CN118119637A - Stable hemoglobin compositions and methods of use thereof - Google Patents

Abstract

The present disclosure provides novel compositions suitable for use in food products, wherein the compositions herein have improved color stability and reduced protein degradation over time. Embodiments disclosed herein provide compositions for non-animal food products, methods of making and methods of using the same, comprising one or more purified heme proteins and one or more antioxidants.

Description

Stable hemoglobin compositions and methods of use thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/271,423, filed on October 25, 2021, the entire contents of which are incorporated herein by reference.

Technical Field

Compositions for non-animal food products, methods of making and using the same, comprising a composition of one or more purified hemoglobins and one or more antioxidants.

Background technique

Consumer demand for alternatives to animal-based foods such as meat, eggs and milk continues to grow due to growing environmental, health and ethical concerns related to the raising and slaughter of livestock animals. To meet these demands, the food and biotechnology sectors have developed many innovative approaches to design animal-free food products; however, many non-animal alternatives to animal protein do not fully mimic the sensory and functional properties of animal protein.

The difficulty of producing non-animal food products at present includes producing products with appropriate color and taste characteristics. As an example, people are very interested in finding non-animal substitutes for hemoglobin (such as myoglobin) commonly found in meat, because these proteins provide ideal red and meaty taste comparable to traditional animal foods. The characteristic red color associated with hemoglobin is determined by the redox state of the protein, which depends on environmental conditions such as pH, ionic strength, temperature and oxygen level. The problem with isolating hemoglobin from plant sources or using cell agriculture methods (such as fermentation) to produce hemoglobin is that the resulting isolated hemoglobin is highly susceptible to chemical degradation during storage and use, and will soon lose the required red and taste characteristics after production. Therefore, it is necessary to develop a non-animal hemoglobin with increased stability, so as to extend its quality and shelf life while maintaining the color and taste characteristics of imitating animal foods (such as muscle meat products).

Summary of the invention

The present disclosure provides novel compositions suitable for use in food products, wherein the compositions herein have improved color stability and reduced protein degradation (eg, aggregation) over time.

Embodiments of the present disclosure provide compositions for use in food products, the compositions comprising one or more purified hemoglobin compositions and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a non-animal source.

In certain embodiments, one or more purified hemoglobin compositions herein may include globulins. In some embodiments, one or more purified hemoglobin compositions herein may include leghemoglobin, non-symbiotic hemoglobin, hemoglobin, erythrocruorin, protoglobulin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, phytoglobulin, or any combination thereof.

In certain embodiments, one or more purified hemoglobin compositions herein may include hemoglobin from a genetically modified non-animal source. In some embodiments, the genetically modified non-animal source herein may include a genetically modified plant, a genetically modified bacterium, a genetically modified yeast, or any combination thereof.

In some embodiments, the present disclosure comprises a hemoglobin composition for a food product, the hemoglobin composition comprising one or more purified hemoglobins and one or more antioxidants. In some embodiments, the one or more purified hemoglobins comprise globulins. In some aspects, the one or more purified hemoglobins comprise leghemoglobin, non-symbiotic hemoglobin, hemoglobin, heme, protoglobulin, cytochrome, cyanobacterial globulin, flavohemoglobin, myoglobin, plant globulin, or any combination thereof. In some embodiments, the hemoglobin composition comprises purified hemoglobin from a genetically modified source. In some aspects, the source comprises a genetically modified plant, a genetically modified bacteria, a genetically modified yeast, or any combination thereof. In some embodiments, the one or more purified hemoglobin compositions comprise polypeptides expressed and/or secreted by a source, wherein the source comprises a plant, a fungus, a bacteria, a yeast, an algae, an archaea, a genetically modified plant, a genetically modified fungus, a genetically modified bacteria, a genetically modified yeast, a genetically modified algae, a genetically modified archaea, or any combination thereof. In some aspects, the polynucleotide sequence encoding the hemoglobin is derived from an animal, a plant, a fungus, a bacterium, a yeast, an algae, an archaea, or any combination thereof.

In certain embodiments, one or more antioxidants herein may include antioxidant vitamins, polyphenols, or any combination thereof. In certain embodiments, one or more antioxidants herein may include vitamin C, vitamin E, any derivative thereof, or any combination thereof. In certain embodiments, one or more antioxidants herein may include flavonoids, or any derivative thereof. In certain embodiments, one or more antioxidants herein may include isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, taxifolin, catechin, gallocatechin, gallocatechin gallate, epicatechin, epigallocatechin, epigallocatechin gallate, theaflavins, theaflavins gallate, thearubigins, or any combination thereof. In certain embodiments, one or more antioxidants herein may have a reduction potential less than about 500mV.

In certain embodiments, the compositions herein may comprise a total amount of one or more purified hemoglobins to a total amount of one or more antioxidants in a ratio by weight of about 1:1 to about 30:1, e.g., about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, or about 30:1. In certain embodiments, the hemoglobin compositions herein may be stable at about 4°C for at least 7 days.

In certain embodiments, the hemoglobin composition herein may include one or more hemoglobins herein, which may include a heme group bound to oxygen, a heme group bound to carbon monoxide, or a combination thereof, for at least 7 days. In certain embodiments, the hemoglobin composition results in an increase in the peak height of the UV-visible absorption spectrum for the hemoglobin composition at one or more of about 550nm and about 582nm. In some embodiments, an increase in the peak height of the UV-visible absorption spectrum for one or more hemoglobin compositions can be detected at one or more of about 550nm and about 582nm for at least about 7 days after adding one or more antioxidants.

In certain embodiments, the hemoglobin compositions herein may include one or more purified hemoglobins from a genetically modified non-animal source and one or more antioxidants herein that stabilize the oxidative state of the one or more purified hemoglobins. In some embodiments, the hemoglobin compositions herein may include one or more purified hemoglobins from a genetically modified non-animal source (such as a genetically modified plant, a genetically modified bacterium, or a genetically modified yeast) and one or more antioxidants herein that stabilize the oxidative state of the one or more purified hemoglobins.

In certain embodiments, one or more purified hemoglobins herein can be recombinant hemoglobins produced from non-animal sources of genetic modification. In certain embodiments, the recombinant hemoglobin herein can be encoded by a polynucleotide, wherein the polynucleotide can include a nucleic acid sequence from a plant, animal, fungus or bacterium encoding hemoglobin. In certain embodiments, the nucleic acid sequence encoding the hemoglobin herein can be derived from a leguminous plant. In certain embodiments, the nucleic acid sequence encoding the hemoglobin herein can be derived from a leopard, a cow or a whale. In certain embodiments, the recombinant hemoglobin herein can be a recombinant globulin. In certain embodiments, the recombinant hemoglobin herein can be a recombinant leghemoglobin, a non-symbiotic hemoglobin, a hemoglobin, a heme, a protoglobulin, a cytochrome, a cyanobacterial globulin, a flavohemoglobin, a myoglobin, a plant globulin or any combination thereof.

In certain embodiments, one or more purified hemoglobins herein may be recombinant hemoglobin produced from a genetically modified yeast source, wherein the recombinant hemoglobin may be encoded by a polynucleotide, wherein the polynucleotide may comprise an endogenous nucleic acid sequence of hemoglobin derived from a legume, horse, leopard, cow, whale, or any combination thereof.

Other embodiments of the present disclosure provide methods for preparing any hemoglobin composition disclosed herein. In certain embodiments, the methods herein can stabilize the oxidation state of one or more purified hemoglobins from non-animal sources herein by combining one or more purified hemoglobins with one or more antioxidants. In some embodiments, the methods herein can stabilize the visual appearance of purified hemoglobin for at least 7 days. In some embodiments, the methods herein can stabilize purified hemoglobin to prevent it from aggregating at a pH ranging from about 5 to about 9. In some embodiments, the methods herein can stabilize the oxidation state of purified hemoglobin for at least 7 days. In certain embodiments, the methods for preparing the hemoglobin compositions herein for use in food products may include obtaining one or more purified hemoglobins from non-animal sources, and combining the one or more purified hemoglobins with at least one or more antioxidants herein. In some embodiments, the methods herein may include recombinantly producing one or more purified hemoglobins from genetically modified non-animal sources. In some embodiments, the methods herein may include combining the hemoglobin herein with one or more antioxidants at about 0.01% to about 10% by weight of the composition. In some embodiments, the methods herein may include combining the antioxidants herein with about 1% to about 99% of one or more purified hemoglobins by weight of the composition. In some embodiments, the methods herein may include combining the antioxidants herein with one or more purified hemoglobins, wherein the hemoglobin composition results in an increase in the peak height of the UV-visible absorption spectrum for one or more hemoglobin compositions detectable at one or more wavelengths of about 550nm and about 582nm for at least about 7 days after adding one or more antioxidants. In some embodiments, the methods herein may further include combining the hemoglobin and antioxidants herein in a buffer solution, wherein the buffer solution may have a pH ranging from about 5 to about 9.

In certain embodiments, the present disclosure also encompasses compositions comprising one or more purified hemoglobin compositions and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a genetically modified non-animal source selected from the group consisting of: a genetically modified plant, a genetically modified bacterium, or a genetically modified yeast; and wherein the composition results in an increase in the peak height of the UV-visible absorption spectrum for one or more hemoglobin compositions detectable at one or more of about 550 nm and about 582 nm for at least about 7 days after the addition of the one or more antioxidants. In certain embodiments, the one or more antioxidants have a reduction potential ranging from about 280 mV to about 500 mV. In certain embodiments, the one or more antioxidants are selected from ascorbic acid, quercetin, taxifolin, Trolox, and combinations thereof. In certain embodiments, the composition comprises a weight ratio of one or more purified hemoglobin to one or more antioxidants of about 1:1 to about 30:1 (e.g., about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, or about 30:1).

In certain embodiments, the hemoglobin of the composition comprising the hemoglobin composition is encoded by a polynucleotide, wherein the polynucleotide comprises an endogenous nucleic acid sequence of hemoglobin derived from a plant, animal, fungal or bacterial source. In certain embodiments, the endogenous nucleic acid sequence of hemoglobin is derived from a leguminous plant. In certain embodiments, the endogenous nucleic acid sequence of hemoglobin is derived from an animal source selected from the group consisting of: horse, leopard, cattle or whale. In certain embodiments, the hemoglobin is a globulin selected from the group consisting of: leghemoglobin, non-symbiotic hemoglobin, hemoglobin, heme, protoglobulin, cytochrome, cyanobacterial globulin, flavohemoglobin, myoglobin and plant globulin. In certain embodiments, wherein the one or more purified hemoglobins are recombinant hemoglobins produced by a genetically modified yeast source, wherein the recombinant hemoglobin is encoded by a polynucleotide, wherein the polynucleotide comprises an endogenous nucleic acid sequence for hemoglobin derived from a leguminous plant, horse, leopard, cattle, whale or any combination thereof. In certain embodiments, the hemoglobin composition comprises myoglobin, and wherein combining one or more purified hemoglobin compositions with one or more antioxidants causes an increase in the relative amounts of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition. In certain embodiments, the increase in the relative amounts of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition is detectable for at least about 7 days after the addition of the antioxidant. In certain embodiments, the increase in the relative amounts of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition ranges from about 1.1 times to about 5 times.

In certain embodiments, the present disclosure encompasses a hemoglobin composition comprising one or more purified hemoglobins and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a genetically modified source selected from the group consisting of: a genetically modified plant, a genetically modified bacteria, a genetically modified fungus, or a genetically modified yeast; and wherein the composition results in an increase in the peak height of the UV-visible absorbance spectrum for the one or more hemoglobin compositions at one or more of about 550 nm and about 582 nm detectable for at least about 7 days after the addition of the one or more antioxidants.

In some embodiments, hemoglobin is encoded by a polynucleotide comprising a nucleic acid sequence from a plant, an animal, a fungus, or a bacterium. In some embodiments, the polynucleotide comprises a nucleic acid sequence derived from a legume, wherein the nucleic acid sequence encodes hemoglobin. In some embodiments, the polynucleotide sequence comprises a nucleic acid sequence derived from the group consisting of: a horse, a cat, a cow, or a whale. In some embodiments, hemoglobin is a globulin selected from the group consisting of: leghemoglobin, non-symbiotic hemoglobin, hemoglobin, heme, protoglobulin, cytochrome, cyanobacterial globulin, flavohemoglobin, myoglobin, and plant globulin. In some embodiments, one or more purified hemoglobins are recombinant hemoglobins produced by a genetically modified yeast source, wherein the recombinant hemoglobin is encoded by a polynucleotide comprising a nucleic acid sequence derived from a legume, a horse, a leopard, a cow, a whale, and encoding hemoglobin.

