WO2023076307A1 - Compositions d'hémeprotéine stabilisées et leurs procédés d'utilisation - Google Patents

Compositions d'hémeprotéine stabilisées et leurs procédés d'utilisation Download PDF

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Publication number
WO2023076307A1
WO2023076307A1 PCT/US2022/047771 US2022047771W WO2023076307A1 WO 2023076307 A1 WO2023076307 A1 WO 2023076307A1 US 2022047771 W US2022047771 W US 2022047771W WO 2023076307 A1 WO2023076307 A1 WO 2023076307A1
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Prior art keywords
hemeprotein
antioxidants
compositions
composition
purified
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PCT/US2022/047771
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English (en)
Inventor
Cheryl CHUNG
Stefan K. Baier
David MCCLEMENTS
Eric Decker
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Motif Foodworks, Inc.
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Priority to CA3233394A priority Critical patent/CA3233394A1/fr
Priority to AU2022375649A priority patent/AU2022375649A1/en
Publication of WO2023076307A1 publication Critical patent/WO2023076307A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/20—Proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A23J3/227—Meat-like textured foods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/46—Addition of dyes or pigments, e.g. in combination with optical brighteners using dyes or pigments of microbial or algal origin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/13—Phenols; Phenolates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/15—Heterocyclic compounds having oxygen in the ring
    • C08K5/151—Heterocyclic compounds having oxygen in the ring having one oxygen atom in the ring
    • C08K5/1535—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/15—Heterocyclic compounds having oxygen in the ring
    • C08K5/156—Heterocyclic compounds having oxygen in the ring having two oxygen atoms in the ring
    • C08K5/1575—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/0008—Organic ingredients according to more than one of the "one dot" groups of C08K5/01 - C08K5/59
    • C08K5/005—Stabilisers against oxidation, heat, light, ozone

Definitions

  • compositions for use in non-animal food products comprising one or more purified hemeproteins and one or more antioxidants, methods of making, and methods of use thereof.
  • the characteristic red color associated with hemeproteins is determined by the protein’s redox state, which depends on environmental conditions, such as pH, ionic strength, temperature, and oxygen levels.
  • the problem in isolating hemeproteins from plant sources or producing them using cellular agriculture approaches (e.g., fermentation), is that the resulting isolated hemeproteins are highly susceptible to chemical degradation during storage and utilization and the desired red color and taste profile is lost soon after production.
  • compositions herein have improved color stability and reduced protein degradation (e.g., aggregation) over time.
  • Embodiments of the present disclosure provide compositions for use in a food product comprising one or more purified hemeprotein compositions and one or more antioxidants, wherein the one or more purified hemeprotein compositions comprise a hemeprotein from a non-animal source.
  • one or more purified hemeprotein compositions herein may comprise a globin.
  • one or more purified hemeproteins compositions herein may comprise a leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, phytoglobin, or any combination thereof.
  • one or more purified hemeproteins compositions herein may comprise a hemeprotein from a genetically modified non-animal source.
  • a genetically modified non-animal source herein may comprise a genetically modified plant, a genetically modified bacteria, a genetically modified yeast, or any combination thereof.
  • the current disclosure comprises hemeprotein composition for use in a food product comprising one or more purified hemeprotein and one or more antioxidants.
  • the one or more purified hemeprotein comprises a globin.
  • the one or more purified hemeprotein comprise leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, phytoglobin, or any combination thereof.
  • the hemeprotein compositions comprise a purified hemeprotein from a genetically modified source.
  • the source comprises a genetically modified plant, a genetically modified bacteria, a genetically modified yeast, or any combination thereof.
  • the one or more purified hemeprotein compositions comprise a polypeptide expressed and/or secreted from a source, wherein the source comprises plants, fungi, bacteria, yeasts, algae, archaea, genetically modified plants, genetically modified fungi, genetically modified bacteria, genetically modified yeasts, genetically modified algae, genetically modified archaea, or any combination thereof.
  • the polynucleotide sequence encoding the hemeprotein is derived from animals, plants, fungi, bacteria, yeasts, algae, archaea, or any combination thereof.
  • one or more antioxidants herein may comprise antioxidant vitamins, polyphenols, or any combination thereof. In some embodiments, one or more antioxidants herein may comprise vitamin C, vitamin E, any derivatives thereof, or any combination thereof. In some embodiments, one or more antioxidants herein may comprise flavonoids or any derivatives thereof.
  • one or more antioxidants herein may comprise isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, taxifolin, catechin, gallocatechin, gallocatechin gallate esters, epicatechin, epigallocatechin, epigallocatechin gallate esters, theaflavin, theaflavin gallate esters, thearubigins, or any combination thereof.
  • one or more antioxidants herein may have a reduction potential less than about 500 mV.
  • compositions herein may comprise a ratio of the total amount by weight of one or more purified hemeproteins to the total amount of one or more antioxidants as 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:l, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, or about 30: 1.
  • hemeprotein compositions herein may be stable for at least 7 days at about 4°C.
  • hemeprotein compositions herein may comprise one or more hemeproteins herein that may comprise a heme group bound to oxygen, a heme group bound to carbon monoxide, or a combination thereof for at least 7 days.
  • hemeprotein composition results in an increase in peak height of the UV-visible absorption spectrum for the heme protein compositions at one or more of about 550 nm and about 582 nm.
  • the increase in peak height of the UV-visible absorption spectrum for the one or more heme protein compositions at one or more of about 550 nm and about 582 nm is detectable for at least about 7 days after addition of the one or more antioxidants.
  • the hemeprotein compositions herein may comprise one or more purified hemeproteins from a genetically modified non-animal source and one or more antioxidants herein that stabilizes the oxidation state of the one or more purified hemeproteins.
  • hemeprotein compositions herein may comprise one or more purified hemeproteins from a genetically modified non-animal source such as genetically modified plant, a genetically modified bacteria, or a genetically modified yeast, and one or more antioxidants herein that stabilizes the oxidation state of the one or more purified hemeproteins.
  • one or more purified hemeproteins herein may be recombinant hemeproteins produced from a genetically modified non-animal source.
  • recombinant hemeproteins herein may be encoded from a polynucleotide, wherein the polynucleotide may comprise an nucleic acid sequence from a plant, animal, fungus, or bacteria, encoding a hemeprotein.
  • the nucleic acid sequence encoding a hemeprotein herein may be derived from a legume.
  • the nucleic acid sequence encoding a hemeprotein herein may be derived from a leopard, a bovine, or a whale.
  • recombinant hemeproteins herein may be recombinant globin. In some embodiments, recombinant hemeproteins herein may be recombinant leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, phytoglobin, or any combination thereof.
  • one or more purified hemeproteins herein may be recombinant hemeproteins produced from a genetically modified yeast source, wherein the recombinant hemeproteins may be encoded from a polynucleotide, wherein the polynucleotide may comprise an endogenous nucleic acid sequence for a hemeprotein derived from a legume, an equine, a leopard, a bovine, a whale, or any combination thereof.
  • methods herein may stabilize an oxidative state of one or more purified hemeproteins from a non-animal source herein by combining the one or more purified hemeproteins with one or more antioxidants.
  • methods herein may stabilize the visual appearance of the purified hemeproteins for at least 7 days.
  • methods herein may stabilize the purified hemeproteins from aggregation at pH ranging from about 5 to about 9.
  • methods herein may stabilize the oxidation state of the purified hemeproteins for at least 7 days.
  • methods of making a hemeprotein composition herein for use in a food product may comprise obtaining one or more purified hemeproteins from a non-animal source and combining the one or more purified hemeproteins with at least one or more antioxidants herein.
  • methods herein may comprise recombinantly producing the one or more purified hemeproteins from a genetically modified non-animal source.
  • methods herein may comprise combining hemeproteins herein with about 0.01% to about 10% by weight of the composition of the one or more antioxidants.
