WO2022197586A1 - Pigments protéiques de cnidaire pour compositions de substitut de viande - Google Patents

Pigments protéiques de cnidaire pour compositions de substitut de viande Download PDF

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Publication number
WO2022197586A1
WO2022197586A1 PCT/US2022/020123 US2022020123W WO2022197586A1 WO 2022197586 A1 WO2022197586 A1 WO 2022197586A1 US 2022020123 W US2022020123 W US 2022020123W WO 2022197586 A1 WO2022197586 A1 WO 2022197586A1
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WIPO (PCT)
Prior art keywords
rcp
cell
meat substitute
meat
protein
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PCT/US2022/020123
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English (en)
Inventor
Hans H. Liao
Tristan LIPKIE
Chris Miller
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Cargill, Incorporated
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Publication date
Application filed by Cargill, Incorporated filed Critical Cargill, Incorporated
Publication of WO2022197586A1 publication Critical patent/WO2022197586A1/fr

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Classifications

    • 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
    • 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
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
    • A23L13/42—Additives other than enzymes or microorganisms in meat products or meat meals
    • A23L13/428—Addition of flavours, spices, colours, amino acids or their salts, peptides, vitamins, yeast extract or autolysate, nucleic acid or derivatives, organic acidifying agents or their salts or acidogens, sweeteners, e.g. sugars or sugar alcohols; Addition of alcohol-containing products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
    • C09B67/0097—Dye preparations of special physical nature; Tablets, films, extrusion, microcapsules, sheets, pads, bags with dyes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present disclosure provides pigment compositions for a meat substitute comprising a red chromogenic protein (RCP) in an amount effective for increasing the red color of a meat substitute.
  • RCP red chromogenic protein
  • the RCP can be a cnidarian RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. S SAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001.
  • the RCP can comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one sequence selected from the group consisting of SEQ ID NOs:l-12 and comprises an XYG chromophore tripeptide.
  • the RCP can have an absorbance spectrum maximum between 450 nm and 600 nm. When the pigment is heated at 80 °C for 20 minutes, the absorbance of light at 580 nm is at least 80% of the absorbance at 580 nm prior to heating or greater than 100% of the absorbance at 580 nm prior to heating.
  • the disclosure further provides a meat substitute comprising a non-meat protein and an RCP.
  • the RCP can be a cnidarian RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. S SAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001.
  • the RCP can comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one sequence selected from the group consisting of SEQ ID NOs:l-12 and comprises an XYG chromophore tripeptide.
  • the RCP can have an absorbance spectrum maximum between 450 nm and 600 nm.
  • the meat substitute can comprise 0.01% to 6%, 0.05% to 5%, 0.1% to 3%, or 0.5% to 2% by weight of the RCP or a pigment composition comprising the RCP.
  • the meat substitute can comprise a plant-based protein, a fungal-derived protein, an insect protein, and in vitro cultured animal cell, or combinations thereof.
  • the non-meat protein can comprise a plant-based protein selected from the group consisting of pea protein, soy protein, corn protein, chickpea protein, and wheat protein.
  • the non-meat protein can comprise a fungal -derived protein, for example, a fungal mycoprotein.
  • the non-meat protein can comprise an insect protein.
  • the meat substitute can comprise an in vitro cultured animal cell.
  • the disclosure further provides a plant, animal, or edible mushroom cell comprising an exogenous polynucleotide encoding an RCP polypeptide comprising a sequence at least 80% identical to at least one of SEQ ID NOs:l-12 and comprising an XYG chromophore tripeptide, wherein the RCP polypeptide has an absorption spectrum maximum between 450 nm and 600 nm.
  • the disclosure also provides a meat substitute comprising said cell.
  • the RCP polypeptide can comprise a sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one of SEQ ID NOs: 1-12 and comprises an XYG chromophore tripeptide.
  • the cell can be a plant cell.
  • the cell can be an edible mushroom cell, such as a Fusarium venenatum cell.
  • the cell can be an animal cell.
  • the cell can be an insect cell.
  • the cell can be an in vitro cultured animal cell.
  • the disclosure also provides a meat substitute comprising said cell(s).
  • the disclosure also provides a method for increasing the red color of a meat substitute comprising adding a pigment composition comprising an RCP to a non-meat protein to form a meat substitute with increased red color prior to cooking relative to an equivalent meat substitute without the pigment composition.
  • the pigment compositing may be any pigment described herein comprising an RCP.
  • the pigment composition can comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the RCP on a dry weight basis
  • the meat substitute comprises a pigment composition comprising the RCP and the pigment composition comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the RCP on a dry weight basis.