In certain embodiments, the present disclosure also encompasses a composition comprising one or more purified hemoglobin compositions and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise: leghemoglobin, non-symbiotic hemoglobin, hemoglobin, heme, protoglobulin, cytochrome, cyanophycoglobulin, flavohemoglobin, myoglobin, phytoglobulin, or any combination thereof; wherein the one or more antioxidants are selected from the group consisting of ascorbic acid, quercetin, taxifolin, Trolox, and combinations thereof; wherein the composition comprises a ratio of about 1:1 to about 30:1 (e.g., about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1 about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1 or about 30:1), and wherein the composition results in an increase in the peak height of the UV-visible absorbance spectrum for the one or more hemoglobin compositions at one or more of about 550 nm and about 582 nm that is detectable for at least about 7 days after the addition of the one or more antioxidants.

Other embodiments of the present disclosure include meat substitute (e.g., meat replica) compositions and methods of making the same. In some embodiments, the method of making a meat substitute comprises combining any composition herein with a meat replica matrix. In some embodiments, the method comprising combining any composition herein with a meat replica matrix enables the composition to impart a meat-like (e.g., beef-like) appearance to the meat substitute herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application document contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. The following drawings form a part of this specification and are included to further illustrate certain embodiments of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. Embodiments of the present inventive concept are described by way of example, wherein like reference numerals represent similar elements, and are as follows.

FIG1 depicts a representative graph of the zeta-potential versus pH curve of horse heart myoglobin (1 mg/mL) measured by electrophoresis.

FIG2 depicts a representative graph of the protein content of horse heart myoglobin (1 mg/mL) at pH 2.5 to 8.5.

FIG. 3A depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of a horse heart myoglobin solution (~1 mg/mL) stored at 4°C for 0 days.

3B depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of horse heart myoglobin solutions (~1 mg/mL) stored at 4°C for 1 day.

FIG. 3C depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of horse heart myoglobin solutions (~1 mg/mL) stored at 4°C for 2 days.

FIG3D depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of horse heart myoglobin solutions (~1 mg/mL) stored at 4°C for 3 days.

FIG. 3E depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of horse heart myoglobin solutions (~1 mg/mL) stored at 4°C for 4 days.

3F depicts representative graphs of the absorbance spectrum and representative images of the visual appearance of horse heart myoglobin solutions (~1 mg/mL) stored at 4°C for 5 days.

FIG. 4A depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solutions combined with (1 mM) ascorbic acid and stored at 4° C. (days 0 to 27 as indicated).

4B depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of samples of horse heart myoglobin solution combined with (1 mM) ascorbic acid and stored at 4° C. over time (from day 0 to day 27 as shown).

4C is a series of representative images of the visual appearance of samples of horse heart myoglobin solutions combined with (1 mM) ascorbic acid and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG4D depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solutions stored at 4° C. in combination with (1 mM) quercetin (from day 0 to day 27 as shown).

4E depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of horse heart myoglobin solution samples bound to (1 mM) quercetin and stored at 4° C. over time (from day 0 to day 27 as shown).

4F is a series of representative images of the visual appearance of samples of horse heart myoglobin solutions combined with (1 mM) quercetin and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG. 4G depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solution (1 mM) bound to epigallocatechin gallate (EGCG) (1 mM) and stored at 4° C. (days 0 to 27 as shown).

FIG. 4H depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of horse heart myoglobin solution samples combined with (1 mM) EGCG and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG4I depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solutions stored at 4° C. (days 0 to 27 as indicated) bound to (1 mM) taxifolin.

FIG. 4J depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of horse heart myoglobin solution samples bound to (1 mM) taxifolin and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG. 4K depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solutions stored at 4° C. in combination with (1 mM) 4-methylcatechol (days 0 to 27 as indicated).

4L depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of horse heart myoglobin solution samples bound to (1 mM) 4-methylcatechol and stored at 4° C. over time (from day 0 to day 27 as shown).

4M is a series of representative images of the visual appearance of samples of horse heart myoglobin solutions (1 mM) stored at 4° C. combined with the antioxidants EGCG, taxifolin, and methylcatechol, respectively, over time (from day 0 to day 27 as shown).

FIG. 4N depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solution (1 mM) combined with Trolox and stored at 4° C. (days 0 to 27 as shown).

FIG. 4O depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of samples of horse heart myoglobin solution (1 mM) combined with Trolox and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG. 4P depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solution (1 mM) bound to caffeic acid and stored at 4° C. (days 0 to 27 as shown).

FIG. 4Q depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of samples of horse heart myoglobin solution (1 mM) conjugated with caffeic acid and stored at 4° C. over time (from day 0 to day 27 as shown).

FIG. 4R depicts representative graphs of absorbance spectra of samples of horse heart myoglobin solution (1 mM) conjugated to gallic acid and stored at 4° C. (days 0 to 27 as shown).

FIG. 4S depicts representative graphs of the absorbance spectral intensity (at 544 nm and 582 nm) of samples of horse heart myoglobin solution (1 mM) conjugated to gallic acid and stored at 4° C. over time (from day 0 to day 27 as shown).

4T is a series of representative images of the visual appearance of samples of horse heart myoglobin solutions (1 mM) stored at 4° C. combined with the antioxidants Trolox, caffeic acid, and gallic acid, respectively, over time (from day 0 to day 27 as shown).

FIG5A depicts representative graphs of the zeta potential of recombinant leopard, bovine, whale, and soy leghemoglobin samples in solutions at pH 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, and 8.5.

5B is a series of representative images of the visual appearance of recombinant leopard, bovine, whale, and soy leghemoglobin samples in solutions at pH 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, and 8.5.

6 depicts representative graphs showing the effect of pH on the solubility of myoglobin solutions (0.5 mg/mL) produced by cell agriculture.

7A depicts a representative graph of the absorbance spectrum of a 1 mg/mL solution of recombinant hemoglobin from Panthera pardus (leopard) stored at 4°C for 27 days.

7B depicts a representative graph of the absorbance spectrum of a 5 mg/mL solution of recombinant hemoglobin from Panthera pardus (leopard) stored at 4° C. for 27 days.

7C is a series of representative images of the visual appearance of 1 mg/mL and 5 mg/mL solutions of recombinant hemoglobin from Panthera pardus (leopard) stored at 4°C for 27 days.

7D depicts a representative graph of the absorbance spectrum of a 1 mg/mL solution of recombinant hemoglobin from Bos taurus (cattle) stored at 4° C. for 27 days.

7E depicts a representative graph of the absorbance spectrum of a 5 mg/mL solution of recombinant hemoglobin from bovine (Bos calves) stored at 4° C. for 27 days.

7F is a series of representative images of the visual appearance of 1 mg/mL and 5 mg/mL solutions of recombinant hemoglobin from bovine (Bovine) stored at 4°C for 27 days.

7G depicts a representative graph of the absorbance spectrum of a 1 mg/mL solution of recombinant hemoglobin from sperm whale (Physeter macrocephalus) (shot whale) stored at 4°C for 27 days.

7H depicts a representative graph of the absorbance spectrum of a 5 mg/mL solution of recombinant hemoglobin from sperm whale (Pithecus cephalus) stored at 4° C. for 27 days.

71 is a series of representative images of the visual appearance of 1 mg/mL and 5 mg/mL solutions of recombinant hemoglobin from sperm whales (Pigeon whales) stored at 4°C for 27 days.

7J depicts a representative graph of the absorbance spectrum of a 1 mg/mL solution of recombinant hemoglobin from soy leghemoglobin stored at 4° C. for 27 days.

7K depicts a representative graph of the absorbance spectrum of a 5 mg/mL solution of recombinant hemoglobin from soy leghemoglobin stored at 4° C. for 27 days.

7L is a series of representative images of the visual appearance of 1 mg/mL and 5 mg/mL solutions of recombinant hemoglobin from soy leghemoglobin stored at 4°C for 27 days.

Detailed ways

The following detailed description refers to the accompanying drawings showing various embodiments of the inventive concept. The drawings and description are intended to describe the embodiments and embodiments of the inventive concept in sufficient detail to enable those skilled in the art to practice the inventive concept. Other components may be utilized and changes may be made without departing from the scope of the inventive concept. Therefore, the following description should not be considered restrictive. The scope of the inventive concept is limited only by the appended claims and the full scope of equivalents to which these claims are entitled.

I. Terminology

The words and terms used herein are for illustrative purposes and should not be considered limiting. For example, the use of singular terms (such as "a") is not intended to limit the number of items. In addition, the use of relational terms such as, but not limited to, "top", "bottom", "left", "right", "above", "below", "below", "above", and "side" in the specification is intended to clarify the specific reference to the drawings and is not intended to limit the scope of the inventive concept or the appended claims.

In addition, since the inventive concept is susceptible to many different forms of embodiments, the present disclosure is intended to be considered as an example of the principles of the inventive concept and is not intended to limit the inventive concept to the specific embodiments shown and described. Any feature of the inventive concept may be used alone or in combination with any other feature. References to the terms "one embodiment", "multiple embodiments" and/or similar references in the specification refer to the one and/or multiple features mentioned being included in at least one aspect of the specification. Separate references to the terms "one embodiment", "multiple embodiments" and/or similar terms in the specification do not necessarily refer to the same embodiment, and are not mutually exclusive unless otherwise specified and/or it is easy for a person skilled in the art to see from the description. For example, the features, structures, processes, steps, actions, etc. described in one embodiment may also be included in other embodiments, but are not necessarily included. Therefore, the inventive concept may include various combinations and/or integrations of the embodiments described herein. In addition, all embodiments of the disclosure described herein are not necessary for its practice. Similarly, other systems, methods, features and advantages of the inventive concept will be or become apparent to those skilled in the art when examining the following drawings and detailed descriptions. All such additional systems, methods, features and advantages are intended to be included in this specification, within the scope of the inventive concept, and covered by the claims.

As used herein, the term "about" can mean ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a recited value (e.g., amount, dosage, temperature, time, percentage, etc.).

The terms "comprising," "including," "covering," and "having" are used interchangeably in this disclosure. The terms "comprising," "including," "covering," and "having" mean including but not necessarily limited to the things so described.

As used herein, the terms "or" and "and/or" should be interpreted as inclusive or meaning any one or any combination. Thus, "A, B or C" or "A, B and/or C" means any of the following: "A", "B" or "C"; "A and B"; "A and C"; "B and C"; "A, B and C". An exception to this definition will only occur if the combination of elements, functions, steps or actions is inherently mutually exclusive to some extent.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

As used herein, "recombinant" refers to cells, nucleic acids, proteins or vectors that have been modified due to the introduction of exogenous nucleic acids or the alteration of native nucleic acids. Nucleic acids can be of genomic, cDNA, semisynthetic and/or synthetic origin, which, by virtue of their origin or manipulation, are not related to all or part of the polynucleotides with which they are naturally associated.

"Transformation" refers to transferring a nucleic acid fragment to a host organism or the genome of a host organism, thereby producing genetically stable inheritance. Host organisms containing transformed nucleic acid fragments are referred to as "recombinant," "transgenic," or "transformed" organisms. Therefore, the isolated polynucleotides of the present invention can be incorporated into a recombinant construct, typically a DNA construct, which can be introduced into a host cell and replicated therein. Such a construct can be a vector comprising a replication system and a sequence capable of transcribing and translating a polypeptide coding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and selectable markers. These vectors can also contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmental or developmental regulation, or position-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.