  • methods herein may comprise combining antioxidants herein with about 1% to about 99% by weight of the composition of the one or more purified hemeproteins.
  • the method herein may comprise combining a comprise combining antioxidants herein with one or more purified hemeproteins wherein the hemeprotein composition results in an increase in peak height of the UV -visible absorption spectrum for the one or more hemeprotein compositions at one or more of about 550 nm and about 582 nm, that is detectable for at least about 7 days after addition of the one or more antioxidants.
  • methods herein may further comprise combining hemeproteins and antioxidants herein with in a buffer solution, wherein the buffer solution may have a pH ranging from about 5 to about 9.
  • the current disclosure also encompasses composition comprising one or more purified hemeprotein compositions and one or more antioxidants, wherein the one or more purified hemeprotein compositions comprise a hemeprotein from a genetically modified non-animal source selected from the group consisting of a genetically modified plant, a genetically modified bacteria, 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 heme protein compositions at one or more of about 550 nm and about 582 nm that is detectable for at least about 7 days after addition of the one or more antioxidants.
  • the one or more antioxidants has a reduction potential ranging from about 280 mv to about 500 mV. In certain embodiments, the one or more antioxidants is selected from ascorbic acid, quercetin, taxifolin, Trolox, and combinations thereof.
  • the composition comprises a weight ratio of the one or more purified hemeprotein to the one or more antioxidants of 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).
  • a weight ratio of the one or more purified hemeprotein to the one or more antioxidants of 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,
  • the hemeprotein of the compositions comprising the hemeprotein composition are encoded from a polynucleotide, wherein the polynucleotide comprises an endogenous nucleic acid sequence for a hemeprotein derived from a plant, animal, fungus, or bacteria source.
  • the endogenous nucleic acid sequence for the hemeprotein is derived from a legume.
  • the endogenous nucleic acid sequence for the hemeprotein is derived from an animal source selected from a group consisting of equine, leopard, bovine, or whale.
  • the hemeprotein is a globin selected from a group consisting of leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, and phytoglobin.
  • the one or more purified hemeproteins are recombinant hemeproteins produced from a genetically modified yeast source, wherein the recombinant hemeproteins are encoded from a polynucleotide, wherein the polynucleotide comprises an endogenous nucleic acid sequence for a hemeprotein derived from a legume, an equine, a leopard, a bovine, a whale, or any combination thereof.
  • the hemeprotein composition comprises a myoglobin and wherein combining the one or more purified hemeprotein compositions with one or more antioxidants causes an increase in the relative amount of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition.
  • the increase in relative amount of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition is detectable for at least about 7 days after addition of the antioxidant.
  • the increase in the relative amount of oxymyoglobin and carboxymyoglobin to metmyoglobin in the composition ranges from about 1.1 -fold to about 5-fold.
  • the current disclosure encompasses a hemeprotein composition comprising one or more purified hemeprotein and one or more antioxidants, wherein the one or more purified hemeprotein compositions comprise a hemeprotein from a genetically modified source selected from the group consisting of a genetically modified plant, a genetically modified bacteria, a genetically modified fungi, 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 heme protein compositions at one or more of about 550 nm and about 582 nm that is detectable for at least about 7 days after addition of the one or more antioxidants.
  • a genetically modified source selected from the group consisting of a genetically modified plant, a genetically modified bacteria, a genetically modified fungi, or a genetically modified yeast
  • the hemeprotein is encoded by a polynucleotide comprising a nucleic acid sequence from a plant, animal, fungus, or bacteria.
  • the polynucleotide comprises a nucleic acid sequence derived from a legume, wherein the nucleic acid sequence encodes a hemeprotein.
  • the polynucleotide sequence comprises a nucleic acid sequence derived from a group consisting of an equine, a feline, a bovine, or a whale.
  • the hemeprotein is a globin selected from a group consisting of leghemoglobin, non-symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, and phytoglobin.
  • the one or more purified hemeproteins are recombinant hemeproteins produced from a genetically modified yeast source, wherein the recombinant hemeproteins are encoded from a polynucleotide, comprising a nucleic acid sequence derived from a legume, an equine, a leopard, a bovine, a whale and encoding a hemeprotein.
  • the current disclosure also encompasses a composition comprising one or more purified hemeprotein compositions and one or more antioxidants, wherein the one or more purified hemeprotein compositions comprise leghemoglobin, non- symbiotic hemoglobin, chlorocruorin, erythrocruorin, protoglobin, cytochrome, cyanoglobin, flavohemoglobin, myoglobin, phytoglobin, or any combination thereof; wherein the one or more antioxidants is 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 hemeprotein to the one or more antioxidants of about 1:1 to about 30:1 (for example: about 1:1, about 2: 1, about 3:l, about 4:1, about 5:l, about 6:l, about 7:l, about 8:l, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1,
  • kits include meat substitute (e.g., meat replica) compositions and methods of making thereof.
  • methods of preparing a meat substitute comprise combining any of the compositions herein with a meat replica matrix.
  • methods that comprise combining any of the compositions herein to a meat replica matrix may result in the composition imparting a meat-like (e.g., a beef-like) appearance to the meat substitutes herein.
  • Fig. 1 depicts a representative graph of zeta-potential versus pH profile of equine heart myoglobin (1 mg/mL) measured by electrophoresis.
  • Fig. 2 depicts a representative graph of protein content of equine heart myoglobin (1 mg/mL) at pH 2.5 to 8.5.
  • Fig. 3A depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for 0 days.
  • Fig. 3B depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for
  • Fig. 3C depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for
  • Fig. 3D depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for
  • Fig. 3E depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for 4 days.
  • Fig. 3F depicts a representative graph of absorption spectra and representative image of visual appearance of equine heart myoglobin solutions ( ⁇ 1 mg/mL) stored at 4°C for
  • Fig. 4A depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions samples stored at 4°C in combination with (1 mM) Ascorbic Acid.
  • Fig. 4B depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution samples stored at 4°C in combination with (1 mM) Ascorbic Acid.
  • Fig. 4C are a series of representative images of visual appearance over time (from day 0 to day 27 as indicated) of equine heart myoglobin solutions samples stored at 4°C in combination with (1 mM) Ascorbic Acid.
  • Fig. 4D depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions samples stored at 4°C in combination with (1 mM) Quercetin.
  • Fig. 4E depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution samples stored at 4°C in combination with (1 mM) Quercetin.
  • Fig. 4F are a series of representative images of visual appearance over time (from day 0 to day 27 as indicated) of equine heart myoglobin solutionssamples stored at 4°C in combination with (1 mM) Quercetin.
  • Fig. 4G depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with (1 mM) Epigallocatechin gallate (EGCG).
  • EGCG Epigallocatechin gallate
  • Fig. 4H depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution samples stored at 4°C in combination with (1 mM) EGCG.
  • Fig. 41 depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions samples stored at 4°C in combination with (1 mM) Taxifolin.
  • Fig. 4 J depicts a representative graph of absorption spectra intensity (at 544 nm and
  • Fig. 4K depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions samples stored at 4°C in combination with (1 mM) 4-Methylcatechol.
  • Fig. 4L depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution samples stored at 4°C in combination with (1 mM) 4-Methylcatechol.
  • Fig. 4M are a series of representative images of visual appearance over time (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with antioxidants EGCG, Taxifolin and Methylcatechol respectively.
  • Fig. 4N depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with Trolox.
  • Fig. 40 depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution (1 mM) samples stored at 4°C in combination with Trolox.
  • Fig. 4P depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with Caffeic Acid.
  • Fig. 4Q depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution (1 mM) samples stored at 4°C in combination with Caffeic Acid.
  • Fig. 4R depicts a representative graph of absorption spectra (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with Gallic Acid.