  • FIG. l is a photo of E. coli expressing DsRED (top streak) or EforRed (bottom streak) red chromogenic proteins.
  • FIG. 2 shows the visible wavelength absorbance data of DsRED (right) and EforRed (left) before and after heating at 80 °C for 20 minutes.
  • FIG. 3 shows the visible wavelength absorbance data for wild-type EforRed and the 2-F2 mutant EforRed before and after heating at 80 °C for 20 minutes.
  • FIG. 4 shows a series of photos of a small lab scale meat substitute application incorporating a beet juice concentrate, a 2-F2 EforRed mutant, and an EforRed pigment before (top, “raw”) and after (bottom, “cooked”) heating on a hot plate set at 130 0 for 90 seconds.
  • FIG. 5 shows a series of photos of a tiny patty meat substitute incorporating a beet juice concentrate, a 2-F2 EforRed mutant, and an EforRed pigment before (top, “raw”) and after (bottom, “cooked”) heating at 80 °C for 20 minutes.
  • Described herein are pigment compositions for meat substitutes that contain a red chromogenic protein. It has been discovered that red chromogenic proteins may be used in a pigment composition having a similar red/pink color to mimicking animal-based meat products. Meat substitutes containing an effective amount of this pigment composition will transition from a red color when raw to a more brown or less red color when cooked. In an aspect, the brown color results from Maillard reactions involving other components of the meat substitute which may become more visible upon cooking the meat substitute.
  • the terms “meat substitute” and “meat substitute composition” are used interchangeably and refer to compositions that mimic the general appearance, nutritional content, and/or taste of natural animal meat or natural animal meat compositions without containing as the majority component tissues or cells from a whole, living vertebrate animal.
  • the meat substitute may be free of, or contain as a minor component, naturally-occurring animal muscle, adipose, or satellite cells from muscle tissues harvested from a whole vertebrate animal (e.g., a cow, a sheep, a pig, a chicken, a turkey, etc.).
  • the meat substitute is free of any animal cells, e.g., any in vivo derived or in vitro cultured animal cells.
  • the meat substitutes and meat substitute compositions described herein include non-meat proteins, plant-based proteins (e.g., pea protein, soy protein, wheat protein, chickpea protein, corn protein, and the like), fungal-based proteins (e.g., mycoproteins derived from fungi such as Fusarium venenatum and the like), in vitro cultured animal cells (e.g., cultured muscle cells, satellite cells, adipose cells, and the like), insect proteins, or combinations thereof.
  • the meat substitute can comprise plant-based proteins including, but not limited to, pea protein, soy protein, wheat protein, chickpea protein, and com protein.
  • the meat substitute can comprise fungal based proteins including, but not limited to, mycoproteins from Fusarium venenatum.
  • the meat substitute can comprise in vitro cultured animal cells including, but not limited to, muscle cells, satellite cells, and adipose cells grown, differentiated and propagated using, for example, fermentation, a bioreactor, scaffold-seeded cell culture, or other artificial methods.
  • the meat substitute can comprise a combination of two or more of plant-based protein, fungal-based proteins, insect proteins and in vitro cultured animal cells.
  • a meat substitute may include a pea protein and a fungal mycoprotein, a soy protein and a cultured bovine muscle cell, a cultured avian adipocyte and a fungal mycoprotein, or any other combination of plant-base protein, fungal-based protein, insect proteins, and in vitro cultured animal cells.
  • the meat substitute comprises plant-based proteins, fungal-based proteins, or combinations thereof and is free of any animal-based proteins or cells.
  • the meat substitute comprises plant-based proteins, fungal-based proteins, insect proteins, and combinations thereof and is free of and any vertebrate animal-based cells or proteins.
  • the meat substitute comprises plant-based proteins and is free of fungal-based, insect, or animal-based cells or proteins.
  • the meat substitutes comprises fungal-based proteins and is free of plant-based, insect, and animal-based cells and proteins.
  • the meat substitute comprises insect proteins and is free of plant-based, fungal-based, and animal-based cells and proteins.
  • the meat substitute comprises in vivo cultured animal cells and is free of plant-based proteins, fungal-based proteins, insect proteins, and in vivo whole animal derived tissues, cells, and proteins.
  • the meat substitute can mimic a beef product, e.g., ground beef, steak, beef jerky, beef ribs, beef patties, beef sausages, and the like.
  • the meat substitute can mimic a pork product, e.g., ground pork, pork chops, ham, smoked pork, bacon, pork sausage, pork patties, pork ribs, and the like.