"Stabilization" or "stabilizing" in the context of the hemoglobin compositions provided herein refers to a composition in which the combination of hemoglobin and an antioxidant causes a sustained increase in the level of oxygenated hemoglobin or carboxyhemoglobin, as indicated by a change in visual appearance, a redder composition, a change in oxidation state (from Met state to oxygen or carbon monoxide binding state), or a change in the UV-visible spectrum, such that the peak height at 550nm (corresponding to CarboxyMb) and/or at 582nm (corresponding to OxyMb) increases compared to a composition without an antioxidant. In some aspects, an increase in peak height can be detected within 1-48 hours after the antioxidant is combined with hemoglobin. This period between the addition of an antioxidant and the increase in detectable peak height is referred to as a lag time or a lag phase. In some aspects, depending on the antioxidant used and storage conditions, the increase in peak height can be sustained or "stabilized" to maintain up to about 0.5 days to about 90 days, although in some embodiments, the absolute peak height can be reduced by this stabilization period.

"Cellular agriculture" is a method of producing animal products from cell cultures rather than using animals that combines biotechnology, tissue engineering, molecular biology, and synthetic biology to create and engineer new methods of producing proteins, fats, and tissues that would otherwise come from traditional agriculture. In the context of this application, in some aspects, hemoglobin can be derived from cellular agriculture and purified.

As used herein, a polynucleotide encoding a hemoglobin that is "derived from" an endogenous polynucleotide or is a "derivative" of an endogenous polynucleotide refers to a polynucleotide that is related in sequence to an endogenous polynucleotide. In some aspects, the polynucleotide may be a variant of an endogenous polynucleotide or a fragment thereof, and may comprise a mutation, insertion, deletion, truncation, modification, or a combination thereof compared to an endogenous polynucleotide. In some aspects, the polynucleotide may comprise a nucleic acid sequence that is at least about 60% identical to an endogenous polynucleotide encoding hemoglobin or a fragment thereof.

As used herein, the term "source" refers to an organism comprising a polynucleotide sequence encoding an endogenous or recombinant hemoglobin that can be purified for use in the compositions and methods disclosed herein. In some aspects, the source may comprise a polynucleotide sequence encoding an endogenous hemoglobin, such as Glycine max (soybean) comprising a polynucleotide sequence encoding soy leghemoglobin that can be purified and used in the compositions and methods disclosed herein. In some aspects, the source may be a genetically modified source. As used herein, the term "genetically modified source" refers to a recombinant organism, such as a genetically modified plant, a genetically modified fungus, a genetically modified bacteria, a genetically modified yeast, a genetically modified algae, a genetically modified archaea, which comprises a polynucleotide sequence encoding hemoglobin and from which hemoglobin can be purified for use in the compositions and methods disclosed herein. In some aspects, the recombinant organism comprises a polynucleotide sequence having at least about 60% identity to an endogenous polynucleotide from a plant or a fragment thereof, or having at least about 60% identity to an endogenous polynucleotide from a cow, horse, cat or whale or a fragment thereof.

It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described unless explicitly stated to the contrary.

II. Composition

The present disclosure provides hemoglobin compositions suitable for food products with improved color stability and reduced protein degradation over time. In some embodiments, the present disclosure results from the surprising result that adding certain antioxidants to these compositions greatly increases the desired properties of hemoglobin. In some embodiments, the compositions herein may have one or more purified hemoglobins and one or more antioxidants.

A. Hemoglobin

In certain embodiments, the hemoglobin compositions suitable for use in food products herein can have one or more purified hemoglobins. As used herein, the term "hemoglobin" includes any polypeptide that can be covalently or non-covalently bound to a heme moiety. In certain embodiments, the hemoglobin herein can be a monomer (i.e., a single polypeptide chain), a dimer, a trimer, a tetramer, a higher order oligomer, or any combination thereof.

In certain embodiments, the hemoglobin herein can be a globulin. The non-limiting example of the globulin that can be covalently or non-covalently bound to the heme part used herein can include male globulin, cell globulin, globulin E, globulin X, globulin Y, hemoglobin, myoglobin, heme, beta hemoglobin, alpha hemoglobin, protoglobulin, cyanobacterial globulin, histamine globulin, neuroglobulin, hemoglobin, truncated hemoglobin (e.g., HbN, HbO, truncated 2/2 globulin, hemoglobin 3 (e.g., Glb3)), cytochrome, or peroxidase. According to certain embodiments herein, globulin can have a globulin fold with a series of about seven to about nine alpha spirals. According to certain embodiments herein, globulin can belong to any category (e.g., Class I, Class II or Class III). According to certain embodiments herein, globulin can transport and/or store oxygen.

In some embodiments, the hemoglobin herein may have an oxidized Fe ⁺ state similar to that of globulin (e.g., myoglobin). In some embodiments, the hemoglobin herein may have an oxidized Fe (iron) ⁺ state higher than that of globulin (e.g., myoglobin). In some embodiments, the hemoglobin herein may have an oxidized Fe+ state of about 10%, 20%, 30%, 40%, 50%, 100% or more compared to globulin ( ^e.g. , myoglobin). In some embodiments, the hemoglobin herein may be similar to oxygenated myoglobin. As used herein, "oxygenated myoglobin" refers to an oxygenated form of myoglobin, which is a single-chain globular protein.

In some embodiments, the hemoglobin herein can be non-symbiotic hemoglobin, leghemoglobin, hemoglobin, heme, protoglobulin, cytochrome, cyanophycoglobulin, flavohemoglobin, myoglobin, phytoglobulin, or any combination thereof.

In some embodiments, the hemoglobin herein can be derived from a non-animal source. Non-limiting examples of non-animal sources include plants, fungi, bacteria, yeast, algae, archaea, genetically modified organisms (such as genetically modified bacteria, plants or yeast), chemical or in vitro synthesizers. In some embodiments, the hemoglobin herein can be a polypeptide derived from a non-animal source. In some embodiments, the hemoglobin herein can be a polypeptide expressed and/or secreted from a non-animal source. In some embodiments, the hemoglobin herein can be a polypeptide expressed and/or secreted from a non-animal source, wherein the polypeptide can be encoded by a polynucleotide derived from an animal, a plant, a fungi, a bacteria, a yeast, an algae, an archaea, or any combination thereof.

In some embodiments, the hemoglobin herein can be isolated from or can be encoded by a polynucleotide derived from a "wild-type" source. The wild-type source of the hemoglobin herein can be a mammal, a fish, a bird, a plant, an algae, a fungus (e.g., a yeast or a filamentous fungus), a ciliate, a bacterium, or any combination thereof.

In some embodiments, the hemoglobin herein can be isolated from or can be encoded by a polynucleotide derived from a mammal belonging to any of 27 mammalian orders, including: Mustela; Carnivora; Artiodactyla; Chiroptera; Hypocrites; Quollida; Dermoptera; Opossums; Diprotodonta; Eulipotyphla; Hyraxes; Lagomorpha; Elephantiformes; Microtheria; Monotremes; Marmosets; Ocellaris; Bandicoots; Perissodactyla; Pholidota; Phalaropoda; Primates; Proboscidea; Rodentia; Tree shrews; Sirenia; and Tubulodonta.

In some embodiments, the hemoglobin herein can be isolated from or can be encoded by a polynucleotide derived from: a human, a non-human primate (e.g., a gibbon, a rhesus monkey, a bonobo, a chimpanzee, a gorilla, an orangutan, a lemur, a slow loris, a tarsier), a bovine (e.g., a cow, a zebu, a bison, a buffalo, a cape buffalo, an antelope), a sheep, a goat, a camel, a canine (e.g., a domestic dog, a wolf, a coyote, a jackal, a fox), a cetacean (e.g., a whale, a dolphin, a porpoise), a feline (e.g., a house cat, a tiger, a lion, a cheetah, a leopard, a jaguar, a bobcat, a wild cat, a margay, a cougar, a serval, a ocelot, a bobcat, a mountain lion), an equine (e.g., a horse, a donkey, a mule, a zebra), a marsupial, or from any other mammal of interest. In some embodiments, the hemoglobin herein can be isolated from or can be encoded by a polynucleotide derived from a mammal, such as a cow, goat, sheep, horse, pig, cattle, mule, rabbit, yak, llama, camel, deer, cat, dog, bear, or any combination thereof.

In some embodiments, the hemoglobin herein can be isolated from or encoded by a polynucleotide derived from a bird. In some embodiments, the hemoglobin herein can be isolated from or encoded by a polynucleotide derived from an Anseriformes (e.g., ducks, swans, geese), Falconiformes (e.g., falcons, eagles, hawks), Galliformes (e.g., chickens, turkeys, pheasants), Struthiformes (e.g., emu, ostrich, kiwi), Passeriformes (e.g., perching birds and songbirds, such as sparrows, larks, crows, swallows, etc.), Sphenisciformes (e.g., penguins), Pelecaniformes (e.g., ibis, herons, pelicans), Strigiformes (e.g., owls), Loons (e.g., loons), Gruiformes (e.g., terrestrial, marsh birds), or any combination thereof.

In some embodiments, the hemoglobin herein can be isolated from a polynucleotide derived from a fish or can be encoded by a polynucleotide derived from a fish. In some embodiments, the hemoglobin herein can be isolated from a polynucleotide or can be encoded by a polynucleotide derived from Scombridae (e.g., tuna), Salmonidae (e.g., salmon), Gadidae (e.g., cod, haddock), Herringidae (e.g., herring, shad, sardine, hilsa, herring), Anchovy (e.g., anchovy), etc. In some embodiments, the hemoglobin herein can be isolated from a polynucleotide or can be encoded by a polynucleotide derived from shrimp, oysters, clams, mussels, etc.

In some embodiments, the hemoglobin herein can be isolated from a polynucleotide derived from a plant or can be encoded by a polynucleotide derived from a plant. In some embodiments, the hemoglobin herein can be isolated from a polynucleotide or can be encoded by a polynucleotide derived from Nicotiana tabacum or Nicotiana sphaerocephala (tobacco); Zea mays (corn), Arabidopsis thaliana, leguminous plants such as soybean (Glycine max) (soybean), chickpea (chick pea or chickpea), pea (pea) varieties such as field pea or sweet snap bean, bean varieties of common beans such as green bean, black bean, white kidney bean, northern bean or piento bean, cowpea varieties (cowpea), mung bean (mung bean), white lupin (lupinus), or alfalfa (alfalfa); Brassica napus (rapa); Triticum spp. (wheat, including wheatberry and spelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania latifolia (wild rice); Helianthus annuus (sunflower); Beta vulgaris (sugar beet); Echinops paniculata (pearl millet); Chenopodium album (quinoa); Sesame (sesame); Flax (flax); or Hordeum vulgare (barley).

In some embodiments, the hemoglobin herein can be isolated from a polynucleotide derived from a fungus or can be encoded by a polynucleotide derived from a fungus. In some embodiments, the hemoglobin herein can be isolated from a polynucleotide or can be encoded by a polynucleotide derived from Saccharomyces cerevisiae, Pichia pastoris, Pyricularia oryzae, Fusarium or Fusarium fussii.

In some embodiments, the hemoglobin herein can be separated from or encoded by polynucleotides derived from bacteria. In some embodiments, the hemoglobin herein can be separated from or encoded by polynucleotides derived from bacteria. In some embodiments, the hemoglobin herein can be separated from or encoded by polynucleotides derived from Escherichia coli, Bacillus subtilis, Synechocystis, hyperthermophiles, Methylacidiphiluminfernorum, or thermophilic bacteria such as Streptococcus thermophilus.

In some embodiments, the hemoglobin herein can be isolated from or encoded by a polynucleotide derived from a non-symbiotic hemoglobin. In some embodiments, the hemoglobin herein can be a non-symbiotic hemoglobin isolated from or encoded by a polynucleotide derived from soybean, sprouted soybean, alfalfa, golden flax, black bean, black-eyed pea, northern bean, chickpea, mung bean, cowpea, pinto bean, pea pod, quinoa, sesame, sunflower, wheat berry, spelt, barley, wild rice, rice, or any combination thereof.

In some embodiments, the hemoglobin described herein may have an amino acid sequence corresponding to a wild-type hemoglobin, a fragment thereof, a truncation, a variant, or a fusion thereof containing a heme binding motif. It will be appreciated by those skilled in the art that the amino acid sequence of any wild-type hemoglobin considered herein can be found in sequence databases such as, but not limited to, the UniProtKB/Swiss-Prot database and the hemoglobin database. In some embodiments, the hemoglobin described herein may have at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) sequence identity with an amino acid sequence corresponding to a wild-type hemoglobin, a fragment thereof, a truncation, a variant, or a fusion thereof containing a heme binding motif. It will be appreciated by those skilled in the art how to determine the percentage identity between two amino acid sequences using methods known in the art. Such methods include, but are not limited to, using the BLAST 2 sequence (Bl2seq) program provided by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), etc.