  • Fig. 4S depicts a representative graph of absorption spectra intensity (at 544 nm and 582 nm) over time (from day 0 to day 27 as indicated) of equine heart myoglobin solution (1 mM) samples stored at 4°C in combination with Gallic Acid.
  • Fig. 4T are a series of representative images of visual appearance over time (from day 0 to day 27 as indicated) of equine heart myoglobin solutions (1 mM) samples stored at 4°C in combination with antioxidants Trolox, Caffeic Acid and Gallic Acid respectively.
  • FIG. 5A depict representative graphs of zeta potential of recombinant leopard, bovine, whale, and soy hemeprotein samples in solutions having a pH of 2.5, 3.5, 4.5, 5.5, 6.5,
  • Fig. 5B are a series of representative images of visual appearance of of recombinant leopard, bovine, whale, and soy hemeprotein samples in solutions having a pH of
  • Fig. 6 depicts a representative graph showing the impact of pH on the solubility of myoglobin solutions (0.5 mg/mL) produced by cellular agriculture.
  • Fig. 7A depicts representative graphs of absorption spectra of a 1 mg/mL solution of recombinant hemeprotein from Panthera pardus (leopard) stored at 4°C for 27 days.
  • Fig. 7B depicts representative graphs of absorption spectra of a 5mg/mL solution of recombinant hemeprotein from Panthera pardus (leopard) stored at 4°C for 27 days.
  • Fig. 7C are a series of representative images of visual appearance of 1 mg/mL and 5mg/mL solutions of recombinant hemeprotein from Panthera pardus (leopard) stored at 4°C for 27 days.
  • Fig. 7D depicts representative graphs of absorption spectra of a 1 mg/mL solution of recombinant hemeprotein from Bos taurus (bovine) stored at 4°C for 27 days.
  • Fig. 7E depicts representative graphs of absorption spectra of a 5mg/mL solution of recombinant hemeprotein from Bos taurus (bovine) stored at 4°C for 27 days.
  • Fig. 7F are a series of representative images of visual appearance of 1 mg/mL and 5mg/mL solutions of recombinant hemeprotein from Bos taurus (bovine) stored at 4°C for 27 days.
  • Fig. 7G depicts representative graphs of absorption spectra of a 1 mg/mL solution of recombinant hemeprotein from Physeter macrocephalus (sperm whale) stored at 4°C for 27 days.
  • Fig. 7H depicts representative graphs of absorption spectra of a 5mg/mL solution of recombinant hemeprotein from Physeter macrocephalus (sperm whale) stored at 4°C for 27 days.
  • Fig. 71 are a series of representative images of visual appearance of 1 mg/mL and 5 mg/mL solutions of recombinant hemeprotein from Physeter macrocephalus (sperm whale) stored at 4°C for 27 days.
  • Fig. 7J depicts representative graphs of absorption spectra of a 1 mg/mL solution of recombinant hemeprotein from soy leghemoglobin stored at 4°C for 27 days.
  • Fig. 7K depicts representative graphs of absorption spectra of a 5mg/mL solution of recombinant hemeprotein from soy leghemoglobin stored at 4°C for 27 days.
  • Fig. 7L are a series of representative images of visual appearance of 1 mg/mL and 5mg/mL solutions of recombinant hemeprotein from soy leghemoglobin stored at 4°C for 27 days.
  • the term “about,” can mean relative to the recited value, e.g , amount, dose, temperature, time, percentage, etc., ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ l%.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) 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 comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • nucleic acid refers to a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid.
  • the nucleic acid can be of genomic, cDNA, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • Transformation refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism, resulting in genetically stable inheritance.
  • Host organisms containing the transformed nucleic acid fragments are referred to as “recombinant”, “transgenic” or “transformed” organisms.
  • isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker.
  • Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally -regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.
  • ‘Stabilization” or “stabilized” in the context of hemeprotein compositions provided herein refers to a composition wherein the combination of hemeprotein with an antioxidant causes a lasting increase in the levels of oxygenated hemeprotein or carboxy hemeprotein, as indicated by a change in visual appearance to a more red composition, a change in the oxidation state (from Met state to oxygen or Carbon monooxide bound state), or a change in UV -Visible spectra such that the peak height at 550 nm (corresponding to Carboxy Mb), and/or at 582nm (corresponding to OxyMb) increases in comparison to a composition without antioxidant.
  • the increase in the peak height can be detected within 1-48 hours of combining the antioxidant with the hemeprotein. This period between adding the antioxidant and a detectible increase in the peak height is referred to as the lag-time or lag-phase. In some aspects, depending on the antioxidant used and the storage conditions, the increase in peak height can last or is “stably” maintained for about 0.5 days to about 90 days, though in some embodiments, the absolute peak height may decrease through this stable period.
  • Cellular Agriculture is a method of producing animal products from cell culture, rather than animals using a combination of biotechnology, tissue engineering, molecular biology, and synthetic biology to create and design new methods of producing proteins, fats, and tissues that would otherwise come from traditional agriculture.
  • hemeproteins can be sourced and purified from cellular agriculture.
  • a polynucleotide encoding a hemeprotein “derived from” or which is a “derivative of’ an endogenous polynucleotide refers to a polynucleotide related to the endogenous polynucleotide by sequence.
  • the polynucleotide may be a variant or comprise a fragment of the endogenous polynucleotide and may comprise mutations, insertions, deletions, truncations, modifications, or combinations thereof compared to an endogenous polynucleotide.
  • the polynucleotide may comprise a nucleic acid sequence at least about 60% identical to an endogenous polynucleotide or a fragment thereof, encoding a hemeprotein.
  • the term “source” refers to an organism that comprises a polynucleotide sequence encoding an endogenous or a recombinant hemeprotein which can be purified for use in the compositions and methods disclosed herein.
  • a source can comprise a polynucleotide sequence that encodes an endogenous hemeprotein, for example Glycine max (soybean) comprises a polynucleotide sequence encoding soy leghemoglobin which can be purified and used in the compositions and methods disclosed herein.
  • the source may be a genetically modified source.
  • the term “genetically modified source” refers to a recombinant organism for example a genetically modified plant, genetically modified fungi, genetically modified bacteria, genetically modified yeast, genetically modified algae, genetically modified archaea comprising a polynucleotide sequence encoding a hemeprotein and from which the hemeprotein can be purified for use in the compositions and methods of the current disclosure.
  • the recombinant organism comprises a polynucleotide sequence that is at least about 60% identical to an endogenous polynucleotide or a fragment thereof from a plant or at least about 60% identical to an endogenous polynucleotide or a fragment thereof from a bovine, equine, feline or whale.
  • compositions suitable for use in food products having improved color stability and reduced protein degradation over time.
  • the current disclosure results from the surprising result that addition of certain antioxidants to these compositions greatly increases the desirable characteristics of the hemeproteins.
  • compositions herein may have one or more purified hemeproteins and one or more antioxidants.
  • hemeprotein compositions suitable for use in food products herein may have one or more purified hemeproteins.
  • hemeprotein includes any polypeptide that can covalently or noncovalently bind to a heme moiety.
  • hemeproteins herein may be a monomer (i.e., a single polypeptide chain), a dimer, a trimer, tetramer, a higher order oligomer, or any combination thereof.
  • hemeproteins herein may be a globin.
  • globins that can covalently or noncovalently bind to a heme moiety for use herein can include an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a histoglobin, a neuroglobin, a chlorocruorin, a truncated hemoglobin (e.g., HbN, HbO, a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3)), a cytochrome, or a peroxidase.
  • HbN, HbO a truncated 2/2 globin
  • a hemoglobin 3 e
  • globins may have a globin fold having a series of about seven to about nine alpha helices.
  • globins may be of any class (e.g., class I, class II, or class III).
