WO2024059578A2 - Biomasse contenant de l'hème dans une composition alimentaire à base de plantes - Google Patents

Biomasse contenant de l'hème dans une composition alimentaire à base de plantes Download PDF

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WO2024059578A2
WO2024059578A2 PCT/US2023/073993 US2023073993W WO2024059578A2 WO 2024059578 A2 WO2024059578 A2 WO 2024059578A2 US 2023073993 W US2023073993 W US 2023073993W WO 2024059578 A2 WO2024059578 A2 WO 2024059578A2
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heme
composition
plant
poultry
containing protein
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PCT/US2023/073993
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English (en)
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WO2024059578A3 (fr
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Philippe PROCHASSON
Dilek Uzunalioglu
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Motif Foodworks, Inc.
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Publication of WO2024059578A2 publication Critical patent/WO2024059578A2/fr
Publication of WO2024059578A3 publication Critical patent/WO2024059578A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present disclosure relates to heme protein-containing compositions and methods of their use in food compositions.
  • Engineered protein expression systems can be used to produce proteins of interest for use in plant-based meat, poultry, and dairy analogs.
  • a nucleic acid sequence encoding a protein of interest is inserted into host cells, typically microbial cells that can be maintained in cell fermentation cultures under suitable conditions and for a time sufficient to produce the desired protein in useful quantity.
  • host cells typically microbial cells that can be maintained in cell fermentation cultures under suitable conditions and for a time sufficient to produce the desired protein in useful quantity.
  • Cost-effective, commercial-scale production, harvest and purification of recombinant proteins using such systems is a growing field, but significant challenges remain. In addition to multiple factors that must be weighed against one another, such as the choice of host cell and the target protein, recovery of recombinant protein from engineered host cells at reasonable yield remains a serious challenge.
  • the current disclosure encompasses hemecontaining protein compositions comprising a biomass comprising a population of microbial cells genetically modified to express a heme-containing protein heterologous to the microbial cells, wherein the titer of the heme-containing protein in the biomass is at least about 15 grams/Liter.
  • the heme-containing protein heterologous to the microbial cells comprises a hemoglobin or a myoglobin or a hemebinding fragment or variant thereof.
  • the heterologous heme-containing protein is encoded by a vertebrate gene for example a mammalian, an avian or a fish gene.
  • the vertebrate gene is a mammalian gene, for example, a bovine, ovine, equine or a porcine gene.
  • the heterologous heme-containing protein is a myoglobin or a heme-binding fragment or variant thereof, wherein the myoglobin comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 2-16.
  • the heterologous heme-containing protein is a bovine myoglobin having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 2.
  • heterologous heme-containing protein comprises an amino acid sequence having no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, or 0% sequence identity with a protein naturally encoded in the genome of the microbial cells
  • the current disclosure also encompasses a plantbased meat, poultry, or seafood analog composition
  • a plantbased meat, poultry, or seafood analog composition comprising (a) a biomass comprising a population of microbial cells genetically modified to express a hemecontaining protein heterologous to the microbial cells, wherein the titer of the hemecontaining protein in the biomass is at least about 15 g/Liter; and (b) a plant-based meat, poultry, or seafood, wherein (a) and (b) are combined to form the plant-based meat, poultry, or seafood analog composition.
  • the heme-containing protein heterologous to the microbial cells comprises a hemoglobin or a myoglobin or a heme-binding fragment or variant thereof.
  • the heterologous heme-containing protein is encoded by a vertebrate gene optionally selected from a mammalian, an avian or a fish gene.
  • the heterologous heme-containing protein is encoded by a mammalian gene for example a bovine, ovine, equine or a porcine gene.
  • the myoglobin comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 2-16.
  • the heme-containing protein is a bovine myoglobin or a heme-binding fragment or variant thereof.
  • the plant-based meat, poultry, or seafood analog composition is substantially free of antibiotics, animal growth hormones, and animal meat.
  • the microbial cells comprise genetically modified yeast, bacteria, or fungi.
  • the microbial cells comprise a yeast cell from the genus Pichia for example Pichia pastoris cells.
  • the genetic modification of the microbial cells comprises an insertion of a nucleic acid sequence encoding the heterologous heme protein into the cell chromosome.
  • the polynucleotide sequence encoding the heterologous heme-containing protein comprises a nucleic acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 1 .