In certain embodiments, the hemoglobin herein can be from a non-animal source of genetic modification. According to certain embodiments herein, the non-animal source of genetic modification can be a genetically modified plant, a genetically modified bacterium, a genetically modified yeast, or any combination thereof. In certain embodiments, the hemoglobin herein can be a recombinant hemoglobin. As used herein, "recombinant hemoglobin" refers to a hemoglobin produced recombinantly using polypeptide expression technology (e.g., using bacterial cells, insect cells, fungal cells such as yeast, plant cells such as tobacco, soybeans, or Arabidopsis, or heterologous expression technology of mammalian cells). In certain embodiments, the recombinant hemoglobin herein can be a polypeptide encoded by a polynucleotide, wherein the polynucleotide can have an endogenous (i.e., wild-type) nucleic acid sequence derived from a hemoglobin of a plant, animal, fish, bird, fungus, or bacterial origin as described herein. According to certain embodiments herein, the endogenous nucleic acid sequence of hemoglobin can be derived from a plant. According to certain embodiments herein, the endogenous nucleic acid sequence of hemoglobin can be derived from a leguminous plant. According to certain embodiments herein, the endogenous nucleic acid sequence of hemoglobin can be derived from a mammal. According to certain embodiments herein, the endogenous nucleic acid sequence of hemoglobin may be derived from a horse, a feline (eg, a leopard), a cow, or a cetacean (eg, a whale).

In some embodiments, standard polypeptide synthesis techniques (e.g., liquid phase polypeptide synthesis techniques or solid phase polypeptide synthesis techniques) can be used to produce any recombinant hemoglobin herein. In some embodiments, in vitro transcription-translation techniques can be used to produce any recombinant hemoglobin herein.

In some embodiments, recombinant hemoglobin can be expressed by a microbial expression system. In some embodiments, the microbial expression system used herein can include at least one expression vector. Microbial expression systems and expression vectors containing regulatory sequences for directing high-level expression of recombinant proteins are well known to those skilled in the art, any of which can be used to produce any gene product (e.g., recombinant hemoglobin) of the polynucleotides disclosed herein. Vectors or boxes for transformation of suitable host cells are well known in the art. "Expression vector" or "expression construct" or "plasmid" or "recombinant DNA construct" refers to a vector for introducing nucleic acid into a host cell. Nucleic acids used herein can be nucleic acids produced via human intervention, including by recombinant means or direct chemical synthesis, with a series of specific nucleic acid elements that allow transcription and/or translation of specific nucleic acids. In some embodiments, the expression vector used herein can be a part of a plasmid, a virus or a nucleic acid fragment or other suitable vector. In some embodiments, the expression vector used herein can also include a nucleic acid to be transcribed that is operably connected to a promoter.

According to certain embodiments herein, a vector comprising a polynucleotide disclosed herein can be introduced into an appropriate microorganism (i.e., a host cell) via a transformation technique to provide a high level of expression of the recombinant hemoglobin used herein. The expression of the polypeptide disclosed herein (e.g., recombinant hemoglobin) can include transient expression and/or constitutive expression (e.g., the development of a stable cell line) in a suitable host cell. The host cell herein can be transformed by any suitable technique, including, for example, a gene gun, electroporation, glass bead transformation, and silicon carbide whisker transformation. Any convenient technique for introducing a transgenic into a microorganism can be used in the present invention. Transformation can be achieved by, for example, the method of D.M.Morrison (Methods in Enzymology 68, 326 (1979)), which increases the permeability of the receptor cell to DNA with calcium chloride (Mandel.M. and Higa, A., J.Mol.Biol., 53, 159 (1970)), etc.

In some embodiments, suitable host cells for producing the recombinant hemoglobin herein can be from genetically modified organisms. In certain embodiments, the genetically modified organisms herein can be bacteria, yeast, fungi, algae, mammalian cells, insect cells, or any combination thereof. In some embodiments, suitable host cells for producing the recombinant hemoglobin herein can be from genetically modified plants, genetically modified bacteria, and/or genetically modified yeast. In certain embodiments, the genetically engineered organisms suitable for producing the recombinant hemoglobin herein can be Acetobacter, Acinetobacter calcoaceticus, Alcaligenes eutrophus, Arxula adeninivorans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, Aurantiochytrium, Bacillus licheniformis, Bacillus methanolicus, Bacillus stearothermophilus, Bacillus subtilis, Candida utilis, Cleomedomonad, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium glutamicum, Escherichia coli, Hansenula polymorpha, Isochrysis, Kluyveromyces lactis, Kluyveromyces marxianus, Lactococcus lactis, Micrococcus lysodeikticus, Nannochloropsis, Ogataea, Paracoccus azotobacter, Pavlova, Penicillium chrysogenum, Pichia guilleriemuni, Pichia pastoris, Pichia stipitis, Pseudomonas, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Streptococcus lactis, Streptomyces, Polystyrene longifolia, Viridophyllum, Thermoanaerobacter, Thermoanaerobacter, Trichoderma reesei, Xanthomonas campestris, and/or Yarrowia lipolytica.

In some embodiments, after introducing a polynucleotide comprising a coding sequence of hemoglobin of the present disclosure, the host cell can be cultured in a conventional nutrient medium appropriately modified to activate the promoter, select transformants and/or amplify the expression of the polynucleotide encoding the polypeptide. In some embodiments, after introducing a polynucleotide comprising a coding sequence of hemoglobin of the present disclosure, the host cell can be cultured by fermentation. Cultivation can be completed in a growth medium with one or more supplements that assist the growth of the culture, including but not limited to aqueous mineral salt medium, organic growth factors, carbon and/or energy materials, molecular oxygen, etc. In some embodiments, the polypeptide (e.g., hemoglobin) can be recovered from the culture (e.g., by centrifugation, purification, etc.), and purified as described herein.

In certain embodiments, the hemoglobin used herein can be purified hemoglobin. As used herein, the term "purified" refers to a polypeptide or protein (e.g., hemoglobin) that has been separated from other components of a source material (e.g., other animals, fish, plants, fungi, algae, bacteria, genetically modified plants, genetically modified bacteria, or genetically modified yeast proteins). In certain embodiments, the purified hemoglobin herein may not contain at least about 2% to about 100% (e.g., about 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) of other components of the source material. The hemoglobin herein can be purified using protein separation methods known in the art, including but not limited to size exclusion chromatography, affinity chromatography, anion exchange chromatography, cation exchange chromatography, ultrafiltration through a membrane, or density centrifugation, isoelectric precipitation, ammonium sulfate precipitation, isoelectric precipitation, surfactant, detergent and solvent extraction.

B. Antioxidants

In certain embodiments, the composition of the food product suitable for use herein can have one or more purified hemoglobins and one or more antioxidants. As used herein, an "antioxidant" is an agent that inhibits oxidation and can therefore be used to prevent the formulation from deteriorating due to the oxidation process. In some embodiments, the antioxidant suitable for use herein can be an antioxidant vitamin, a polyphenol, or any combination thereof.

In some embodiments, the antioxidant herein can be a naturally occurring or synthetic form of a vitamin with antioxidant properties. In some embodiments, the antioxidant vitamin can be vitamin C, its derivatives and/or its analogs. According to certain embodiments herein, the antioxidant vitamin can be ascorbic acid, L-ascorbic acid, ethylated L-ascorbic acid, vitamin C or its isoascorbic acid isomer, or a salt or ester thereof. In some embodiments, the antioxidant vitamin can be vitamin E, its derivatives and/or its analogs. Vitamin E is a group of eight fat-soluble compounds, including four tocopherols and four tocotrienols. According to certain embodiments herein, the antioxidant vitamin can be alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocopheryl acetate, RRR-alpha-tocopherol, SSR-alpha-tocopherol, alpha-tocotrienol, vitamin E, or a salt or ester thereof.

In some embodiments, the antioxidant herein can be a naturally occurring or synthetic form of polyphenols with antioxidant properties. Polyphenols are common ingredients in plant-derived foods and are the main antioxidants in the diet. The main dietary sources of polyphenols include, but are not limited to, fruits, vegetables, and beverages (e.g., coffee). Non-limiting examples of antioxidants are polyphenolic compounds, chlorogenic acid, flavonoids, tocopherols, dibasic or tribasic carboxylic acids (such as citric acid), EDTA (ethylenediaminetetraacetic acid), ascorbic acid (vitamin C), anthocyanidins, catechins, quercetin, resveratrol, rosmarinic acid, carnosol, Maillard reaction products, enzymes such as superoxide dismutase, certain proteins, amino acids, and protein hydrolysates, etc. Thousands of different polyphenols have been identified in food. In some embodiments, the antioxidant polyphenol herein can be a naturally occurring or synthetic form of flavonoids with antioxidant properties. Non-limiting examples of flavonoids include quercetin (present in onions, tea, apples), catechins (tea, fruits), hesperidin (citrus fruits), and anthocyanidins (red fruits). In some embodiments, the antioxidant flavonoids herein can be isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, taxifolin, catechin, gallocatechin, gallocatechin gallate, epicatechin, epigallocatechin, epigallocatechin gallate, theaflavins, theaflavins gallate, thearubigins, or any combination thereof. In some embodiments, the antioxidant polyphenols herein can be naturally occurring or synthetic forms of phenolic acids having antioxidant properties. Non-limiting examples of phenolic acids include caffeic acid present in many fruits and vegetables. Caffeic acid, most commonly esterified with quinic acid (such as chlorogenic acid), is the main phenolic compound in coffee.

In some embodiments, the antioxidant herein can be ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite, as well as other materials known to those of ordinary skill in the art.

In some preferred embodiments, the antioxidant herein can be ascorbic acid, quercetin, epigallocatechin gallate, EGCG, trolox, taxifolin, 4-methylcatechol, caffeic acid, gallic acid, or any combination thereof.

In some embodiments, the antioxidant used herein can have one or more characteristics that impart the desired properties of the compositions described herein. In some embodiments, the antioxidant used herein can have a strong reduction potential. The standard reduction potential describes the ability of a compound to accept electrons. In some embodiments, the antioxidant used herein can have a reduction potential less than about 500mV (e.g., about 0.5, 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500mV). In some embodiments, the antioxidant used herein can have a reduction potential less than about 200, or 250, or 300, or 350, or 400, or 450, or 500mV.

C. Hemoglobin Composition

In some embodiments, the hemoglobin compositions described herein may include one or more purified hemoglobins. In some embodiments, by composition weight, the compositions described herein may include about 1% to about 99% (e.g., about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%) of the weight of the composition as disclosed herein One or more purified hemoglobin compositions. As used herein, "hemoglobin compositions" include hemoglobin in hydrated or solution form. In certain embodiments, hemoglobin content can be calculated based on dry basis, which means that hemoglobin content and concentration are measured when liquid is removed from the hemoglobin composition. In such embodiments, the compositions described herein may comprise, on a dry weight basis, from about 1% to about 99% (e.g., about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%) of one or more purified hemoglobins, by weight of the composition.

In some embodiments, the compositions described herein may include about 0.01% to about 10% (e.g., about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) of one or more antioxidants disclosed herein by weight of the composition. In some embodiments, the compositions herein may include about 1% to about 99% (e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of one or more purified hemoglobins herein by weight of the composition and about 0.01% to about 10% (e.g., about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of one or more antioxidants disclosed herein by weight of the composition. In some embodiments, the compositions herein may comprise a ratio of total hemoglobin amount to total antioxidant amount. In some embodiments, the compositions herein may have a weight ratio of total hemoglobin composition content to antioxidant content ranging from about 1:1 to about 30:1. In certain embodiments, the eight ratios of total hemoglobin content to total antioxidant content are about 1:1 to about 30:1, for example, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1 or about 30:1.