  • globins may transport and/or store oxygen.
  • hemeproteins herein may have an oxygenated Fe + state similar to that of globin (e.g., myoglobin). In some embodiments, hemeproteins herein may have an oxygenated Fe (iron) + state higher than globin (e.g., myoglobin). In some embodiments, hemeproteins herein may have an oxygenated Fe + state about 10%, 20%, 30%, 40%, 50%, 100% or higher than globin (e.g., myoglobin). In some embodiments, hemeproteins herein may be similar to oxymyoglobin. As used herein “oxymyoglobin” refers to the oxygenated form of myoglobin which is a single chain globular protein.
  • hemeproteins herein may be a non-symbiotic hemoglobin, a leghemoglobin, a chlorocruorin, an erythrocruorin, a protoglobin, a cytochrome, a cyanoglobin, a flavohemoglobin, a myoglobin, a phytoglobin, or any combination thereof.
  • hemeproteins herein may be derived from non-animal sources.
  • non-animal sources include plants, fungi, bacteria, yeasts, algae, archaea, genetically modified organisms such as genetically modified bacteria, plants, or yeast, chemical or in vitro synthesis.
  • hemeproteins herein may be a polypeptide derived from non-animal sources.
  • hemeproteins herein may be a polypeptide expressed and/or secreted from a non-animal source.
  • hemeproteins herein may be a polypeptide expressed and/or secreted from a non-animal source wherein the polypeptide may be encoded from a polynucleotide derived from animals, plants, fungi, bacteria, yeasts, algae, archaea, or any combination thereof.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a “wild-type” source.
  • a wild-type source of hemeproteins herein may be mammals, fish, birds, plants, algae, fungi (e.g., yeast or filamentous fungi), ciliates, bacteria, or any combination thereof.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a mammal belonging to any of the 27 orders of mammalian species, the orders including: Afrosoricida; Carnivora; Cetartiodactyla; Chiroptera; Cingulata; Dasyuromorphia; Dermoptera; Didelphimorphia; Diprotodontia; Eulipotyphla; Hyracoidea; Lagomorpha; Macroscelidea; Microbiotheria; Monotremata; Notoryctemorphia; Paucituberculata; Peramelemorphia; Perissodactyla; Pholidota; Pilosa; Primates; Proboscidea; Rodentia; Scandentia; Sirenia; and Tubulidentata.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from human, non-human primate (e.g., gibbon, rhesus macaque, bonobo, chimpanzee, gorilla, orangutan, lemur, loris, tarsier), bovinae (e.g., cow, zebu, bison, water buffalo, African buffalo, antelopes), ovine, caprine, camelid, canine (e.g., domestic dog, wolves, coyotes, jackals, foxes), cetacean (e.g., whales, dolphins, porpoises), feline (e.g., domestic cat, tiger, lion, cheetah, leopardjaguar, bobcat, caracal, margay, oncilla, cougar, serval, ocelot, lynx, puma), e
  • non-human primate
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a mammal such as a cow, goat, sheep, horse, pig, ox, mule, rabbit, yak, llama, camel, deer, cat, dog, bear, or any combination thereof.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a bird. In some embodiments, hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from Anseriformes (e.g., ducks, swans, geese), Falconiformes (e.g., falcons, eagles, hawks) Galliformes (e.g.
  • Anseriformes e.g., ducks, swans, geese
  • Falconiformes e.g., falcons, eagles, hawks
  • Galliformes e.g.
  • Struthioniformes e.g., emus, ostriches, kiwis
  • Passeriformes e.g., perching birds and songbirds such as sparrows, larks, crows, swallows, and the like
  • Sphenisciformes e.g., penguins
  • Pelecaniformes e.g., Ibis, herons, pelicans
  • Strigiformes e.g., owls
  • Gaviiformes e.g., loons
  • Gruiformes e.g., terrestrial, marsh birds
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a fish.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a Scombridae (e.g., tuna), Salmonidae (e.g., salmon), Gadidae (e.g., cod, haddock), Clupeidae (e.g., herrings, shads, sardines, hilsa, menhadens), Engraulidae (e.g., anchovies), and the like.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from shrimp, oysters, clams, mussels, and the like.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from a plant.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (com), Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicer arietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such as garden peas or sugar snap peas, Phaseolus vulgaris varieties of common beans such as green beans, black beans, navy beans, northern beans, or pinto beans, Vigna unguiculata varieties (cow peas), Vigna radiate (Mung beans), Lupinus albus (lupin), or Medicago sativa (alfalfa); Brassica napus (cano
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from fungi. In some embodiments, hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, or Fusarium oxysporum.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from bacteria. In some embodiments, hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from Escherichia coli, Bacillus subtilis, Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or thermophilic bacteria such as Thermophilus.
  • hemeproteins herein may be isolated from or may be encoded from a polynucleotide derived from non-symbiotic hemoglobin.
  • hemeproteins herein may be non-symbiotic hemoglobins isolated from or encoded from a polynucleotide derived from soybean, sprouted soybean, alfalfa, golden flax, black bean, black eyed pea, northern, garbanzo, moong bean, cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheat berries, spelt, barley, wild rice, rice, or any combination thereof.
  • hemeproteins described herein may have an amino acid sequence corresponding to a wild-type hemeprotein, fragments, truncations, variants or fusions thereof that contain a heme-binding motif.
  • amino acid sequences of any of the wild-type hemeproteins contemplated herein can be found in sequence databases such as, but not limited to, the UniProtKB/Swiss-Prot database and the Heme Protein Database.
  • hemeproteins described herein may have an amino acid sequence with at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence corresponding to a wild-type hemeprotein, fragments, truncations, variants or fusions thereof that contain a hemebinding motif.
  • a BLAST 2 Sequences B12seq
  • National Center for Biotechnology Information www.ncbi.nlm.nih.gov
  • hemeproteins herein may be from a genetically modified non-animal source.
  • a genetically modified non-animal source may be a genetically modified plant, a genetically modified bacteria, a genetically modified yeast, or any combination thereof.
  • hemeproteins herein may be recombinant hemeproteins.
  • recombinant hemeproteins refers to hemeproteins recombinantly produced using polypeptide expression techniques (e.g., heterologous expression techniques using bacterial cells, insect cells, fungal cells such as yeast, plant cells such as tobacco, soybean, or Arabidopsis, or mammalian cells).
  • recombinant hemeproteins herein may be a polypeptide encoded from a polynucleotide, wherein the polynucleotide may have an endogenous (i.e., wild-type) nucleic acid sequence for a hemeprotein derived from a plant, animal, fish, bird, fungus, or bacteria source as described herein.
  • an endogenous nucleic acid sequence for the hemeprotein may be derived from a plant.
  • an endogenous nucleic acid sequence for the hemeprotein may be derived from a legume.
  • an endogenous nucleic acid sequence for the hemeprotein may be derived from a mammal.
  • an endogenous nucleic acid sequence for the hemeprotein may be derived from an equine, feline (e.g., leopard), bovine, or cetacean (e.g., whale).
  • standard polypeptide synthesis techniques e.g., liquid-phase polypeptide synthesis techniques or solid-phase polypeptide synthesis techniques
  • in vitro transcription-translation techniques may be used to produce any of the recombinant hemeproteins herein
  • recombinant hemeproteins may be expressed by a microbial expression system.
  • microbial expression systems for use herein may comprise at least one expression vector.
  • Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of recombinant proteins are well known to those skilled in the art, any of which may be used to produce the any one of the gene products (e.g., recombinant hemeproteins) of the polynucleotides disclosed herein.
  • Vectors or cassettes useful for the 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 vehicle for introducing a nucleic acid into a host cell.
  • a nucleic acid for use herein can be one that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid.
  • expression vectors for use herein can be part of a plasmid, virus, or nucleic acid fragment, or other suitable vehicle.