  • the current disclosure also encompasses methods of making a plant-based meat, poultry, or seafood analog composition, comprising: combining (a) a biomass comprising a population of microbial cells genetically modified to express a heme-containing protein heterologous to the microbial cells, wherein the titer of the heme-containing protein in the biomass is at least about 15 grams/Liter; and (b) a plant-based meat, poultry, or seafood analog dough, to form the plant-based meat, poultry, or seafood analog composition.
  • the plant-based meat, poultry, or seafood analog dough composition comprises at least one plant-derived protein and one or more components selected from a fat, a dietary fiber, a carbohydrate, amino acids, enzymes, a stabilizer, a thickener, a vitamin, and a dietary mineral.
  • the plant-based meat, poultry, or seafood analog composition has a form, for example of, ground meat, ground poultry, ground seafood meat patties, poultry patties, meat sausage, poultry sausage, eggs, meat balls, poultry balls, a meat filet, poultry filet, fish fillet, seafood cutlets, seafood pies, salmon burgers, fish sticks, crab cakes, fish burgers, fish cakes, chowder, bisques, rolls, or seafood stews.
  • the current disclosure also encompasses use of a heme-containing protein composition to prepare a plant-based meat, poultry, or seafood analog composition
  • the heme-containing protein composition comprises a biomass comprising a population of microbial cells genetically modified to express a heme-containing protein heterologous to the microbial cells, wherein the titer of the heme-containing protein in the biomass is at least about 15 grams/Liter.
  • the microbial cells comprise genetically modified yeast, bacteria, or fungi, for example Pichia pastoris.
  • the plant-based meat, poultry, or seafood composition is in the form of a gravy, sauce, puree, broth, soup, paste or spread.
  • FIG. 1 is a series of color photographs of heme-containing yeast cell paste either in raw, thawed or dried form.
  • FIG. 2 is a series of color photographs of heme-containing yeast cell pastes: fresh yeast cell paste, spray dried paste and freeze-dried paste.
  • FIG. 3 is a series of color photographs of raw and cooked burger patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after day 1 of the initial freezing.
  • FIG. 4 is a series of color photographs of raw and cooked burger patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after day 14 of the initial freezing.
  • FIG. 5 is a series of color photographs of raw and cooked burger patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after day 14 of the initial freezing and additional 7 days of refrigeration.
  • FIG. 6 is a series of color photographs of raw and cooked burger patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after day 14 of the initial freezing and additional 14 days of refrigeration.
  • FIG. 7A is a bar graph depicting the L-value of the raw patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • L-value is a measure of color along a black to white (dark/light) axis.
  • FIG. 7B is a bar graph depicting the a-value of the raw patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • the a-value is a measure of degree of redness.
  • FIG. 7C is a bar graph depicting the b-value of the raw patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • the b-value is a measure along a yellow to blue value.
  • FIG. 8A is a bar graph depicting the L-value of the cooked patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • L-value is a measure of color along a black to white (dark/light) axis.
  • FIG. 8B is a bar graph depicting the a-value of the cooked patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • the a-value is a measure of degree of redness.
  • FIG. 8C is a bar graph depicting the b-value of the cooked patties with heme protein (C279018), fresh yeast cell paste, spray dried paste and freeze-dried paste after storage under provided conditions.
  • the b-value is a measure along a yellow to blue value.
  • FIG. 9 is a bar graph of pH values of cooked patties evaluated over a period of frozen storage period at days 1 , 14, and 7, and 14 days of refrigerated storage after 14 days of frozen storage.
  • FIG. 10 is a bar graph of water retention values of cooked patties evaluated over a period of frozen storage period at days 1 , 14, and 7, and 14 days of refrigerated storage after 14 days of frozen storage.
  • FIG. 11 shows effect of increasing concentration of heme yeast cell paste (“HYCP”) in vegetable broth.
  • HYCP heme yeast cell paste
  • the present disclosure provides compositions and methods relating to use of a heme-containing biomass in food compositions, including but not limited to plant-based meat, poultry, or seafood analog compositions.
  • a biomass as described herein comprises a population of microbial host cells genetically modified to express a heme-containing protein which is heterologous to the microbial cells.
  • the present disclosure is a result of extensive experimentation and optimization establishing that use of heme-containing biomass at containing relatively high concentrations of at least about 15g/L can be prepared to provide improved taste, color and texture characteristics in food compositions.
  • biomass as described herein can be combined directly with a plant-based food composition, without the need to extract the heme protein from the microbial cells.
  • the cells in the biomass can be lysed.
