three-dimensional network structures indicated that rheological parameters could be used to predict sensory, texture and functionality of comminuted meat products. The dynamic rheological technique is widely used for the evaluation of gelation of myofibrillar proteins. Viscoelastic properties of storage (G' ), loss (G' ') modulus and tan delta (=) between acid and alkaline extractions were determined upon heating and cooling.
Changes in storage modulus, loss modulus and tan = during heating is given in Figure 11 A, B and C. Proteins isolated at different pH values showed a similar trend for both G' and G' ' values. However, the G' values were considerably higher in magnitude than the G' ' values indicating the formation of more elastic gels. The G' ' value is an estimation of energy dissipated as heat per sinusoidal cycle and is used to evaluate the gel viscosity.

During the heating phase, the G' showed only marginal change, until the temperature reached 36 C, where onset of gelation occurred (Figure 11A).
Storage modulus (G') is a measure of the energy stored in material and recovered from it per cycle of sinusoidal shear deformation and indicates solid or elastic characteristics. The increase in G' has been attributed to the ordered protein aggregation and formation of three-dimensional network with water entrapment in the matrix. The gelation starts with unfolding of myosin molecules at 35 - 40 C. The same increasing pattern was observed in loss modulus for heat induced gelation of dark chicken meat protein isolates indicating the formation of a viscoelastic network. G' values increased until temperature reached 56 - 58 C; further increase in temperature caused weakening of the gels as shown by decrease in G' values. This decrease might be due to the result of denaturation of light meromyosin, leading to increased fluidity. The maximum increase in G' value was in the temperature range of 40 to 56.6 C.
The forces which are responsible for the formation of the gel network include hydrophobic interactions, disulphide cross bridges and hydrogen bonds. Overall, the patterns of slopes for acid and alkaline extracted MSTM proteins were similar, excluding pH
10.5.

Tan = values indicated a major transition point at temperature of 47.3 C for proteins extracted at pH 2.5, 3.5, 11.5 and 51.9 C for proteins extracted with pH 10.5 (Figure 11 Q. This transition point refers to the denaturation of the myosin molecule.
This is consistent with rheological analysis of alkali-extracted proteins from dark chicken meat. This may be attributed to the transition temperature at 50.1 O C
to the denaturation of myosin. One minor transition point was observed for acid extracted samples at around 65 C, which corresponds to the denaturation point of collagen.
Above 35 C tan = values were found to be decreasing until the temperature reached 47 C for pH 2.5, 3.5, 11.5 and 52 C for the pH 10.5. In general, a decrease in tan indicates the formation of an ordered gel network. The use of tan = to estimate the gel characteristics has the advantage of incorporating the contributions of both G' and G' ' into a single parameter to evaluate the final network.

Storage modulus values of different protein isolates at various temperatures (5 0, 56.6 0 and 80 C) is given in Figure 12. The highest (P < 0.0001) G ' value (at 5 0, 56.6 0 and 80 C) was obtained for the sample extracted with pH 3.5. The lowest was observed with pH 10.5 extracted samples, while G' for proteins extracted at more extreme pH of 2.5 and 11.5 was not significantly different from each other.
The same trend was observed with increasing temperature to 56.6 C (the peak value for storage modulus). At 80 C protein extracted with pH 3.5 possessed significantly (P =
0.0005) higher G' compared to pH 2.5 and 11.5.

On cooling from 80 0 to 5 C, all samples showed an increase in G' and G' as interactions between the proteins become stronger with the decrease in temperature (Figure 13). However, a notable difference was observed for pH 10.5 extracted proteins, where G' and G' ' showed the lowest values. During cooling the highest value was reached at the end of the gelation process. The increase in storage and loss modulus is attributed to the formation of hydrogen bonds during cooling. High G' value during cooling is also an indication of the formation of a firm gel structure.
Conclusion The present study indicated that functional properties and rheological characteristics of MSTM could be greatly improved by extraction procedures.
Emulsion activity index of myofibrillar proteins was better at extraction pH
of 11.5.
Proteins extracted at pH 3.5 showed higher ability to form a viscoelastic gel network.
Acid extractions were more efficient in heme pigment removal, which resulted in better color characteristics than alkali treated samples. The study revealed that acid and alkaline processing can be the alternatives for recovering functional proteins from MSTM. In conclusion, proteins extracted at pH 3.5 were found to be the most suitable considering the rheological characteristics as well as pigment removal.