  • expression vectors for use herein may further include a nucleic acid to be transcribed operably linked to a promoter.
  • vectors comprising polynucleotides disclosed herein can be introduced into appropriate microorganisms (i.e., host cells) via transformation techniques to provide high-level expression of the recombinant hemeproteins for use herein.
  • Expression of a polypeptide (e.g., recombinant hemeprotein) of the disclosure may include transient expression and/or constitutive expression (e.g., developing of a stable cell line) in a suitable host cell.
  • Host cells herein may be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation and silicon carbide whisker transformation.
  • Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel. M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the like.
  • a suitable host cell for production of recombinant hemeproteins herein may be from a genetically modified organism.
  • a genetically modified organism herein may be a bacterium, a yeast, a fungus, an algae, a mammalian cell, an insect cell, or any combination thereof.
  • a suitable host cell for production of recombinant hemeproteins herein may be from a genetically modified plant, a genetically modified bacteria, and/or a genetically modified yeast.
  • a genetically engineered organism suitable for production of recombinant hemeproteins herein may be Acetobacter, Acinetobacter calcoaceticus, Alcaligenes eutropha, Arxula adeninivorans , Aspergillus nidulans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium spp., Bacillus licheniforms , Bacillus methanolicus , Bacillus stearothermophilus, Bacillus subtilis, Candida utilis, Chlamydomonas reinhardtii, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium glutamicum, Escherichia coli, Hansenula polymorpha, Isochrysis spp., Kluyveromyces lactis, Kluyveromyces marxianus, Lactococc
  • a host cell following introduction of a polynucleotide comprising the coding sequence for a hemeprotein of the disclosure, a host cell may be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, and/or amplifying expression of a polypeptide-encoding polynucleotide.
  • a host cell following the introduction of a polynucleotide comprising the coding sequence for a hemeprotein of the disclosure, may be cultured by fermentation.
  • Culturing may be accomplished in a growth medium having one or more supplements to aid in culture growth including, but not limited to, aqueous mineral salts medium, organic growth factors, carbon and/or energy source material, molecular oxygen, and the like.
  • aqueous mineral salts medium including, but not limited to, aqueous mineral salts medium, organic growth factors, carbon and/or energy source material, molecular oxygen, and the like.
  • polypeptides e.g., hemeproteins
  • hemeproteins for use herein may be purified hemeproteins.
  • purified refers to a polypeptide or protein (e.g., a hemeprotein) that has been separated from other components of the source material (e.g., other animal, fish, plant, fungal, algal, bacterial, genetically modified plant, genetically modified bacteria, or genetically modified yeast proteins).
  • purified hemeproteins herein may be free of 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 the other components of the source material.
  • Hemeproteins herein can be purified using methods of protein separation known in the art, including but not limited to, size exclusion chromatography, affinity chromatography, anion exchange chromatography, cation exchange chromatography, ultrafiltration through membranes, or density centrifugation, isoelectric precipitation, ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, and solvent extraction.
  • compositions suitable for use in food products herein may have one or more purified hemeproteins and one or more antioxidants.
  • antioxidants are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process.
  • antioxidants suitable for use herein may be an antioxidant vitamin, a polyphenol, or any combination thereof.
  • an antioxidant herein may be a naturally occurring or a synthetic form of a vitamin having antioxidant properties.
  • an antioxidant vitamin may be vitamin C, a derivative thereof, and/or an analogue thereof.
  • an antioxidant vitamin may be ascorbic acid, L- ascorbic acid, ethylated L-ascorbic acid, vitamin C, or of the erythorbic acid isomer thereof, or salts or esters thereof.
  • an antioxidant vitamin may be vitamin E, a derivative thereof, and/or an analogue thereof.
  • Vitamin E is a group of eight fat soluble compounds that include four tocopherols and four tocotrienols.
  • an antioxidant vitamin may be alpha-Tocopherol, beta-Tocopherol, gamma-Tocopherol, delta-Tocopherol, Tocopheryl acetate, RRR-alpha-tocopherol, SSR- alpha-tocopherol, alpha-tocotrienol, vitamin E, or salts or esters thereof.
  • an antioxidant herein may be a naturally occurring or a synthetic form of a polyphenol having antioxidant properties.
  • Polyphenols are common constituents of foods of plant origin and contribute the major antioxidants found in diets.
  • the main dietary sources of polyphenols include, but are not limited to, fruits, vegetables, and beverages (e.g., coffee).
  • antioxidants are polyphenolic compounds, chlorogenic acids, flavonoids, tocopherols, di- or tri-carboxylic acids (such as citric acid), EDTA (ethylenediaminetetraacetic acid), ascorbic acid (vitamin C), anthocyanins, catechins, quercetin, resveratrol, rosmarinic acid, camosol, Maillard reaction products, enzymes such as superoxide dismutase, certain proteins, amino acids, and protein hydrolyzates, etc.
  • antioxidant polyphenols herein may be a naturally occurring or a synthetic form of a flavonoid having antioxidant properties.
  • flavonoids include quercetin (found in onion, tea, apple), catechin (tea, fruit), hesperidin (citrus fruits), and cyanidin (red fruits).
  • antioxidant flavonoids herein may be isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, taxifolin, catechin, gallocatechin, gallocatechin gallate esters, epicatechin, epigallocatechin, epigallocatechin gallate esters, theaflavin, theaflavin gallate esters, thearubigins, or any combination thereof.
  • antioxidant polyphenols herein may be a naturally occurring or a synthetic form of a phenolic acids having antioxidant properties.
  • Anon-limiting example of a phenolic acid includes caffeic acid which is present in many fruits and vegetables. Caffeic acid, most often esterified with quinic acid as in chlorogenic acid, is the major phenolic compound in coffee.
  • an antioxidant herein may be ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite and other materials known to one of ordinary skill in the art.
  • an antioxidant herein may be ascorbic acid, quercetin, epigallocatechin gallate, EGCG, trolox, taxifolin, 4-methycatechol, caffeic acid, gallic acid, or any combination thereof.
  • antioxidants for use herein may have one or more characteristics that impart a desirable property to the compositions described herein.
  • antioxidants for use herein may have a strong reducing potential. Standard reduction potential describes the ability of a compound to accept electrons.
  • antioxidants for use herein may have reduction potential less than about 500 mV (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, 500 mV).
  • antioxidants for use herein may have reduction potential less than about 200, or 250, or 300, or 350, or 400, or 450, or 500 mV.
  • hemeprotein compositions as described herein may include one or more purified hemeproteins.
  • the compositions described herein may comprise 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%) by weight of the composition of one or more purified hemeprotein compositions, as disclosed herein.
  • a “hemeprotein composition” includes the hemeprotein hydrated or in solution.
  • the hemeprotein content can be calculated on a dry basis, meaning the hemeprotein content and concentration is determined with the liquid from the hemeprotein composition removed.
  • the compositions described herein may comprise 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%) by weight of the composition of one or more purified hemeproteins on a dry weight basis.
  • compositions described herein may comprise 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%) by weight of the composition of one or more antioxidants disclosed herein.
  • compositions herein may comprise about 1% to about 99% (e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) by weight of the composition of one or more purified hemeproteins disclosed herein 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%) by weight of the composition of one or more antioxidants disclosed herein.
  • compositions herein may comprise a ratio of total hemeprotein amount to total antioxidant amount.
  • compositions herein may have a weight ratio of total hemeprotein composition content to antioxidant content ranging from about 1 :1 to about 30: 1.
  • the eight ratio of total hemeprotein content to total antioxidant content is 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: l, 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..
  • the composition comprises a leghemoglobin and one or more antioxidants.
  • the composition comprises a soy leghemoglobin and one or more antioxidant selected from any one of Quercetin, Ascorbic acid, EGCG, Trolox or Taxifolin.
  • the compositions 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..