In certain embodiments, the composition comprises leghemoglobin and one or more antioxidants. In certain embodiments, the composition comprises soy leghemoglobin and one or more antioxidants selected from any one of quercetin, ascorbic acid, EGCG, Trolox or Taxifolin. In certain embodiments, the composition herein can have a weight ratio of total soy leghemoglobin content to total antioxidant content of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1 or about 20:1. In some other embodiments, the composition comprises myoglobin and one or more antioxidants. In some embodiments, the composition comprises one or more myoglobin selected from horses, cattle, cats (e.g., leopards) or whales (e.g., sperm whales) and one or more antioxidants selected from any of quercetin, ascorbic acid, EGCG, Trolox or Taxifolin. In some embodiments, the composition herein may have a weight ratio of total soy leghemoglobin content to total antioxidant content of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1 or about 20:1.

In some embodiments, the hemoglobin composition herein may have one or more purified hemoglobin disclosed herein and one or more antioxidants disclosed herein in a buffered solution. The buffered solution used herein may be any solution suitable for use in a food product. In some embodiments, the hemoglobin composition herein may also include a buffered solution having or having a plurality of buffers, wherein a "buffer" is a compound for resisting pH changes when diluting or adding an acid or base. Buffers used herein may include, for example but not limited to, potassium metaphosphate, potassium phosphate, monobasic sodium acetate, and anhydrous and dihydrated sodium citrate and other materials known to those of ordinary skill in the art. In some embodiments, any food grade organic or inorganic buffer may be used. In some embodiments, the composition disclosed herein may include at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% of the total weight of one or more buffers. In some embodiments, the amount of one or more buffers may depend on the pH level required for the composition herein. In some embodiments, the buffered solutions herein can have a pH ranging from about 5 to about 9 (e.g., about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.4, 8.5, 9). In some embodiments, the compositions disclosed herein can have a pH ranging from about 4 to about 9 (e.g., about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9).

In some embodiments, the hemoglobin composition herein may comprise additional components, such as binders, flavor enhancers, oligosaccharides, stabilizers, pH adjusters, preservatives, non-hemoglobin, dietary fiber, gelling agents, surfactants, water, fats, oil emulsifiers, starches, colorants, and combinations thereof. In some embodiments, the additional components may include, for example, one or more of the following: glucose, fructose, ribose, arabinose, glucose-6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, sucrose, maltodextrin, glycogen, nucleotide-binding sugars, molasses, phospholipids, lecithin, inosine, inosine monophosphate (IMP), guanosine monophosphate (GMP), pyrazine, adenosine monophosphate (AMP), lactic acid, succinic acid, glycolic acid, thiamine, creatine, pyrophosphate, vegetable oil, algae oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, linseed oil, rice bran oil, cottonseed oil, sunflower oil, rapeseed oil, olive oil, free fatty acids, cysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, threonine, tryptophan, valine, arginine, threonine, threonine, thiamine, creatine, pyrophosphate, vegetable oil, algae oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, linseed oil, rice bran oil, cottonseed oil, sunflower oil, rapeseed oil, olive oil, free fatty acids, cysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, threonine, threonine, thiamine, creatine, histidine, threonine, threonine, thiamine, creatine, histidine, threonine amino acids, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, glutathione, amino acid derivatives, protein hydrolysates, malt extract, yeast extract, vitamins, dietary fiber (e.g., plant fiber from carrots, bamboo, peas, broccoli, potatoes, sweet potatoes, corn, whole grains, alfalfa, kale, celery, celery root, parsley, cabbage, pumpkin, green beans, common beans, black beans, red beans, white beans, beets, cauliflower, nuts, apple peels, oats, wheat or plantains, or mixtures thereof), onion spice, garlic spice or herb spice, basil, celery leaf, chervil, chives, cilantro, parsley, oregano, tarragon, thyme, spice extracts, spice oils, natural smoke solutions, natural smoke extracts, yeast extracts and shiitake mushroom extracts.

In some embodiments, the hemoglobin compositions herein comprising one or more antioxidants described herein and one or more hemoglobins described herein can be formulated, for example, as a liquid, gel, paste, sauce, powder or cube, component of a seasoning packet, seasoning packet, or shaker. In some embodiments, the compositions herein can be formulated into or added to, for example, a soup or stew base, broth, or bouillon.

D. Characteristics of Hemoglobin Composition

The present disclosure provides hemoglobin compositions suitable for use in food products having improved color stability and reduced protein degradation over time.

In certain embodiments, the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins. The iron atom in the heme group of hemoglobin may be in a ferrous (Fe ²⁺ ) oxidation state to support the binding and transport of oxygen and other gases. Hemoglobin in normal red blood cells is protected by a reduction system to stabilize these states. Initially oxidized to a ferric iron (Fe ³⁺ ) state in the absence of oxygen converts hemoglobin into a "methemoglobin" that cannot bind oxygen. For example, hemoglobin myoglobin may exist in an oxygen-bound ferrous (Fe ²⁺ ) state (referred to herein as oxygenated myoglobin (OxyMb)), in a deoxygenated state (referred to herein as deoxygenated myoglobin (DeoxyMb)), in a state combined with carbon monoxide (referred to herein as carboxymyoglobin (CarboxyMb)), or in a ferric iron state (referred to as metmyoglobin (MetMb)). The change in the oxidation state of hemoglobin may be determined, for example, from the UV-visible absorption spectrum of a solution containing a composition disclosed herein. For example, the absorption spectrum may include peaks consistent with CarboxyMb (550 nm), OxyMb (582 nm), and MetMb (503 nm and 632 nm).

In some embodiments, changes in oxidation state can also result in changes in the appearance of solutions containing compositions disclosed herein. For example, solutions containing higher amounts of CarboxyMb or OxyMb are characterized by brighter red, while MetMb results in a more undesirable brown coloration. In some embodiments provided herein, the presence of an antioxidant results in a change and/or stabilization of the oxidation state of hemoglobin.

In certain embodiments, the hemoglobin composition herein may include an antioxidant to serve as a reduction system for changing and/or stabilizing the oxidation state of one or more purified hemoglobins. In the context of the hemoglobin composition provided herein, "stabilization" or "stabilizing ... " refers to a composition wherein the combination of hemoglobin and an antioxidant causes a persistent increase in the level of oxygenated hemoglobin or carboxyhemoglobin, such as a more red composition indicated by a change in appearance, or a change in the UV-visible spectrum, such that the increase in the peak height at 550nm (corresponding to CarboxyMb) and/or 582nm (corresponding to OxyMb) compared to a composition without an antioxidant. In some aspects, the increase in peak height can be detected within 1-48 hours after the antioxidant is combined with hemoglobin. This period of time between the addition of an antioxidant and the detectable increase in peak height is referred to as a lag time or a lag phase. In some aspects, depending on the antioxidant used and storage conditions, the increase in peak height can be sustained or "stabilized" to maintain up to about 0.5 days to about 90 days, although in some embodiments, the absolute peak height can be reduced by this stable period. Guidance on some exemplary antioxidants and their effects on peak height is provided in the Examples included herein.

In some embodiments, the present disclosure encompasses a hemoglobin composition comprising hemoglobin and an antioxidant, wherein the antioxidant causes an increase in the relative amount of oxidized or carboxylated hemoglobin in the composition compared to the oxidized Met state. In certain embodiments of the compositions disclosed herein, the hemoglobin is myoglobin and the antioxidant causes an increase in the relative amount of oxymyoglobin to metmyoglobin in the composition, as measured by changes in the UV-visible spectrum of the composition solution. In some embodiments, the increase in the relative amount of oxymyoglobin to metmyoglobin in the composition is detectable after adding an antioxidant and storing at refrigerated temperatures (e.g., about 2°C to about 8°C) for about 0.5 to about 90 days, or about 0.5 to 1 day, or about 1-10 days, or about 10-20 days, or about 20-30 days, or about 30-40 days, or about 40-50 days. In some embodiments, the relative amount of oxymyoglobin to metmyoglobin in the composition increases by at least about 1.1 to about 5 times, or about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, or about 5 times. In some embodiments, the antioxidant causes a change in the peak height increase at about 550 nm and about 582 nm in the UV-visible absorption spectrum from about 4 hours to about 50 days, or about 0.5 to about 1 day, or about 1-10 days, or about 10-20 days, or about 20-30 days, or about 30-40 days, or about 40-50 days after the addition of the antioxidant and refrigeration (e.g., about 2° C. to about 8° C.).

In some embodiments, the hemoglobin compositions herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, the compositions herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for at least about 7 days (e.g., about 0.5, 1, 2, 3, 4, 5, 6, 7 days).

In some embodiments, when the composition is stored at a temperature below freezing (e.g., below 0°C), the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 days to about 30 days. In some embodiments, when the composition is refrigerated (e.g., about 2°C to about 8°C), the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, when the composition is stored at room temperature (e.g., about 22°C to about 27°C), the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, when the composition is stored at about -30°C to about 30°C (e.g., about -30°C, about -20°C, about -10°C, about 0°C, about 10°C, about 20°C, about 30°C, about 40°C), the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, when the composition is stored at about 4°C, the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, when the hemoglobin composition is stored at about 4°C, the hemoglobin composition herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 7 days. In some embodiments, the hemoglobin compositions herein may include an antioxidant for stabilizing the oxidation state of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days when the composition is stored sequentially at two or more temperatures ranging from about -30°C to about 40°C.

In some embodiments, the hemoglobin compositions herein may comprise an antioxidant and hemoglobin, wherein the heme group of the hemoglobin is bound to oxygen, carbon monoxide, or a combination thereof. In some embodiments, the hemoglobin compositions herein may comprise an antioxidant and hemoglobin, wherein the heme group of the hemoglobin is bound to oxygen, carbon monoxide, or a combination thereof for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, the hemoglobin compositions herein may comprise an antioxidant and hemoglobin wherein a higher portion of the heme groups of the hemoglobin are bound to oxygen, carbon monoxide, or a combination thereof for at least about 0.5 days, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, at least about 12 days, or at least about 13 days, or at least about 14 days, or at least about 15 days, or at least about 16 days, or at least about 17 days, or at least about 18 days, or at least about 19 days, or at least about 20 days or more. In some embodiments, the compositions herein may comprise an antioxidant and hemoglobin, wherein a higher portion of the heme groups of the hemoglobin are bound to oxygen, carbon monoxide, or a combination thereof for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days when the composition is stored at about -30°C to about 40°C (e.g., about -30°C, -20°C, -10°C, 0°C, 10°C, 20°C, 30°C, 40°C).

In some embodiments, the hemoglobin compositions herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins. As used herein, the stable visual appearance of hemoglobin herein refers to the appearance of a red composition under visible light. As used herein, the unstable visual appearance of hemoglobin herein refers to the appearance of a brown composition under visible light. In some embodiments, the hemoglobin compositions herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins for about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, the hemoglobin compositions herein may comprise an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins for at least about 0.5 days, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, at least about 12 days, or at least about 13 days, or at least about 14 days, or at least about 15 days, or at least about 16 days, or at least about 17 days, or at least about 18 days, or at least about 19 days, or at least about 20 days, or at least about 21 days, or at least about 22 days, or at least about 23 days, or at least about 24 days, or at least about 25 days, or at least about 26 days, or at least about 27 days, or at least about 28 days, or at least about 29 days, or at least about 30 days or more. In some embodiments, when the hemoglobin composition is stored at about -30°C to about 40°C (e.g., about -30°C, about -20°C, about -10°C, about 0°C, about 10°C, about 20°C, about 30°C, about 40°C), the hemoglobin composition herein may include a stabilizing agent for the visual appearance of one or more purified hemoglobins for at least about 0.5 day, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days. or at least about 24 days, or at least about 25 days, or at least about 26 days, or at least about 27 days, or at least about 28 days, or at least about 29 days, or at least about 30 days or more.

In some embodiments, the hemoglobin composition herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins, wherein the red intensity of the composition is slowly reduced over time compared to a composition comprising only hemoglobin. In some embodiments, the hemoglobin composition herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins, wherein the red intensity of the composition is reduced by about 0.5% to about 70% after about 30 days (e.g., about 0.5 days, about 1 day, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days). In some embodiments, the hemoglobin composition herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins, wherein after preparing the composition according to the method herein, the red intensity of the composition is reduced in a linear manner every day. In some embodiments, the hemoglobin composition herein may include an antioxidant for stabilizing the visual appearance of one or more purified hemoglobins, wherein after preparing the composition according to the method herein, the red intensity of the composition is reduced by about 0.001% to about 0.5% every day.