III. Preparation of Films Protein Solubilization and Extraction To purify myofibrillar protein isolates, certain amounts of ground chicken thigh meat or mechanically separated poultry meat was tempered at 4 C
overnight and then minced in a commercial-grade food processor with ice water. Figure 14 shows an exemplary method for solubilizing and isolating a poultry protein.
The resulting meat slurry was placed in a large mixing beaker and the pH was adjusted to pH 10.5 or 2.5 for protein solubilization using alkali (NaOH) or acids (HC1 and citric acid) with constant mixing (Figure 15). After 30 minutes, the entire contents of the mixing beaker were centrifuged at 13,000 rpm for 20 minutes. Three layers were formed after centrifugation: an upper fat layer, a middle aqueous layer of soluble myofibrillar protein and a bottom sediment layer (Figure 16). The middle protein supernatant layer was carefully removed and without further dewatering, the protein solution was used in the protein film formation.

Protein Film Formation Biopolymer films were prepared from casting a film-forming solution based on mechanically separated and thigh meat protein extracts resulting from the solubilization and extraction steps described above. The protein concentration of the film-forming solution was 1.0% and glycerol as a plasticizer was added at 50%
(w/w) of protein. The film forming solution was cast on to Teflon coated trays or silicone resin plates set on a level surface and air blown for 12 hours at room temperature prior to further drying in an environmental chamber at 25 C and 50% relative humidity for 24 hours. The resulting film was manually pealed off of the Teflon coated tray.
Figure 17 shows the resulting biopolymer film.

IV. Crosslinking of Protein Compositions With mechanical stirrer, 5.0% myofibrillar protein solution produced using the techniques described herein was prepared at pH 11.5. Into the sticky protein solutions, 1.0% of the following crosslinkers was added: formaldehyde, glutaraldehyde or glyoxal. After vigorous stirring, the gelation processes were observed. The use of glyoxal provided the best physical properties. The pH
used to form the protein gels were change from pH 11.5 to pH 7.5-8.5. Glyoxal crosslinked the myofibrillar protein to form strong gel network, which has applications for making films.
V. Temperature Effects on Film Production In order to improve the film performance, the temperature effect was evaluated on the formulation of 1.6% myofibrillar protein and 0.7% glycerol at pH
11.5. The results are shown in Tables 8-10. With the increase of temperature, water vapor permeability (WVP) increased and tensile strength (TS) decreased (Tables and 9). High temperatures did not promote protein crosslinking, but degraded the larger protein molecules to small ones. Therefore, a weaker film structure was formed and water vapor can readily pass through the film. Regarding the temperature influence on elongation at break of the films, no obvious pattern was seen (Table 10).

Table 8 Temperature effect on Water Vapor Permeability (WVP) T ( C) WVP (g/m per s per Pa) Stdev 22 8.97E-11 4.55E-12 45 8.80E-11 2.22E-12 70 9.16E-11 4.13E-12 90 1.09E-10 5.90E-12 Table 9 Temperature effect on Tensile Strength (TS) T ( C) TS (MPa) Stdev 22 3.46 0.82 45 3.32 0.58 70 2.85 0.70 90 1.96 0.52 Table 10 Temperature effect on Elongation at Break (EAB) T ( C) EAB (%) Stdev 22 132.6 43.3 45 116.4 49.8 70 125.3 52.0 90 87.3 47.1 VI. Preparation of Noodles using Protein Formulations An aqueous solution of 1.6% myofibrillar protein extracted from chicken thigh meat with 1.0% sodium alginate and 0.7% glycerol was prepared. Next, a 5.0%
calcium chloride solution was prepared. With the 30 ml syringe without needle, the homogenous thick protein solution was extruded into the calcium chloride solution to produce a high protein and alginate fiber enriched noodle. After water rinse, the noodle could be consumed wet or oven-dried depending on the end-use. The protein solution can contain various flavors and colorants.