  • the composition comprises a myoglobin and one or more antioxidants.
  • the composition comprises one or more myoglobin selected from an equine, bovine, feline (for example leopard) or whale (for example, sperm whale) and one or more antioxidant selected from any one of Quercetin, Ascorbic acid, EGCG, Trolox or Taxifolin.
  • compositions 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.
  • hemeprotein compositions herein may have one or more purified hemeproteins as disclosed herein and one or more antioxidants as disclosed herein in a buffer solution.
  • a buffer solution for use herein may be any solution suitable for use in a food product.
  • hemeprotein compositions herein may also include a buffer solution having or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents for use herein can include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art.
  • compositions disclosed herein may comprise 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% total amount of one or more buffering agents by total weight of the composition.
  • the amount of one or more buffering agents may depend on the desired pH level of compositions herein.
  • buffer solutions herein may 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).
  • compositions disclosed herein may 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).
  • the hemeprotein compositions herein may comprise additional components for example, binding agents, flavor enhancers, oligosaccharides, stabilizing agents, pH regulators, preservatives, non-heme proteins, dietary fibers, gelling agents, surfactants, water, fats, oils emulsifiers, starches, coloring agents and combinations thereof.
  • the additional components may include for example, one or more of, glucose, fructose, ribose, arabinose, glucose-6-phosphate, fructose 6-phosphate, fructose 1,6-di phosphate, inositol, maltose, sucrose, maltodextrin, glycogen, nucleotide-bound sugars, molasses, a phospholipid, a lecithin, inosine, inosine monophosphate (IMP), guanosine monophosphate (GMP), pyrazine, adenosine monophosphate (AMP), lactic acid, succinic acid, glycolic acid, thiamine, creatine, pyrophosphate, vegetable oil, algal oil, com oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cottonseed oil, sunflower oil, canola oil, olive oil, a free fatty acid, cysteine, methi
  • the hemeprotein compositions herein comprising one or more of the antioxidants described herein and one or more of the hemeproteins described herein can be formulated into for example, liquids, gels, pastes, sauces, powder or cubes, ingredients of flavor packets, seasoning packets or shakers.
  • the compositions herein may be formulated into or added to, for example, soup or stew bases, bouillon or broths.
  • the present disclosure provides for hemeprotein compositions suitable for use in food products having improved color stability and reduced protein degradation over time.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins.
  • the iron atom in the heme group of a hemeprotein can be in the ferrous (Fe 2+ ) oxidation state to support oxygen and other gases’ binding and transport. Hemoglobin in normal red blood cells is protected by a reduction system to stabilize these states. Initial oxidation to the ferric (Fe 3+ ) state without oxygen converts hemoglobin into “methemoglobin” which cannot bind oxygen.
  • the hemeprotein myoglobin can exist in an oxygen bound ferrous (Fe 2+ ) state referred herein as Oxymyoglobin (OxyMb), in a deoxygenated state referred herein as Deoxymyoglobin (DeoxyMb), a state bound to carbon monoxide referred herein as Carboxymyoglobin (CarboxyMb) or in the ferric state referred to as Metmyoglobin (MetMb).
  • Changes in the oxidative states of hemeprotein can be determined from the UV -visible absorption spectra, for example of a solution comprising the composition disclosed herein.
  • the absorption spectra may comprise peaks consistent with CarboxyMb (550 nm), OxyMb (582 nm), and MetMb (503 nm and 632 nm).
  • change in the oxidative states can also result in change in the appearance of the solution comprising the composition disclosed herein.
  • a solution comprising higher amounts of CarboxyMb or OxyMb is characterized by a brighter red color, while MetMb results in a more undesirable brown coloration.
  • presence of antioxidant results in change and/or stabilization of the oxidative state of the hemeprotein.
  • hemeprotein compositions herein may comprise antioxidants to act as a reduction system for changing and/or stabilization of the oxidation state of the one or more purified hemeproteins.
  • “Stabilization” or “stabilized” in context of hemeprotein compositions provided herein refers to a composition wherein the combination of hemeprotein with an antioxidant causes a lasting increase in the levels of oxygenated hemeprotein or carboxy hemeprotein, as indicated by a change in appearance to a more red composition, or a change in UV-Visible spectra such that the peak height at 550 nm (corresponding to CarboxyMb), and/or at 582nm (corresponding to OxyMb) increases in comparison to a composition without antioxidant.
  • the increase in the peak height can be detected within 1-48 hours of combining the antioxidant with the hemeprotein. This period between adding the antioxidant and a detectible increase in the peak height is refered to as the lag-time or lag-phase. In some aspects, depending on the antioxidant used and the storage conditions, the increase in peak height can last or is “stably” maintained for about 0.5 days to about 90 days, though in some embodiments, the absolute peak height may decrease through this stable period. A guidance for some exemplary antioxidants and their impact on peak heights is provided in the Examples included herein.
  • the current disclosure encompasses hemeprotein compositions comprising a hemeprotein and antioxidant, wherein the antioxidant causes an increase in relative amount of oxygenated or carboxygenated hemeprotein compared to the oxidized Met state in the composition.
  • the hemeprotein is a myoglobin and the antioxidant causes an increase in relative amount of oxymyoglobin to metmyoglobin in the composition as measured by the change in the UV-visible spectrum of a solution of the composition.
  • the increase in relative amount of oxymyoglobin to metmyoglobin in the composition is detectable at about 0.5 to about 90 days, or at 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 after addition of the antioxidant and stored at refrigeration temperatures (e.g., about 2°C to about 8°C).
  • the increase in the relative amount of oxymyoglobin to metmyoglobin in the composition is at least about 1.1 to about 5-fold, or about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 3, about 4 or about 5-fold.
  • the antioxidant causes a change in the UV-visible absorption spectrum with an increase in peak height at about 550 nm and about 582 nm, at 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 addition of the antioxidant and stored in refrigeration (e.g., about 2°C to about 8°C).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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.
  • compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins for at least about 7 days (e.g., about 0.5, 1, 2, 3, 4, ,5 ,6, 7 days).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 temperatures below freezing (e.g., below 0°C).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 in refrigeration (e.g., about 2°C to about 8°C).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 room temperature (e.g., about 22°C to about 27°C).
  • compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 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).
  • antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 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
  • compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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 4°C.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins for about 7 days when the hemeprotein composition is stored at about 4°C.
  • compositions herein may comprise antioxidants for stabilization of the oxidation state of the one or more purified hemeproteins 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.
  • hemeprotein compositions herein may comprise antioxidants and hemeproteins wherein the heme group of the hemeprotein is bound to oxygen, carbon monoxide, or a combination thereof.
  • hemeprotein compositions herein may comprise antioxidants and hemeproteins wherein the heme group of the hemeprotein 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.
  • hemeprotein compositions herein may comprise antioxidants and hemeproteins wherein the heme group of a higher fraction of hemeprotein is 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, or at least about 9, or at least about 10, or at least about 11, at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20 or more days.
  • compositions herein may comprise antioxidants and hemeproteins wherein the heme group of a higher fraction of the hemeprotein 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 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).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins.
  • a stabilized visual appearance of a hemeprotein herein refers to the appearance of a red composition in visible light.
  • a destabilized visual appearance of a hemeprotein herein refers to the appearance of a brown composition in visible light.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins 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.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins 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, or at least about 9, or at least about 10, or at least about 11, at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30 or more days.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins 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, or at least about 9, or at least about 10, or at least about 11, at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30 or more days, when the hemeprotein 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
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins, wherein intensity of the red color of the composition decreases slowly over time compared to compositions only comprising hemeproteins.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins, wherein intensity of the red color of the composition decreases by about 0.5% to about 70% after about 30 days (e.g., about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30 days).
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins, wherein intensity of the red color of the composition decreases in a linear manner per day after making the compositions according to the methods herein.