  • the biomass confers on the plant-based food composition such beneficial features as color, taste and texture which closely mimic those of real meat, poultry, and seafood.
  • plant-based meat, poultry, or seafood dough compositions can be made which are compellingly meat-like, poultry-like or seafood-like, without the time and expense needed to extract or separate the recombinant heme-containing protein from the host cells.
  • the biomass can be added to gravies, sauces, puree, broth, soups, pastes, and spreads to add color, taste, and texture.
  • the present disclosure provides food compositions comprising heme-containing biomass.
  • the food composition can be any edible composition including but not limited to plant-based meat analogs, plant-based poultry, or plant-based seafood analogs, sauces, gravies, soups, purees, broths, pastes, and spreads.
  • the plant-based food compositions described herein comprise a biomass combined with a plant-based meat analog dough, a plant-based poultry analog dough, or a plant-based seafood analog dough or any combination thereof.
  • the term “meat” encompasses any animal eaten as food including but is not limited to beef, lamb, pork, and goat.
  • the term “poultry” encompasses any bird eaten as food including but not limited to chicken, turkey, duck, and goose.
  • the term “seafood” encompasses all fresh and saltwater fish, crustaceans, and shellfish, including tu not limited to anchovy, bass, bluefish, carp, catfish, char, clams, cod, flounder, haddock, halibut, herring, orange roughy, mahi-mahi, sardines, salmon, trout and tuna, and crab, crayfish, lobster, mussels, oysters, prawns, and shrimp and echinoderms.
  • the present disclosure relates in part to their respective plant-based analogs.
  • Recombinant production of a protein in a host cell can provide a desirable vehicle for producing the protein in commercially relevant quantities.
  • the recombinant production of a protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein is placed under the expression control of a promoter from a regulated gene.
  • the expression cassette is introduced into the host cell, usually by plasmid-mediated transformation. Production of the protein is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
  • the resulting biomass comprises a population of microbial cells genetically modified to express a heme-containing protein heterologous to the microbial cells.
  • the heme-containing protein is for example a hemoglobin, myoglobin, neuroglobin, cytoglobin, or a leghemoglobin or a heme-binding fragment or variant thereof.
  • the heme containing protein is a myoglobin or a heme-binding fragment or variant thereof.
  • the myoglobin is a vertebrate’s myoglobin or a heme-binding fragment or variant thereof.
  • the myoglobin is a mammalian, or an avian myoglobin, or a fish myoglobin, or hemebinding fragments or variants thereof.
  • the microbial cells may comprise genetically modified yeast, bacteria, or fungi.
  • a population of the modified microbes, a modified yeast in one example is then cultivated for a time and under conditions sufficient for the microbial biomass to express the heme-containing protein in a fermentation system in an amount of at least 15 g/Liter, or at least about 15 g/Liter of the heme-containing protein.
  • a genetically modified yeast strain may be prepared from a yeast strain capable of expressing a heterologous heme-containing protein. Genetic modification of yeast cells can be achieved by various means known in the art, for example by harnessing the yeast’s homologous recombination repair mechanism.
  • a target site in the yeast chromosomal DNA is selected, and an expression construct is introduced into the cells.
  • the expression construct comprises heterologous nucleic acid (e.g., DNA) to be inserted under the control of a promoter compatible with the host species.
  • the target site in the yeast chromosome is chosen for having regions of nucleic acid sequence homology with the nucleic acid for insertion.
  • the process of introducing the expression construct results in homologous recombination events which incorporate the heterologous DNA into the yeast chromosome.
  • the heterologous DNA encodes a protein of interest, such as in the present disclosure a heme-containing protein as described in further detail below.
  • expression vectors can comprise additional regulatory sequences (e.g., termination sequence, translational control sequence, etc.), as well as selectable marker sequences. Plasmids are known in the art, including those based on pBR322, PUC, and so forth. Viral vectors may also be used to provide intracellular expression of the cell lysis protein or peptide. Suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, herpes virus vectors, and so forth.
  • the microbial cells may comprise a yeast strain.
  • the yeast strain may be from the genus Pichia, such as Pichia pastoris, a eukaryotic, methylotrophic, non-pathogenic, and non-toxigenic microorganism which can be used to produce recombinant proteins.
  • Use of P. pastoris in the food industry is well known. Genetic typing of P. pastoris led to a re-naming of the strain to Komagatella phaffii. The name P. pastoris is however still commonly used and is used throughout this disclosure and is to be understood as interchangeable with Komagatella phaffii in referring to the same organism.