In some embodiments, the hemoglobin compositions herein comprising one or more antioxidants herein and one or more hemoglobins herein can have almost no protein degradation of hemoglobin. In some embodiments, the hemoglobin compositions herein comprising one or more antioxidants herein and one or more hemoglobins herein can have protein degradation of about 0.01% to about 15% (e.g., about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%) of hemoglobin. In some embodiments, the hemoglobin compositions herein comprising one or more antioxidants herein and one or more hemoglobins herein can have about 0.01% to about 15% (e.g., about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%) protein degradation of hemoglobin after about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days. In some embodiments, the hemoglobin compositions herein comprising one or more antioxidants herein and one or more hemoglobins herein can have about 0.01% to about 15% (e.g., (e.g., about 0.01%, about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14, about 15%)) of protein degradation of hemoglobin after about 1 to about 120 days, about 2 to about 100 days, about 3 to about 80 days, about 4 to about 60 days, or about 7 to about 30 days when the composition is stored at about -30°C to about 40°C (e.g., about -30°C, about -20°C, about -10°C, about 0°C, about 10°C, about 20°C, about 30°C, about 40°C).

III. How to use

The present disclosure provides food products and methods for preparing such food products using the hemoglobin compositions herein. In certain embodiments, the hemoglobin compositions herein can be used to produce meat substitute food products ("meat imitations" or "meat analogs"). As used herein, the term "meat imitations" or "meat analogs" has the same meaning as commonly understood by those of ordinary skill in the art, and includes but is not limited to plant-derived meat, vegan meat, meat substitutes, simulated meat, meat substitutes, artificial meat, vegetarian meat, fake meat or artificial meat. The term "meat imitations" or "meat analogs" refers to food products that are intended to have a realistic meat appearance without containing animal ingredients. In certain embodiments, the compositions herein can be used as materials in methods for making meat imitations, and the meat imitations include but are not limited to minced meat imitations (e.g., minced beef, minced chicken, minced turkey, minced lamb or minced pork), and imitations of meat and fish cuts.

In some embodiments, the method of preparing a food product (e.g., a meat imitation) may include combining the hemoglobin composition of the present invention with a non-animal-based fat, a non-animal-based matrix, a non-animal-based edible fiber component, or any combination thereof. In some embodiments, the method of making a food product (e.g., a meat imitation) may include combining the composition of the present invention with a non-animal-based fat, a non-animal-based matrix, a non-animal-based edible fiber component, or any combination thereof, wherein the addition of the composition of the present invention provides a meat-like appearance to the meat substitute. In some embodiments, the method of preparing a food product (e.g., a meat imitation) may include combining the composition of the present invention with a non-animal-based fat, a non-animal-based matrix, a non-animal-based edible fiber component, or any combination thereof, wherein the addition of the composition of the present invention provides a meat-like taste to the meat substitute. In some embodiments, the method of preparing a food product (e.g., a meat imitation) may include combining the composition of the present invention with a non-animal-based fat, a non-animal-based matrix, a non-animal-based edible fiber component, or any combination thereof, wherein the addition of the composition of the present invention provides a meat-like smell to the meat substitute. According to some embodiments of the present invention, the meat-like appearance, taste or smell can be a beef-like appearance, taste or smell, a poultry-like appearance, taste or smell, a seafood-like appearance, taste or smell, a game-like appearance, taste or smell, a pork-like appearance, taste or smell, a lamb-like appearance, taste or smell, or any combination thereof.

In some embodiments, methods of preparing food products may include combining the compositions herein with a non-animal based meat-like, poultry-like, or seafood-like base dough that can be used in meat imitation products sold in forms such as: "ground meat", burgers/patties, or other forms, such as with Burger (from Impossible ^TM Foods), Beyond/> (From Beyond/> ）、Veggie Chik/> (From Morningstar/> ) and plant-based patties from Good&Gather ^TM . Other examples of poultry, meat and seafood analog products that can contain the compositions provided herein include: such as from Morningstar/> Veggie Meal/> products such as Veggie CHIK'N Nugget, Veggie Popcorn CHIK'N, Veggie CHIK'N Strips, Veggie/> Veggie Buffalo; Beyond/> Beef analog products produced by products such as Beyond/> Debris, Beyond/> Ground Beef and Beyond/> sausages; or fish analogue products such as salmon burgers, fish sticks, fish fillets, crab cakes, fish burgers and fish cakes produced by GoodCatch.

In some embodiments, the method of making a food product may comprise combining a liquid, gel, paste, sauce, powder or cube, ingredients of a spice packet, seasoning packet or spice bottle, soup or stew base, broth or bouillon with the food product before, during, or after cooking of the edible food product. In some embodiments, the compositions herein may be used to adjust the flavor and/or aroma characteristics of a variety of edible food products.

In some embodiments, the food products herein (e.g., meat imitations) may contain from about 1% to about 99% (e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of any hemoglobin composition herein by weight. In some embodiments, the food products herein (e.g., meat imitations) may contain from about 0.01% to about 99% (e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of any antioxidant herein by weight. In some embodiments, the food products herein (e.g., meat replicas) may comprise from about 1% to about 99% (e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) by weight of any of the hemoglobins herein.

Examples

The following examples are included to illustrate preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the examples that follow represent techniques that the inventors have found to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes of its practice. However, it will be appreciated by those skilled in the art based on the present disclosure that many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present disclosure and still obtaining the same or similar results.

The following Examples 1-4 were performed using the exemplary techniques provided herein for guidance, although equivalent techniques known to those skilled in the art may be used.

Exemplary Techniques

In the exemplary methods disclosed in Examples 1-4 below, the following techniques are performed as detailed below.

Preparation of horse heart myoglobin solution. A 50 mg/ml horse heart myoglobin solution was prepared by dispersing weighed protein powder into a buffer solution (2.5 mM sodium phosphate/2.5 mM histidine, pH 9.0). The resulting myoglobin solution was stirred continuously for at least 1 hour to ensure that the protein was completely dissolved. The conversion of myoglobin to metmyoglobin was carried out according to the method described by Tang et al., Journal of Food Science, 69 (9) (2004), with some slight modifications. In brief, potassium ferrocyanide (5 mg/mL) was added to the myoglobin solution, and the mixture was then stirred in an ice bath (4 ° C) for 1 hour. Any residual ferrocyanide was then removed from the mixture using a desalting column (Sephadex G-25PD-10).

Metmyoglobin (MetMb) was then reduced to oxymyoglobin (OxyMb) by adding sodium bisulfite or antioxidants: ascorbic acid, caffeic acid, EGCG, gallic acid, quercetin, taxifolin, Trolox, and 4-methylcatechol. The desalted MetMb solution was diluted to 2 mg Mb/mL with a buffer solution (2.5 mM sodium phosphate/2.5 mM histidine, pH 9.0) and then reduced by adding sodium bisulfite (0.5 mg/mL) or antioxidant (1 mM) solution. This was achieved by adding a weighed amount of reducing agent to the OxyMb solution and mixing thoroughly. Any excess sodium bisulfite was subsequently removed using a desalting column (Sephadex G-25 PD-10). To add antioxidants to the myoglobin solution (~2.0 mgMb/mL – desalted), water-insoluble antioxidants (caffeic acid, quercetin, or taxifolin) were first added to ethanol (5% to 10% of the total volume) before addition to the MetMb solution. The reduction potential and solubility of the antioxidants used are listed in Table 1. For all systems, the pH of the myoglobin solution was measured after at least 2 h of incubation. All samples were stored at 4°C in screw-capped glass vials or capped 1.5 mL plastic cuvettes for up to 52 days.

Preparation of cellular agricultural hemoglobin solutions. Powdered heme samples obtained using cellular agricultural methods (e.g., fermentation) were used to prepare protein solutions: bovine, leopard, and sperm whale myoglobin and soybean leghemoglobin. Weighed amounts of each myoglobin powder were dissolved in a buffered aqueous solution (2.5 mM sodium phosphate/2.5 mM histidine, pH 9.0) to obtain a range of protein concentrations (0.5, 1.0, and 5 mg Mb/mL). The resulting mixture was then stirred continuously for at least 1 hour to completely dissolve the protein. All solutions were prepared fresh daily or stored at 4°C for up to 1 day prior to analysis.

Appearance. Use a digital camera to capture the appearance of the myoglobin sample.

Electrical properties. The zeta potential (ζ-potential) of protein solutions (1.0 or 5.0 mg/mL) was measured using a particle electrophoresis instrument (Zetasizer Pro, Malvern Instruments Ltd., Worcestershire, UK). The ζ-potential was measured in the pH range of 8.5 to 2.5 by titrating the initial solution with acid (0.1 to 0.25 M HCl) or base (0.25 M NaOH) solutions under continuous stirring.

Solubility. The pH dependence of protein solubility in myoglobin solution was determined by measuring the soluble protein and total protein concentrations within a certain pH range. Myoglobin solution (0.5 mg/mL) was stored overnight (4 ° C) and then the protein concentration was measured using a Bradford assay kit. The total protein concentration (c _total ) was determined by directly analyzing the myoglobin solution, while the soluble protein concentration (c _soluble ) was determined by centrifugation at 13,000 rpm for 15 minutes and then analyzing the supernatant. Protein concentration measurements were performed according to the instructions provided by the Bradford assay kit. In brief, 5 μL of protein sample (centrifuged or non-centrifuged) was moved into a well of a 96-well microplate (Costar, Corning, Corning, New York, USA), and 250 μL of Bradford reagent was added to each well, and the solution was mixed well. The mixture was then incubated at room temperature for 5 minutes and then analyzed. The absorbance of the sample was then measured at 595 nm using a UV-visible spectrophotometer (SpectraMax M2e, Sunnyvale, California, USA). The standard curve was drawn by measuring the absorbance of a series of bovine serum albumin (BSA) solutions with different protein concentrations (0.125 mg/mL to 1 mg/mL), and a linear calibration curve with R ² = 0.995 was obtained. Myoglobin solubility was calculated using the following expression: Solubility % = 100× _cSoluble / _cTotal

Absorption spectroscopy measurements. The absorption spectra of myoglobin solutions (1 and 5 mg/mL) were measured in the range of 450 to 750 nm using a UV-visible spectrophotometer (Cary 100, Agilent Technologies, Santa Clara, CA, USA) (Tang et al., 2004). All samples were placed in cuvettes (volume = 1.5 mL; optical path = 1 cm) and stored at 4°C throughout the storage study. The myoglobin redox state ratios (deoxymyoglobin (DeoMb), oxymyoglobin (OxyMb), and metmyoglobin (MetMb)) in the myoglobin system containing antioxidants were calculated according to the modified Krzywicki equation, similar to the method described in Tang et al., Journal of Food Science, 69 (9) (2004).

Computational modeling. Molecular docking analysis of horse heart myoglobin and selected antioxidants was performed to obtain some insights into the nature of the molecular interactions between antioxidants and proteins. Molecular docking was performed using Glide software (version 2019-3, Schrödinger LLC, New York, NY) to obtain some additional insights into the nature of the binding interactions between antioxidants and myoglobin. The myoglobin crystal structure (PDB ID 1MNH) was used as the starting configuration and processed and optimized using the Protein Preparation Wizard, with PROPKA set to pH = 8.0. The 3D structures of the four ligands (ascorbic acid, quercetin, gallic acid, and 4-methylcatechol) were constructed using Maestro 3DBuilder. All ligand structures were then prepared using LigPrep with the OPLS3 force field. The ligands maintained chirality and possible states at a target pH of 8.0 ± 1.0 were generated. A docking grid centered on the heme group was created to cover the entire myoglobin by adjusting the size of the box in the midpoint of the ligand diameter, and no other constraints were imposed. Docking experiments were then performed using ligand docking in the extra-precise (XP) mode. The number of poses reported was not restricted, but post-docking minimization was applied to 20 poses per ligand, including a strain correction term. All reported results were then ranked by docking score, and poses with docking scores above zero were excluded. Only the specific configurations (binding sites and binding poses) with the highest (most negative) glide docking scores from the docking experiments are discussed and described in the Results section.