  • hemeprotein compositions herein may comprise antioxidants for stabilization of the visual appearance of the one or more purified hemeproteins, wherein intensity of the red color of the composition decreases by about 0.001% to about 0.5% per day after making the compositions according to the methods herein.
  • hemeprotein compositions herein comprising one or more antioxidants herein and one or more hemeproteins herein may have almost no protein degradation of the hemeprotein.
  • hemeprotein compositions herein comprising one or more antioxidants herein and one or more hemeproteins herein may 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 the hemeprotein.
  • hemeprotein compositions herein comprising one or more antioxidants herein and one or more hemeproteins herein may 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 the hemeprotein 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.
  • hemeprotein compositions herein comprising one or more antioxidants herein and one or more hemeproteins herein may 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%)) protein degradation of the hemeprotein 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).
  • about 0.01% to about 15% e.g., protein degradation of the hemeprotein
  • hemeprotein compositions herein can be used for producing meat substitute food products (“meat replicas” or “meat analogs”).
  • meat substitute food products meat substitute food products
  • the term “meat replica” or “meat analog” has the same meaning as commonly understood by one of ordinary skill in the art and includes but is not restricted to plant-derived meat, vegan meat, meat substitute, mock meat, meat alternative, imitation meat, vegetarian meat, fake meat or faux meat.
  • the terms “meat replica” or “meat analog” refer to a food product aiming to have a realistic meat-like appearance without containing an animal-based component.
  • compositions herein can be used as a materials in and in methods of making meat replicas, including, but not limited to ground meat replicas (e.g., ground beef, ground chicken, ground turkey, ground lamb, or ground pork), as well as replicas of cuts of meat and fish.
  • ground meat replicas e.g., ground beef, ground chicken, ground turkey, ground lamb, or ground pork
  • methods of making food products may include combining the hemeprotein compositions herein with non-animal-based fat, nonanimal-based matrixes, non-animal-based edible fibrous components, or any combination thereof.
  • methods of making food products may include combining the compositions herein with non-animal-based fat, non-animal-based matrixes, non-animal-based edible fibrous components, or any combination thereof wherein the addition of the composition herein provides a meat-like appearance to the meat substitute.
  • methods of making food products may include combining the compositions herein with non-animal-based fat, non-animal-based matrices, non-animal-based edible fibrous components, or any combination thereof wherein the addition of the composition herein provides a meat-like taste to the meat substitute.
  • methods of making food products may include combining the compositions herein with non-animal-based fat, non-animal-based matrixes, non-animal- based edible fibrous components, or any combination thereof wherein the addition of the composition herein provides a meat-like smell to the meat substitute.
  • a meat-like appearance, taste, or smell may 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 lamblike appearance, taste, or smell, or any combination thereof.
  • the methods of making food-products may include combining the compositions herein with non-animal-based meat-like, poultry-like, or seafoodlike base dough, that can be used in meat replicas sold in a form such as “ground meat”, burgers/patties, or other forms, for example comparable to Impossible® Burger (from ImpossibleTM Foods), Beyond Burger® (from Beyond Meat®), Veggie Chik Patty® (from Morningstar Farms®), and Plant-Based Patties from Good & GatherTM.
  • Impossible® Burger from ImpossibleTM Foods
  • Beyond Burger® from Beyond Meat®
  • Veggie Chik Patty® from Morningstar Farms®
  • Plant-Based Patties from Good & GatherTM for example comparable to Impossible® Burger (from ImpossibleTM Foods), Beyond Burger® (from Beyond Meat®), Veggie Chik Patty® (from Morningstar Farms®), and Plant-Based Patties from Good & GatherTM.
  • poultry, meat and seafood analog products that may include compositions provided herein include products like Veggie Meal Starters® from Morningstar Farms®, such as Veggie CHIK’N Nugget, Veggie Popcorn CHIK’N, Veggie CHIK’N Strips, Veggie Grillers®, Veggie Buffalo, beef analogue products made by Beyond Meat® products such as Beyond Beef® Crumbles, Beyond Beef® Ground Beef, and Beyond Beef® Sausage, or fish analog products made by Good Catch like salmon burgers, fish sticks, fish fillets, crab cakes, fish burgers and fish cakes.
  • Veggie Meal Starters® from Morningstar Farms®
  • Veggie CHIK’N Nugget Nugget
  • Veggie Popcorn CHIK’N Veggie CHIK’N Strips
  • Veggie Grillers® Veggie Buffalo
  • beef analogue products made by Beyond Meat® products such as Beyond Beef® Crumbles, Beyond Beef® Ground Beef, and Beyond Bee
  • the methods of making food-products may include combining the liquids, gels, pastes, sauces, powder or cubes, ingredients of flavor packets, seasoning packets or shakers, soup or stew bases, bouillon or broths into a food product before, during, or after cooking of the consumable food product.
  • the compositions herein can be used to modulate the flavor and/or aroma profile for a variety of consumable food products.
  • food products herein may comprise about 1% to about 99% (e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of any of the hemeprotein compositions herein by weight.
  • food products herein e.g., meat replicas
  • food products herein may comprise about 1% to about 99% (e.g., about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%) of any of the hemeproteins herein by weight.
  • equine heart myoglobin solution 50 mg/ml of equine heart myoglobin solution was prepared by dispersing a weighed mass of the protein powder into a buffer solution (2.5 mM sodium phosphate/2.5 mM histidine, pH 9.0). The resulting myoglobin solution was stirred continually for at least 1 hour to ensure the protein was fully 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 ferricyanide (5 mg/mL) was added to the myoglobin solution and then the mixture was stirred for 1 hour in an ice bath (4°C). Any residual ferricyanide was then removed from the mixture using a desalting column (Sephadex G-25 PD-10).
  • MetMb metmyoglobin
  • OxyMb oxymyoglobin
  • the desalted MetMb solution was diluted to 2 mg Mb/mL with buffer solution (2.5 mM sodium phosphate/2.5 mM histidine, pH 9.0) and then reduced by adding either sodium hydrosulfite (0.5 mg/mL) or antioxidant (1 mM) solutions. This was achieved by adding a weighed mass of the reducing agent to the OxyMb solution and mixing well.
  • any excess sodium hydrosulfite was subsequently removed using a desalting column (Sephadex G-25 PD-10).
  • a desalting column Sephadex G-25 PD-10
  • non-water-soluble antioxidants caffeic acid, quercetin, or taxifolin
  • ethanol 5 to 10% total volume
  • the reduction potential and solubility of antioxidants used are listed in Table 1.
  • the pH of the myoglobin solutions was measured after incubation for at least 2 hours. All samples were stored at 4°C in screw-capped glass vials or 1.5 mL plastic cuvettes with a lid for up to 52 days.
  • the zeta-potential ( ⁇ -potential) of the protein solutions was measured using a particle electrophoresis instrument (Zetasizer Pro, Malvern Instruments Ltd., Worcestershire, UK).
  • the ⁇ -potential was measured from pH 8.5 to 2.5 by titrating the initial solutions with either acid (0.1 to 0.25 M HC1) or alkaline (0.25 M NaOH) solutions with continuous stirring.
  • Solubility The pH-dependence of the solubility of the proteins in the myoglobin solutions was determined by measuring the soluble and total protein concentrations over a range of pH values. The myoglobin solutions (0.5 mg/mL) were stored overnight (4°C) and then the protein concentrations were measured using a Bradford assay kit. The total protein concentration (c to tai) was determined by directly analyzing the myoglobin solutions, whereas the soluble protein concentration (c so iubie) was determined by centrifuging them at 13,000 rpm for 15 minutes and then analyzing the supernatant. The protein concentration measurements were performed according to the instructions provided with the Bradford assay kit.