  • strain CBS7435 (synonymous with NRRL Y-11430), first deposited in the CBS and NRRL culture collections (Phillips Petroleum).
  • strain CBS7435 (NRRL Y-11430) is identical to the type strain of K. phaffii (NRRL Y-7556).
  • Genotypic analyses (Brady et al., (2020)), have demonstrated that strain NRRL Y-11430 and NRRL Y-7556 differ by a single nucleotide polymorphism.
  • a production strain of P. pastoris can for example be constructed by genetically modifying a strain of P. pastoris using techniques and tools such as described by the Organization for Economic Cooperation and Development (OECD) criteria for Good Industrial Large-Scale Practice (GILSP) microorganisms (OECD, 1992, 1993), and criteria for safe production microorganisms (Pariza and Foster, 1983; Pariza and Johnson, 2001 ).
  • OECD Organization for Economic Cooperation and Development
  • GILSP Good Industrial Large-Scale Practice
  • a parental P. pastoris strain can be genetically modified to overexpress the proteins of the native heme biosynthetic pathway of P. pastoris.
  • the heme biosynthetic pathway involves eight steps, each catalyzed by an enzyme that is highly conserved across plant, animal, and fungal species.
  • Genes encoding all eight enzymes can be generated by DNA synthetic techniques as well known in the art, and transformed into the P. pastoris parental strain also using transformation techniques as well known in the art.
  • antibiotic resistance cassettes can be used to identify successfully transformed cells and eliminate untransformed cells.
  • Antibiotic resistance cassettes can be removed from the strains after each round of transformation. This transformation process yields a stable intermediate P.
  • the resulting P. pastoris strain which contains extra copies of each of the native Pichia heme biosynthesis enzymes.
  • the resulting P. pastoris strain can then be further modified by introduction of nucleic acid sequences encoding a vertebrate heme-containing protein for example a mammalian, an avian or a fish heme-containing protein or a heme-binding fragment or variant thereof, with a promoter compatible with the P. pastoris strain.
  • the P. pastoris strain can then be further modified to express the Bos taurus myoglobin gene.
  • the heme-containing protein gene is codon-optimized for expression in the host organism, for example codon-optimized for expression in P. pastoris.
  • multiple copies of a heme-containing protein gene are stably integrated into the host organism, under the control of one or more promoters.
  • the modified host strain can be further modified by inserting an additional copy of the gene encoding a transcription factor native to the organism, e.g., a transcription factor native to P. pastoris.
  • the copies of the heme-containing protein gene are stably integrated into the host organism along with an antibiotic resistance cassette, which is later removed. Phenotyping and/or whole genome sequencing is performed to confirm removal of all antibiotic resistance genes introduced during construction of the production strain.
  • production strains can be created without plasmid sequences being present, so that no plasmid sequences are expected to be capable of being transferred from the production strain to non-related organisms.
  • the nucleic acid sequence encoding the heterologous protein may optionally be linked to a selectable marker, such as a nucleic acid sequence encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), and/or glutamine synthase (GS), wherein any of HPRT, DHFR, and/or GS can be used as an amplifiable selectable marker.
  • the nucleic acid sequence encoding the heterologous protein may optionally be codon optimized to facilitate and/or promote expression in a heterologous system (/.e., a bacterial or yeast system).
  • modified P. pasto s cells that can be used according to the present disclosure to produce a heme-containing protein at sufficiently high titer include but are not limited to cells as described in WO/2022/051696A1 and WO/2022/108839A1 .
  • Downstream processing of recombinant production strain such as yeast can be expensive and time consuming, typically requiring multiple steps. The process become increasingly more expensive with every additional step closer to extraction of the recombinant protein.
  • biomass compositions requiring minimal downstream processing, that can be added to food compositions.
  • the biomass compositions as used herein may comprise hemecontaining, genetically modified microbial cells or cell lysates.
  • the biomass composition may comprise heme-containing yeast cells, for example methylotrophic yeast cells such as but not limited to Pichia pastohs.
  • the biomass composition may comprise live or dead whole cells or both.
  • the cells after fermentation production of a heme-containing protein by the cell, the cells are lysed such that the biomass comprises lysed cells.
  • the cells are lysed by dehydration, freeze drying, heat, spray drying or lyophilization.
  • the biomass may thus comprise dehydrated, freeze dried, heat killed, spray dried or lyophilized whole cells or a combination thereof.