  • the meat substitute can mimic a chicken product, e.g., ground chicken, chicken breast, check legs, chicken thighs, chicken wings, chicken patties, chicken tenders, chicken nuggets, chicken sausage, and the like.
  • the meat substitute can mimic a turkey product, e.g., ground turkey, turkey sausage, turkey patties, and the like.
  • the meat substitute can mimic a shellfish product, e.g., crab, lobster, shrimp, crayfish, clams, scallops, oysters, mussels, and the like.
  • the meat substitute can mimic a cured, salted, or processed meat product, e.g., charcuterie, salami, summer sausage, prosciutto, bologna, kielbasa, and the like.
  • non-meat protein refers to protein sourced from plants, fungus, insects, dairy products, or in vitro cultured animal cells, and excludes in vivo vertebrate animal derived tissues, cells, or proteins.
  • non-meat proteins may include plant-based proteins, fungal-based proteins, insect proteins, milk proteins (e.g., casein and whey), proteins from in vitro cultured animal cells, or combinations thereof.
  • RCP red chromogenic protein
  • PCP pink chromogenic protein
  • RCP may also be referred to in the art as “red fluorescent proteins.”
  • RCPs suitable for use in the pigments and meat substitutes described herein include, but are not limited to, cnidarian RCPs, for example, from a species selected from the group consisting of Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. SSAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, and Zoanthus sp. SAL-2001.
  • the RCP polypeptides described herein are characterized by the chromophore forming tripeptide X-Y-G.
  • the chromophore Upon spontaneous cyclization and oxidation of the X-Y-G tripeptide, the chromophore includes a phenol ring derived from the Tyr residue of the tripeptide and an N- acylimine formed by desaturation of the Ca-N double bond derived from the Try backbone and formation of a double bond between Ca and Ob of the Tyr. Chromophore formation and how the RCP chromophore differs from, for example, green and blue chromophores are known and described in the art. See, for example, Miyawaki et al.
  • polypeptide and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties.
  • enzyme or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function.
  • a summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.
  • thermostable RCP refers to an RCP polypeptide that, when heated at 80 °C for 20 minutes, retains at least 80% absorbance at its absorbance spectrum maximum as compared to the absorbance at the absorbance spectrum maximum prior to heating.
  • EforRed has an absorbance spectrum maximum at 580 nm and upon heating at 80 °C for 20 minutes, the heated EforRed retains at least 80% of the absorbance at 580 nm compared to the absorbance prior to heating.
  • the absorbance after heating can be greater than 100% of the absorbance prior to heat.
  • the thermostable RCP may have a higher absorbance at its absorbance spectrum maximum after heating than it did before.
  • EforRed refers to the RCP from the coral Echinopora forskaliana. GenBank ID EU498726.1. The wild-type polypeptide sequence of EforRed is provided in SEQ ID NO:l and the MYG chromophore tripeptide is underlined.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 1 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the MYG tripeptide of residues 62-64 of SEQ ID NO: 1.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:2 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the DYG tripeptide of residues 67-69 of SEQ ID NO:2.
  • asPink refers to the RCP from Ammonia sulcate. GenBank ID EF587182.1.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the MYG tripeptide of residues 63-65 of SEQ ID NO:3.
  • spike sPink refers to the RCP from Stylophora pistillata. GenBank ID DQ206398.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:4 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the KYG tripeptide of residues 66-68 of SEQ ID NO:4.
  • DsRed refers to the RCP from Discosoma sp. GenBank ID AF168419.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:5 and including an XYG chromophore, said RCP haivng an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the QYG tripeptide of residues 66-68 of SEQ ID NO:5.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the DYG tripeptide of residues 66-68 of SEQ ID NO:6.
  • ceriantRFP refers to the RCP from Cerianthus sp. GenBank ID AY296063.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 7 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the QYG tripeptide of residues 61-63 of SEQ ID NO:7.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 8 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore tripeptide is the QYG tripeptide of residues 66-68 of SEQ ID NO:8.
  • eqFP611 refers to the RCP from Entacmaea quadricolor. GenBank ID AY130757.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 9 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore is the MYG chromophore of residues 63-65 of SEQ ID NO:9.
  • KO refers to the RCP from Lithophyllon concinna. GenBank ID AB128820.
  • the wild-type polypeptide sequence of KO is provided in SEQ ID NO: 10 and the CYG chromophore tripeptide is underlined.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 10 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore is the CYG chromophore of residues 64-66 of SEQ ID NO: 10.
  • pporRFP refers to the RCP from Porites porites. GenBank ID DQ206380.