Statistical analysis. For each myoglobin system, replicate analyses were performed, and at least three replicates were measured for each analytical test. Means and standard deviations of the combined data were calculated based on six or more experimental data points using Microsoft Excel 2016.

Example 1. Effect of pH on the properties of horse heart myoglobin

In an exemplary method, initial characterization of the physical properties of an exemplary myoglobin (horse myoglobin), including charge, solubility, and stability, was performed at different pHs.

Power:

The electrical properties of horse heart myoglobin were determined by measuring the pH dependence of its ζ-potential values. In particular, the isoelectric point (pI) of each protein was determined from the point at which its net charge is zero. The isoelectric point of horse heart myoglobin (1 mg/mL) is approximately pH 5.5 (Figure 1). Visual observation showed that a thin sediment layer formed at the bottom of the samples around pH 5.5 and below (data not shown), which can be attributed to aggregation of the protein.

Protein solubility

The effect of pH on the solubility of horse heart myoglobin was determined by measuring the soluble and total protein content of the solutions using the Bradford assay. The protein was highly soluble over the entire pH range studied, with solubility consistently greater than 90% (Figure 2).

Example 2. Effect of adding antioxidants on the stability of oxymyoglobin

In another exemplary method, the effects of various natural and synthetic antioxidants on converting metmyoglobin to oxymyoglobin in solution form rather than in meat muscle form were examined, thereby improving the color characteristics of myoglobin solutions for food product development. The oxidized form of myoglobin (oxymyoglobin; OxyMb) plays a key role in determining the desirable bright red color of many meat products. However, it is very susceptible to oxidation to metmyoglobin (MetMb), resulting in a color change from red to brown.

The UV-visible absorption spectra of the antioxidant-free horse heart myoglobin (OxyMb and MetMb) solutions are shown in Figure 3A. The oxymyoglobin spectrum has two different absorption peaks near 544 and 582 nm, which is why these solutions are bright red. In contrast, metmyoglobin has relatively small absorption peaks only near 503 and 632 nm, which is why it is brown. The oxymyoglobin solution only remains bright red for about 1 day, then turns brown and exhibits a UV-visible spectrum similar to that of metmyoglobin. Figures 3B-3F show that the concentration of oxymyoglobin gradually decreases over time, and the metmyoglobin concentration increases to about 100% on the 5th day.

Antioxidants with different reduction potential values (E ^ó ) were investigated for their potential to convert metmyoglobin to oxymyoglobin, thereby producing the desired red color in the system. Table 1 provides exemplary antioxidants used herein.

Table 1

It was found that the addition of 1 mM ascorbic acid (~2 mg/mL) to a solution of metmyoglobin converted MetMb to OxyMb, as shown in UV-visible light absorption and color measurements (Figures 4A-4C). After OxyMb was formed and reached a peak concentration (99.9%) on the first day, the OxyMb fraction gradually decreased over 13 days in the presence of ascorbic acid, and only 2 to 3 days in the absence of ascorbic acid (Figures 4A-4C). Interestingly, there was a "lag time" of about 4 to 24 hours for the OxyMb concentration to reach a maximum, and the color of the solution turned bright red (Figures 4A-4C). The source of this effect may be due to the delayed ability of ascorbic acid to reach the heme group. Therefore, the observed lag time may be due to the time required for ascorbic acid molecules to move from the aqueous solution to the active site, where they can reduce the heme group. Without wishing to be bound by theory, the longer lag times observed in the samples herein may be because they were stored at 4°C, which may have slowed down the reaction kinetics. In addition, it was observed that the lag time of the myoglobin solution herein may be related to the "bloom" time observed in fresh cuts of meat, i.e., the time when the interior of the meat changes from brown to red due to the oxidation of myoglobin. Figure 4A also shows that the concentration of deoxymyoglobin (DeoMb) increases on day 10 and beyond. This may occur due to the depletion of oxygen around the myoglobin molecules and the formation of a low oxygen partial pressure environment, as ascorbic acid is an oxygen scavenger, and the resulting conversion from MetMb to DeoMb.

Quercetin, a natural hydrophobic antioxidant, has also been studied for its potential to stabilize oxymyoglobin. 1 mM quercetin (initially dissolved in ethanol) reduced metmyoglobin to oxymyoglobin, with 70% oxymyoglobin formed on day 1, which then gradually decreased over 21 days (Figures 4D-4F). The lag time for red stabilization was approximately 2 to 10 hours (data not shown). Quercetin was observed to have a shorter lag time.

Epigallocatechin gallate (EGCG), which is soluble in water and organic solvents, was also tested. The addition of EGCG (1 mM) can also effectively reduce metmyoglobin to oxymyoglobin (about 40%) and turn the protein solution into a light red color (Figures 4G, 4H, and 4M). The 40% oxymyoglobin formed is not very stable and reverts to 10% metmyoglobin after 24 hours, causing the solution to quickly turn brown.

Adding the hydrophobic antioxidant tachyphyllin (1 mM) to the solution converted MetMb to OxyMb, and the red color remained relatively stable over 27 days (Figures 4I, 4J, and 4M). It took about 24 hours of lag time for oxymyoglobin to be completely stable and reach a peak concentration of about 65% (Figure 4I) and form a bright red color in the system. The characteristic absorption peaks and red intensity associated with OxyMb slowly decreased over 27 days, with the OxyMb concentration gradually decreasing to 22%, and the MetMb state reaching 72% concentration.

Addition of 1 mM 4-methylcatechol to myoglobin did not produce the characteristic double peak spectrum typical of oxymyoglobin, with the OxyMb state reaching only 10% to 20% (Figures 4K-4M). This suggests that 1 mM 4-methylcatechol is not an effective reducing agent or antioxidant. Instead, the absorbance measured at lower wavelengths increased substantially (Figure 4K), suggesting the formation of some pigment molecules or colloidal particles that absorb or scatter light, respectively. A "blood red" color was observed in these samples, which may have applications in certain food systems.

Adding 1 mM Trolox (a water-soluble analog of vitamin E) to the protein solution was also able to reduce MetMb to OxyMb (52%), which then remained stable for up to 26 days (Figures 4N, 4O, and 4T). This system had a 24-hour lag time before the first observation of the characteristic absorption spectrum, the maximum concentration of OxyMb, and the red color associated with OxyMb (Figure 4T). Over 26 days, the OxyMb concentration gradually decreased from 52% to 30%, and the MetMb fraction reached 61%. From day 7 onwards, the deoxymyoglobin concentration increased almost linearly, which may be due to oxygen depletion (Figure 4O).

The addition of 1 mM caffeic acid, a hydrophobic antioxidant, was able to reduce the metmyoglobin solution to only about 25% oxygenated myoglobin after 24 hours. On day 1, only a slight red color was observed in the solution, which then turned brown (Figures 4P, 4Q, and 4T). The 24-hour lag time saw only a slight red color develop, which may be due to the relatively low reducing site capacity of caffeic acid. Interestingly, the metmyoglobin concentration also decreased over time after reaching a peak of 78% on day 4, while the DeoMb fraction increased linearly over 15 days starting from day 0 (Figure 4P). Similarly, the conversion from MetMb to DeoMb may be due to the free radical scavenging properties of caffeic acid, which leads to the formation of low oxygen partial pressure in the system.

The addition of 1 mM gallic acid (water soluble) was able to reduce metmyoglobin and form about 35% oxymyoglobin (Figures 4R, 4S, and 4T). The reddish myoglobin solution observed on day 0 turned dark brown after 1 day of storage, and the OxyMb concentration decreased to 14% only after 24 hours (day 1). The lower conversion of MetMb to OxyMb may be due to the relatively high reduction potential of gallic acid among the other antioxidants used (Table 1). About 20% DeoMb was measured in the system throughout the storage period (Figure 4R).

Overall, the results of these exemplary methods indicate that different natural and synthetic antioxidants have different abilities to convert MetMb to OxyMb and subsequently stabilize the oxymyoglobin form. In particular, ascorbic acid and quercetin, which have relatively low reduction potentials (Table 1), can both effectively stabilize the color of oxymyoglobin. These insights are important for the application of myoglobin in food products that are expected to have a long shelf life and thus retain color for a long time.

Example 3. Molecular docking experiment

In another exemplary method, molecular docking experiments were performed on selected antioxidants using Glide (Schrodinger Suite) to gain insight into the key molecular interactions involved in the binding of antioxidants to myoglobin. The docking results showed that ascorbic acid, quercetin, and gallic acid had similar ligand binding sites near the heme group, and the distances between these antioxidant ligands and the heme group were approximately 10 to 12 angstroms.

Ascorbic acid: For ascorbic acid, three lysines (K42, K47 and K98) and one aspartic acid (D44) on myoglobin participate in the binding interaction. In terms of the nature of the bonds, salt bridges and five hydrogen bonds are formed between the hydroxyl/carbonyl groups on ascorbic acid and these amino acids on the protein.

Quercetin: For quercetin, two lysines (K96 and K98), one aspartic acid (D44), and one glutamic acid (E41) are involved in the binding interaction. In this case, several hydrogen bonds are formed between the hydroxyl/carbonyl groups on quercetin and the amino acids on the protein.

Gallic acid: For gallic acid, three lysines (K42, K96, and K98) and one aspartic acid (D44) participate in the binding interaction. In this case, two salt bridges are formed between the carboxylate on gallic acid and the K42/K98 side chains. In addition, three hydrogen bonds are formed between the hydroxyl group on gallic acid and the K47/D44 side chains and the K96 carbonyl group on the protein.

4-Methylcatechol: Molecular docking experiments with 4-methylcatechol were also performed, since this compound did not produce a typical spectrum of oxymyoglobin when added to the myoglobin solution (Figures 4K and 4L). In fact, this compound binds to a different site on the protein molecule, close to the K34 and E52 residues, which are far away from the heme group. Only two hydrogen bonds are formed between the hydroxyl groups on 4-methylcatechol and the K34/E52 side chains on the protein. The different binding sites of 4-methylcatechol could be the reason for the different absorption spectra measured in the mixed system.

Example 4. Characterization of myoglobin produced by cellular agriculture

In another exemplary method, four different myoglobin samples (leopard, cattle, sperm whale, and soybean) produced using cellular agriculture (fermentation) methods were characterized. Specifically, the charge, color, solubility, and redox stability of the protein were measured using methods similar to those discussed in Examples 1 and 2 and described in detail herein for horse heart myoglobin.

Charge. The ζ-potential and pH curves of four different myoglobin samples were measured to determine their isoelectric points (pI) and aggregation stability under different solution conditions (Figure 5A). Leopard myoglobin, bovine myoglobin, and soybean leghemoglobin all have similar isoelectric points (pH 4.5), while sperm whale myoglobin has a higher isoelectric point (pH 6.5), and the isoelectric point of horse myoglobin is between these four samples, that is, pH 5.5. When the pH is reduced from 8.5 to 2.5, the ζ-potential of leopard myoglobin changes from about -18 to +14mV, bovine myoglobin and soybean leghemoglobin change from -30 to +17mV, and sperm whale myoglobin changes from -18 to +28mV.

Appearance. All four myoglobin solutions changed from red to brown when the pH was decreased from 8.5 to 2.5, an effect that was most pronounced for the animal-derived myoglobin samples (Figure 5B), including horse heart myoglobin. This color change can be attributed to the unfolding of the protein and the exposure of the heme groups at low pH, which results in the loss of oxygen molecules bound to the heme iron. Visually, the soy and leopard myoglobin solutions appeared to retain their intense red color longer than the other samples, which could be an advantage for food applications.

Protein Solubility. Visually, some protein aggregation was observed around and below the isoelectric point of the myoglobin solution, which may be attributed to electrostatic repulsion and reduced protein unfolding effects. However, overall, the four myoglobin samples had relatively high solubility (>78%) across the entire pH range studied (Figures 7A-7D). This suggests that there are strong repulsive forces (electrostatic or steric) or weak attractive forces (van der Waals or hydrophobic) between myoglobin molecules. For comparison, the average solubility of the proteins across the pH range was calculated: soybean (101%) > leopard (97%) > sperm whale (94%) > cattle (91%). Overall, these results indicate that all hemoglobins have high aqueous solubility across the pH range, but there are some differences between them. These differences may arise from differences in the primary structure (amino acid sequence) of the proteins, resulting in differences in their conformation, surface charge, and hydrophobicity.