  • the myoglobin redox state proportions (deoxygenated myoglobin (DeoMb), oxygenated myoglobin (OxyMb), and metmyoglobin (MetMb)) in the myoglobin systems containing antioxidant were calculated according to the modified Krzywicki equations by similar to that described in Tang et al., Journal of Food Science, 69(9) (2004).
  • the 3D structures of four ligands were built using Maestro 3D Builder. All the ligand structures were then prepared with LigPrep with OPLS3 force field. Ligand chirality was maintained, and possible states at the target pH 8.0 ⁇ 1.0 were generated. A docking grid was created and centered on the heme group, which enabled the entire myoglobin to be covered by adjusting the size of ligand diameter midpoint box, and no other constraint was applied. Docking experiments were then performed using ligand docking with extra precision (XP) mode.
  • XP extra precision
  • the number of poses to be reported was not limited, but post-docking minimization was applied with 20 poses per ligand included with strain correction terms. Then, all the reported results were ranked by docking scores, and poses with a docking score above zero were excluded. Only the specific configurations (binding site and binding pose) with the highest Glide docking scores (most negative) in docking experiments are discussed and illustrated in the Results section.
  • an initial characterization of the physical properties, including electrical charge, solubility and stability of an exemplary myoglobin was conducted at different pH.
  • the electrical characteristics of equine heart myoglobin were determined by measuring the pH-dependence of their ⁇ -potential values.
  • the isoelectric point (pl) of each protein was determined from the point where their net charge was zero.
  • the isoelectric point of the equine heart myoglobin (1 mg/mL) was around pH 5.5 (Fig. 1). Visual observation indicated that a thin sediment layer formed at the bottom of the samples around and below pH 5.5 (data not shown), which can be attributed to aggregation of the protein.
  • Fig. 3A The UV-visible absorption spectra of equine heart myoglobin (OxyMb and MetMb) solutions without antioxidant are shown in Fig. 3A.
  • the oxymyoglobin spectrum had two distinct absorption peaks around 544 and 582 nm, which are responsible for the bright red color of these solutions.
  • the metmyoglobin only had relatively small absorption peaks around 503 and 632 nm, which are responsible for its brown color.
  • the oxymyoglobin solutions only remained bright red for about 1 day before turning brown and exhibiting a UV-visible spectrum similar to that of metmyoglobin.
  • Figs. 3B-3F shows the concentration of oxygenated myoglobin decreased gradually over time and metmyoglobin concentration increased to about 100% on day 5.
  • the origin of this effect may have been due to the delay in the ability of the ascorbic acid to reach the heme group. Consequently, the observed lag-time may have been due to the time taken for the ascorbic acid molecules to move from the aqueous solution to the active site where they could reduce the heme group. Without wishing to be bound by theory, the longer lag-time observed in the samples herein may have been because they were stored at 4°C, which could have slowed down the reaction kinetics. Moreover, the lag-time time observed the myoglobin solutions herein may be related to the “bloom” time observed in freshly cut meat, i.e., the time for the interior of the meat to turn from brown to red due to oxygenation of the myoglobin. Fig.
  • Deoxygenated myoglobin (DeoMb) concentration was increasing at day 10 and onward. This occurrence could be due to depletion of oxygen around the myoglobin molecules and formation of a low partial oxygen pressure environment, as ascorbic acid is an oxygen scavenger, and hence the DeoMb conversion from MetMb.
  • Quercetin a natural hydrophobic antioxidant, was also studied for its potential in stabilizing the oxymyoglobin.
  • 1 mM quercetin originally dissolved in ethanol
  • the lag-time for the red color to stabilize was around 2 to 10 hours (data not shown).
  • the quercetin was observed to have a shorter lag-time.
  • EGCG Epigallocatechin gallate
  • Ascorbic acid' For ascorbic acid, three lysines (K42, K47, and K98) and an aspartic acid (D44) on the myoglobin were involved in the binding interaction. In terms of the nature of the bonds, a salt-bridge and five hydrogen bonds were formed between hydroxy 1/carbonyl groups on the ascorbic acid and these amino acids on the protein.
  • Quercetin' For quercetin, two lysines (K96 and K98), an aspartic acid (D44), and a glutamic acid (E41) participated in the binding interactions. In this case, several hydrogen bonds were formed between the hydroxy 1/carbonyl groups on the quercetin and the amino acids on the protein.
  • Gallic acid' For gallic acid, three lysines (K42, K96, and K98) and an aspartic acid (D44) participated in the binding interaction. In this case, two salt-bridges were formed between the carboxylate on the gallic acid and the K42/K98 side chains. In addition, three hydrogen bonds were formed between the hydroxyl groups on the gallic acid and the K47/D44 side chains and K96 carbonyl on the protein.
  • 4-methylcatechol Also performed was a molecular docking experiment for 4- methylcatechol, since this compound did not yield a typical oxymyoglobin spectra when added to the myoglobin solution (Figs. 4K and 4L). Indeed, this compound was bound to a different site on the protein molecule, near to the K34 and E52 residues, which is far away from the heme group. There were only two hydrogen bonds formed between the hydroxyl groups on the 4-methylcatechol and the K34/E52 side chains on the protein. The different binding site for the 4- methylcatechol may be the reason for the different absorption spectra measured in the mixed system.
  • myoglobin samples (leopard, bovine, sperm whale, and soy legume) generated using a cellular agriculture (fermentation) approach were characterized.
  • the electrical charge, color, solubility, and redox stability of the proteins was measured using similar methods as for the equine heart myoglobin discussed in Examples 1 and 2 and described in detail herein.
  • the ⁇ -potential changed from about -18 to +14 mV for leopard myoglobin, -30 to +17 mV for bovine myoglobin and soy leghemoglobin, and -18 to +28 mV for sperm whale myoglobin.
  • the bovine myoglobin had slightly beter oxidative stability than the leopard myoglobin, with the characteristic double-peak spectra still being observed at day 21.
  • the bovine myoglobin solutions did not, however, display a strong red color even on Day 0, and over time the solutions turned brown. This change in color was consistent with the gradual change of the absorption spectra from Carboxy Mb/Oxy Mb to MetMb, with a prominent peak gradually forming at 632 nm over time (Figs. 7D-7F).
  • Fig. 7G show that the initial sperm whale myoglobin had a slightly different redox state than the other proteins examined herein.
  • the absorption spectrum contained peaks consistent with CarboxyMb (550 nm), Oxy Mb (582 nm), and Metmyoglobin (503 nm and 632 nm). Over about 5 days storage, however, the absorption spectrum gradually became more similar to that of pure MetMb, with the solution turning from red to brown (Figs. 7G-7I). This study suggested that the sperm whale sample used in this exemplary study may have undergone some autoxidation.
  • Soy leghemoglobin produced solutions herein with a much stronger bright cherry red color than the three animal myoglobin solutions. Initially, the absorption spectrum of this sample had peaks that were consistent with the presence of CarboxyMb and Oxy Mb (Fig. 7J- JL) A double-peak absorption spectrum was still observed in the soy leghemoglobin at Day 52, suggesting that this hemeprotein was highly resistant to oxidation. Consequently, the soy leghemoglobin used in this study may have applications in food products that have a long shelf life.

Abstract

La présente divulgation concerne de nouvelles compositions appropriées pour être utilisées dans des produits alimentaires, les compositions selon l'invention ayant une stabilité de couleur améliorée et une dégradation de protéine réduite dans le temps. Des modes de réalisation de l'invention concernent des compositions destinées à être utilisées dans des produits alimentaires non animaux comprenant une ou plusieurs hemeprotéines purifiées et un ou plusieurs antioxydants, leurs procédés de fabrication et leurs procédés d'utilisation.
PCT/US2022/047771 2021-10-25 2022-10-25 Compositions d'hémeprotéine stabilisées et leurs procédés d'utilisation WO2023076307A1 (fr)

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