  • the wild-type polypeptide sequence for pporRFP is provided in SEQ ID NO: 11 and the MYG chromophore is underlined.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 11 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore is the MYG chromophore of residues 67-69 of SEQ ID NO: 11.
  • zoan2RFP refers to the RCP from Zoanthus sp. SAL-2001.
  • a pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 12 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm.
  • the XYG chromophore is the DYG chromophore of residues 66-68 of SEQ ID NO: 12.
  • variants or modified sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include or greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein. [0053] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm.
  • Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches.
  • An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters.
  • NCBI National Center for Biological Information
  • Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected.
  • Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only.
  • a sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.
  • Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.”
  • wild-type is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms.
  • a “variant, “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule.
  • a variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.
  • amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence.
  • a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule.
  • conservative amino acid substitutions are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide.
  • amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and/or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. There phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • the DNA polynucleotides may be a cDNA or a genomic DNA sequence.
  • a polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • polynucleotides i.e., polynucleotides encoding an EforRed polypeptide
  • the polynucleotides may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell.
  • polypeptides encoded by polynucleotide sequences found in coral are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used.
  • polynucleotides encoding an RCP described herein may encode any of the RCP polypeptides described herein, for example, the polynucleotide may encode a polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to any one of SEQ ID NOs: 1-12 and an absorbance spectrum maximum between 450 nm and 600 nm.
  • polypeptides described herein may be provided as part of a construct.
  • the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies.
  • the construct may be a vector including a promoter operably linked to the polynucleotide encoding the thermolabile EforRed polypeptide.
  • the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked.
  • the vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.
  • the cell may be a procaryotic cell or a eukaryotic cell.
  • Suitable procaryotic cells include bacteria cell, for example, Escherichia coli and Bacillus subtilis cells.
  • Suitable eukaryotic cells include, but are not limited to, fungal cells, plant cells, and animal cells.
  • Suitable fungal cells include, but are not limited to, Fusarium venenatum , Pichia pastoris , Saccharomyces cerevisiae, Kluyveromyces lactis , Yarrow ia lipolytica , Trichomderma reesei, Issatchenkia orientalis , and Aspergillus niger cells.
  • Suitable plant cells include, but are not limited to, a pea cell ( Pisum sativum ), a corn cell (Zea mays), a soybean cell ( Glycine max), and a wheat cell ( Triticum sp.).
  • Suitable animal cells include, but are not limited to, muscle cells (e.g., myocytes, myoblasts, myosatellite, and satellite cells) and fat cells (e.g., adipocytes or adipocyte progenitor cells such as mesenchymal stem cells).
  • Suitable animal cells may be mammalian (e.g., bovine, porcine, and ovine), avian (e.g., poultry), crustacean (e.g., shrimp, lobster, and crab), mollusk (e.g., clam, mussel, scallop, and oyster) or insect cells.
  • the cell is an edible mushroom cell, which refers to a mushroom that is safe for human consumption.
  • the edible mushroom cell can be a Fusarium venenatum , Agaricus bisporus, Lentinula edodes, or Volvariella volvacea cell.
  • pigment compositions containing an RCP polypeptide and meat substitutes including such pigment compositions.
  • the pigment compositions disclosed herein can be used to provide color to a meat substitute that is similar to the color of natural animal meat when raw.
  • the pigment composition may provide a pink and/or red color to a raw, uncooked meat substitute.
  • the pigment composition may provide a pink and/or red color to a cured meat product.
  • the pigment compositions described herein include an RCP.
  • Suitable RCPs include, but are not limited to cnidarian RCPs, for example, an RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. SSAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001.
  • the pigment composition may include a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs:l-12 and an absorption spectrum maximum between 450 nm and 600 nm.
  • the pigment composition can be included in a meat substitute at a level that provides increased or improved pink and/or red color in the meat substitute, while also providing increased or improved brown color in the meat substitute after cooking.
  • the pigment composition is used at a level of at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.25%, or 1.5% on a wet (total) weight basis in a meat substitute composition.
  • the pigment composition may be used at a level in the range of 0.01% to 6%, 0.05% to 5%, 0.1% to 3%, or 0.5% to 2% by weight in a meat substitute composition.
  • the pigment composition may additionally include a carrier or a diluent.
  • the pigment composition may also include a blend of the RCP polypeptide with another color or pigment.
  • the pigment composition may include the RCP polypeptide and a fruit or vegetable extract-based pigment composition.
  • the pigment composition described herein can be used as a pigment in any meat substitute composition.