Oxidative Stability. Absorption spectra of freshly prepared myoglobin solutions (1 and 5 mg/mL) were measured to determine their initial redox state. Panther myoglobin solutions appeared red, and the color intensity increased with increasing protein concentration. These solutions had distinct absorption peaks at 550 and 582 nm (Figure 6); however, the spectrum was somewhat different from the expected spectrum for oxymyoglobin (Figure 4A). In particular, the peak for panther myoglobin was strongest at 550 nm, while the peak for reduced horse heart myoglobin was strongest at 582 nm. When myoglobin binds to carbon monoxide, it produces a bimodal spectrum very similar to that of OxyMb, but with a more prominent peak near 541 nm. OxyMb and CarboxyMb also have fairly similar bright cherry red colors. Therefore, a mixture of OxyMb and CarboxyMb, as well as some MetMb (a small peak near 635 nm), was likely formed in the initial panther myoglobin system. This may have occurred because some carbon monoxide attached to myoglobin during the production, isolation, and dehydration of the protein. Over time, OxyMb and CarboxyMb gradually oxidized to MetMb, as shown by the changes in the absorption spectra, i.e., the formation of peaks around 503 and 632 nm (Figures 7A-7C). For more concentrated myoglobin solutions (5 mg/mL), higher absorbance intensity and longer redox stability were observed, which is expected because there are more active compounds.

The absorption spectrum of the bovine myoglobin solution also showed that it contained a mixture of CarboxyMb, OxyMb, and MetMb, with peaks observed at 554 and 630 nm (Figures 7D-7F). The oxidative stability of bovine myoglobin was slightly better than that of leopard myoglobin, and the characteristic double-peak spectrum was still observed on day 21. However, even on day 0, the bovine myoglobin solution did not show a strong red color, and the solution turned brown over time. This color change was consistent with the gradual shift in the absorption spectrum from CarboxyMb/OxyMb to MetMb, with a prominent peak gradually forming at 632 nm over time (Figures 7D-7F).

Figure 7G shows that the initial sperm whale myoglobin has a slightly different redox state than the other proteins examined herein. The absorption spectrum contains peaks consistent with CarboxyMb (550nm), OxyMb (582nm), and Metmyoglobin (503nm and 632nm). However, over the course of about 5 days of storage, the absorption spectrum gradually became more similar to pure MetMb, with the solution turning from red to brown (Figures 7G-7I). This study suggests that the sperm whale samples used in this exemplary study may have undergone some auto-oxidation.

The soy leghemoglobin solution produced in this paper has a bright cherry red color that is much stronger than the three animal myoglobin solutions. Initially, the absorption spectrum of this sample has peaks consistent with the presence of CarboxyMb and OxyMb (Figure 7J-JL). At day 52, a double peak absorption spectrum was still observed in soy leghemoglobin, indicating that this hemoglobin has a high antioxidant activity. Therefore, the soy leghemoglobin used in this study may be suitable for food products with a longer shelf life.

Compared to horse heart myoglobin without added antioxidants, the myoglobin produced by cell agriculture has a longer oxidative stability (compare Figure 3 and Figures 7A-7L). This may be related to the method used to produce these myoglobins. Nevertheless, all of these systems have typical oxymyoglobin/carboxymyoglobin absorption spectra.

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The foregoing discussion of the present disclosure is presented for the purpose of illustration and description. The foregoing is not intended to limit the present disclosure to one or more forms disclosed herein. Although the description of the present disclosure has included a description of one or more embodiments and certain variations and modifications, other variations and modifications are also within the scope of the present disclosure, for example, within the skills and knowledge of those skilled in the art after understanding the present disclosure. It is intended to obtain the right to include alternative embodiments within the scope allowed, including alternative, interchangeable and/or equivalent structures, functions, scopes or steps claimed, regardless of these alternative, interchangeable and/or equivalent structures, functions, scopes disclosed herein some steps or steps, and it is not intended to publicly dedicate any patentable subject matter.

Claims (36)

1. A composition for use in a food product, the composition comprising one or more purified hemoglobin compositions and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a non-animal source.

2. The composition of claim 1, wherein the one or more purified hemoglobin compositions comprise globulin.

3. The composition of claim 1, wherein the one or more purified hemoglobin compositions comprise leghemoglobin, non-symbiotic hemoglobin, hemocyanin, heme-like, tropoglobulin, cytochrome, cyanobacterioglobin, flavohemoglobin, myoglobin, phytoglobulin, or any combination thereof.

4. The composition of claim 1, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a genetically modified non-animal source.

5. The composition of claim 4, wherein the genetically modified non-animal source comprises a genetically modified plant, a genetically modified bacterium, a genetically modified yeast, or any combination thereof.

6. The composition of claim 1, wherein the one or more purified hemoglobin compositions comprise polypeptides expressed and/or secreted from non-animal sources, wherein the non-animal sources comprise plants, fungi, bacteria, yeasts, algae, archaebacteria, genetically modified plants, genetically modified fungi, genetically modified bacteria, genetically modified yeasts, genetically modified algae, genetically modified archaebacteria, or any combination thereof.

7. The composition of claim 6, wherein the polypeptide expressed and/or secreted from a non-animal source is encoded by a polynucleotide derived from an animal, plant, fungus, bacteria, yeast, algae, archaebacteria, or any combination thereof.

8. The composition of claim 1, wherein the one or more antioxidants comprise vitamins, polyphenols, and any combination thereof.

9. The composition of claim 1, wherein the one or more antioxidants comprise vitamin C and any derivatives thereof, vitamin E and any derivatives thereof, and any combinations thereof.

10. The composition of claim 1, wherein the one or more antioxidants comprise flavonoids or any derivatives thereof.

11. The composition of claim 10, wherein the one or more antioxidants comprise isorhamnetin, kaempferol, myricetin, procyanidins, quercetin, rutin, douglas fir, catechin, gallocatechin gallate, epicatechin, epigallocatechin gallate, theaflavins gallate, thearubigins, or any combination thereof.

12. The composition of claim 1, wherein the one or more antioxidants have a reduction potential ranging from about 150mV to about 500 mV.

13. The composition of claim 1, comprising a weight ratio of the one or more purified hemoglobin compositions to the one or more antioxidants ranging from about 1:1 to about 30:1.

14. The composition of claim 13, comprising a weight ratio of the one or more hemoglobin compositions to the one or more antioxidants ranging from about 2:1 to about 10:1.

15. The composition of claim 1, wherein the composition results in an increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582 nm.

16. The composition of claim 15, wherein the increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582nm is detectable within at least about 7 days after the addition of the one or more antioxidants.

17. A composition for a food product, the composition comprising one or more purified hemoglobin compositions and one or more antioxidants, wherein the one or more purified hemoglobin compositions comprise hemoglobin from a non-animal source, wherein,

The non-animal source includes plants, fungi, bacteria, yeasts, algae, archaebacteria, genetically modified plants, genetically modified fungi, genetically modified bacteria, genetically modified yeasts, genetically modified algae, genetically modified archaebacteria, or any combination thereof;

The hemoglobin is expressed and/or secreted from a non-animal source, wherein the polypeptide is encoded by a polynucleotide comprising a nucleic acid derived from an animal, plant, fungus, bacteria, yeast, algae, archaebacteria, or any combination thereof;

the polypeptide is selected from the group consisting of: soy hemoglobin, non-symbiotic hemoglobin, hemocyanin, heme-like, tropoglobulin, cytochrome, cyanobacteria globulin, flavohemoglobin, myoglobin, and phytoglobulin; and

Wherein the one or more antioxidants have a reduction potential ranging from about 280mV to about 500mV,

Wherein the composition comprises a weight ratio of the one or more purified hemoglobin compositions to the one or more antioxidants of about 1:1 to about 30:1.

18. The composition of claim 17, wherein the polypeptide is selected from the group consisting of leghemoglobin and myoglobin.

19. The composition of claim 17, wherein the one or more antioxidants are selected from the group consisting of: ascorbic acid, quercetin, taxifolin and Trolox.

20. The composition of claim 17, wherein the composition comprises a weight ratio of the one or more purified hemoglobin compositions to the one or more antioxidants of about 2:1 to about 10:1.

21. The composition of claim 17, wherein the composition results in an increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582 nm.

22. The composition of claim 21, wherein the increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582nm is detectable at least about 7 days after the addition of the one or more antioxidants.

23. A composition comprising one or more purified hemoglobin compositions and one or more antioxidants,

Wherein the one or more purified hemoglobin compositions comprise genetically modified non-animal derived hemoglobin from a group selected from the group consisting of: a genetically modified plant, a genetically modified bacterium, a genetically modified fungus, or a genetically modified yeast; and wherein the composition results in an increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582nm that is detectable for at least about 7 days after the addition of the one or more antioxidants.

24. The composition of claim 23, wherein the one or more antioxidants have a reduction potential ranging from about 150mV to about 500 mV.

25. The composition of claim 23, wherein the one or more antioxidants are selected from the group consisting of ascorbic acid, quercetin, taxifolin, trolox, and combinations thereof.

26. The composition of claim 24, wherein the composition comprises a weight ratio of the one or more purified hemoglobin compositions to the one or more antioxidants of about 2:1 to about 10:1.

27. The composition of claim 23, wherein the purified hemoglobin is encoded by a polynucleotide comprising a nucleic acid sequence from a plant, animal, fungus, or bacterium.

28. The composition of claim 27, wherein the polynucleotide comprises a nucleic acid sequence derived from a leguminous plant encoding hemoglobin.

29. The composition of claim 28, wherein the polynucleotide comprises a nucleic acid sequence derived from any one of a horse, leopard, cow, or whale.

30. The composition of claim 27, wherein the one or more purified hemoglobin is a globulin selected from the group consisting of: leghemoglobin, non-symbiotic hemoglobin, hemocyanin, heme-like, tropoglobulin, cytochrome, cyanobacteria globulin, flavohemoglobin, myoglobin, and phytoglobulin.

31. The composition of claim 27, wherein the one or more purified hemoglobin is recombinant hemoglobin produced from a genetically modified yeast source, wherein the recombinant hemoglobin is encoded by a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence derived from a legume, a horse, a leopard, a cow, a whale, or any combination thereof and encoding hemoglobin.

32. The composition of any of claims 1, 17, or 23, wherein the hemoglobin composition comprises myoglobin, and wherein combining the one or more purified hemoglobin compositions with one or more antioxidants causes an increase in the relative amount of oxymyoglobin and conversion of carbomyoglobin to ferrimyoglobin in the composition.

33. The composition of claim 32, wherein an increase in the relative amount of oxymyoglobin and conversion of carbomyoglobin to ferrimyoglobin in the composition is detectable for at least about 7 days after the antioxidant is added.

34. The composition of claim 33, wherein the relative amount of oxymyoglobin and the increase in conversion of carbooxymyoglobin to ferrimyoglobin in the composition ranges from about 1.1-fold to about 5-fold.

35. A composition comprising one or more purified hemoglobin compositions and one or more antioxidants,

Wherein the one or more purified hemoglobin compositions comprise leghemoglobin, non-symbiotic hemoglobin, hemocyanin, heme-like, tropoglobulin, cytochrome, cyanobacterioglobin, flavohemoglobin, myoglobin, phytoglobulin, or any combination thereof;

Wherein the one or more antioxidants are selected from the group consisting of: ascorbic acid, quercetin, taxifolin, trolox, and combinations thereof;

Wherein the composition comprises a weight ratio of the one or more purified hemoglobin compositions to the one or more antioxidants of about 2:1 to about 10:1, and

Wherein the composition results in an increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582nm that is detectable for at least about 7 days after the addition of the one or more antioxidants.

36. A method of making a meat substitute, the method comprising combining the composition of any one of claims 1, 17, 23, or 35 with a meat replica matrix, wherein the composition of any one of claims 1, 17, 23, or 35 results in an increase in peak height of the UV-visible absorption spectrum for the one or more hemoglobin compositions at one or more of about 550nm and about 582nm that is detectable for at least about 7 days after the addition of the one or more antioxidants.