It is to be understood that the above-described compositions and modes of application are only illustrative of preferred embodiments of the present invention.
Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements.
Thus, while the present invention has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, variations in size, materials, shape, form, function and manner of operation, assembly and use may be made without departing from the principles and concepts set forth herein.

Claims (27)

1. A composition comprising a poultry protein and a plasticizer.

2. The composition of claim 1, wherein the poultry protein comprises from 50 wt% to 70 wt% of the composition.

3. The composition in any preceding claim, wherein the poultry protein comprises an isolated poultry protein.

4. The composition in any preceding claim, wherein the poultry protein comprises a poultry myofibrillar protein.

5. The composition in any preceding claim, wherein the poultry protein comprises a chicken myofibrillar protein, a turkey myofibrillar protein, duck myofibrillar protein, an ostrich myofibrillar protein, quail myofibrillar protein, guinea fowl myofibrillar protein, geese myofibrillar protein, pigeon myofibrillar protein, swan myofibrillar protein, or any combination thereof.

6. The composition in any preceding claim, wherein the plasticizer comprises from 30 wt% to 40 wt% of the biopolymer film.

7. The composition in any preceding claim, wherein the plasticizer comprises glycerol, acetylated monoglycerides, trioctyl citrate, trihexyl citrate, sorbitol, polyethylene glycol 400, or any combination thereof.

8. The composition in any preceding claim, further comprising fish oil, flaxseed oil, or a combination thereof.

9. The composition of claim 8, wherein the fish oil, flaxseed oil, or a combination thereof comprise essential fatty acids.

10. The composition of claim 9, wherein the essential fatty acids comprise omega-3 fatty acids, omega-6 fatty acids, or a combination thereof.

11. The composition of claim 8, wherein the fish oil, flaxseed oil, or a combination thereof comprises 0.5 to 10 wt% of the composition.

12. The composition in any preceding claim, further comprising an anti-microbial agent, an anti-viral agent, an anti-oxidant, a release agent, a time-release agents, a colorant, a flavorant, a crosslinking agent, or any combination thereof.

13. The composition in any preceding claim, wherein the composition comprises a crosslinking agent, and the crosslinking agent comprises a transglutaminase, ferulic acid, glyoxal, glutaraldehyde, or any combination thereof.

14. The composition in any preceding claim, wherein the composition further comprises a lipid.

15. The use of the composition in any preceding claim to produce an edible film..

16. The use of the composition in any preceding claim to produce a biodegradable packaging film.

17. The use of the composition in any preceding claim to produce a poultry surimi.

18. The use of a meat protein to produce an extruded food article.

19. The use of claim 18, wherein the extruded food article is a noodle.

20. The use of a meat protein as a meat extender.

21. The use of claim 20, wherein meat extender is added to restructured meat products, comminuted meat products, emulsified meat products, marinated meat products, or any combination thereof.

22. The use of claim 20, wherein meat extender is injected into the meat.

23. The use of a meat protein as a nutritional supplement.

24. The use in any of claims 18-23, wherein the meat protein is a poultry protein, a fish protein, a porcine protein, a bovine protein, or any combination thereof.

25. The use in any of claims 18-23, wherein the meat protein is a poultry protein.

26. A composition comprising a meat protein and a crosslinker.

27. The composition of claim 26, wherein the meat protein is poultry protein and the crosslinker is glyoxal.