  • An exemplary, but non-limiting, meat substitute composition is a composition which comprises: plant protein (e.g., textured pea protein and/or pea protein), water, vegetable oil, flavor ingredients, salt, sugar, binders, and the pigment composition described herein.
  • the pigment composition described herein can also be used in food applications other than meat substitutes.
  • Meat substitutes described herein may include one or more cells comprising an exogenous polynucleotide encoding an RCP as described herein.
  • the meat substitutes may include a fungal, plant, or animal cell as described herein comprising an exogenous polynucleotide encoding an RCP described herein.
  • the method for increasing the red color of a meat substitute may include adding a pigment composition comprising an RCP to a non-meat protein to form a meat substitute with increased red color prior to cooking relative to an equivalent meat substitute without the pigment composition.
  • Suitable RCPs include, but are not limited to, any RCP described herein.
  • the RCP may be a polypeptide at least 80% identical to at least one of SEQ ID NOs: 1-12.
  • the method for increasing the red color of a cured meat substitute may include adding a pigment composition comprising an RCP to a non-meat protein to form a cured meat substitute with increased red color prior relative to an equivalent cured meat substitute without the pigment composition.
  • Suitable RCPs include, but are not limited to, any RCP described herein.
  • the RCP may be a polypeptide at least 80% identical to at least one of SEQ ID NOs: 1-12.
  • EforRed was expressed in E. coli cells using a His6 tag with a protease cleavage site (MGS SHHHHHHS SGLVPRGSH, SEQ ID NO: 13) on the N-terminus of the EforRed sequence of SEQ ID NO:l.
  • Both red chromogenic proteins EforRed and DsRed are red/pink in color (FIG. 1) and demonstrate the corresponding absorbance peak at the 580 nm wavelength.
  • EforRed and DsRed Upon heating at 80 °C for 20 minutes, both EforRed and DsRed remained stable, maintaining the absorbance peak and 580 nm, with DsRed showing an increase in absorbance at 580 nm following heating (FIG. 2).
  • EforRed was expressed in E. coli as outlined in Example 1.
  • the 2-F2 mutant was mutagenized in E. coli using the expression system outlined in Example 1.
  • each of the beet juice concentrate, the EforRed 2-F2 mutant, and the wild-type EforRed pigments increased the red color of the raw meat substitute composition (FIG. 4).
  • the red color of the EforRed 2-F2 mutant pigment was reduced while the beet juice and wild-type EforRed pigments retained the red color.
  • Hunter colorimetry data for each of meat substitute pigmentations are reported in Table 3.

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Abstract

Sont divulguées dans la présente invention des compositions de pigments pour substituts de viande et des substituts de viande comprenant de telles compositions de pigments. Les compositions de pigments comprennent une protéine chromogène rouge provenant de corail. Les compositions de pigments fournissent une couleur rose et/ou rouge à une composition de substitut de viande. Par exemple, la protéine chromogène rouge peut être un cnidaire RCP à partir de Montipora efflorescens, Echinopora forskaliana, Anemonia sulcate, Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. SSAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, ou Zoanthus sp. SAL-2001.
PCT/US2022/020123 2021-03-15 2022-03-14 Pigments protéiques de cnidaire pour compositions de substitut de viande WO2022197586A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053119A1 (fr) * 2002-12-09 2004-06-24 University Of Miami Proteines fluorescentes issues d'especes aquatiques
US20060107351A1 (en) * 2001-03-02 2006-05-18 Florigene Limited Cell visual characteristic-modifying sequences
WO2014110539A1 (fr) * 2013-01-11 2014-07-17 Maraxi, Inc. Procédés et compositions utilisables en vue de la production de produits comestibles
WO2020097370A2 (fr) * 2018-11-08 2020-05-14 Triton Algae Innovations, Inc. Procédés de surproduction de protoporphyrine ix dans des algues et compositions à base de celle-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060107351A1 (en) * 2001-03-02 2006-05-18 Florigene Limited Cell visual characteristic-modifying sequences
WO2004053119A1 (fr) * 2002-12-09 2004-06-24 University Of Miami Proteines fluorescentes issues d'especes aquatiques
WO2014110539A1 (fr) * 2013-01-11 2014-07-17 Maraxi, Inc. Procédés et compositions utilisables en vue de la production de produits comestibles
WO2020097370A2 (fr) * 2018-11-08 2020-05-14 Triton Algae Innovations, Inc. Procédés de surproduction de protoporphyrine ix dans des algues et compositions à base de celle-ci

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* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. EF587182.1
MIYAWAKI ET AL.: "Red fluorescent proteins: chromophore formation and cellular applications", CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 22, 2012, pages 679 - 688

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