  • the biomass comprises dehydrated, freeze dried, spray dried or lyophilized cell lysate.
  • Cell lysis of a recombinant production strain such as a yeast is normally required to harvest recombinant protein produced within the cells, as is a recombinant heme-containing protein such as myoglobin.
  • Cell lysis can be expensive and time-consuming, typically requiring as it does multiple steps involving the application of mechanical, chemical and/or enzymatic disruption methods.
  • the present disclosure obviates the necessity for cell lysis by incorporating cell biomass into a product which can be added to another product such as a plant-based meat, poultry, or seafood analog dough as described herein.
  • a cell biomass can be prepared as a liquid, gel, semisolid, paste, powder or a solid composition which can then be incorporated into a plant-based meat, poultry, or seafood analog composition.
  • a biomass once separated from a fermentation broth liquid can be prepared as a paste composition which can then be incorporated into a plant-based meat, poultry, or seafood analog composition as disclosed herein.
  • the whole biomass can be lysed and then dried to be incorporated into biomass compositions that can then be incorporated into food.
  • a fermentation broth of a yeast e.g., a P. pastoris fermentation which produces at least 15, 16, 17, 18, 19, or 20 g/L heme in broth is filtered or centrifuged to reduce the liquid content and produce a pellet having at least about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80 mg heme/g yeast, wet cell weight.
  • a yeast e.g., a P. pastoris fermentation which produces at least 15, 16, 17, 18, 19, or 20 g/L heme in broth is filtered or centrifuged to reduce the liquid content
  • the pellet has at least about 25 to about 80 mg heme/g yeast, wet cell weight. In some aspects, the pellet has at least about 30 to about 70 mg heme/g yeast, wet cell weight. In some aspects, the pellet has at least about 30 mg heme/g yeast, wet cell weight. In one aspect, the pellet is not dried. The pellet is then used to prepare a composition such as a gel, semisolid or paste composition which can then be incorporated in a meat, poultry, or seafood analog composition. For example, the pellet may be washed with water or an aqueous solution and mixed with a sufficient amount of the water or a suitable liquid formulation to form a paste.
  • a composition such as a gel, semisolid or paste composition which can then be incorporated in a meat, poultry, or seafood analog composition.
  • the pellet may be washed with water or an aqueous solution and mixed with a sufficient amount of the water or a suitable liquid formulation to form a paste.
  • the suitable liquid formulation may comprise one or more of water, buffering agent, oils, preservatives, antioxidants, or color preservers.
  • the paste can be added directly to food compositions or suitably processed, packaged, and stored for future use.
  • the pellet may be dehydrated and packaged.
  • the dehydrated pellet has at least about 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129,
  • the dehydrated pellet has at least about 72 to about 280 mg heme/g yeast, dry cell weight. In some aspects, the dehydrated pellet has at least about 90 to about 245 mg heme/g yeast dry cell weight. In some aspects, the dehydrated pellet has about 105 mg heme/g yeast, dry cell weight. In some aspects, the dehydrated pellet may be mixed with additional ingredients, for example preservatives, buffering agents, color preservers prior to packaging. In some aspects the cell pellet may be further processed, for example lysed prior to addition to food compositions.
  • the lysed pellet can be dehydrated, for example using spray drying or freeze-drying technologies and packaged.
  • additional ingredients may be added to the pellet before lysing, after lysing or after drying.
  • the dehydrated lysate can be directly added to food compositions, suitably processed, and packaged for future use.
  • the dehydrated lysate can be stored as for example powder, pellets, or cubes.
  • the heterologous heme-containing protein is encoded by a vertebrate gene.
  • the heterologous heme-containing protein is encoded by a mammalian, or an avian or a fish gene, which may be for example a bovine or a porcine gene.
  • the heme-containing protein is for example a hemoglobin, myoglobin, neuroglobin, cytoglobin, or a leghemoglobin or a heme-binding fragment or variant thereof.
  • the heme containing protein may be a bovine, equine, ovine, chicken, turkey, goose, emu, whale, eel (for example, Swamp Eel), tuna (for example, Bullet or Bluefin Tuna), perch (for example, European Perch), mackerel (for example, Chub Mackerel) or marlin (for example, Atlantic Blue Marlin) protein or a heme-binding fragment or variant thereof.
  • the heme-containing protein may be a myoglobin, for example a bovine myoglobin encoded by a bovine myoglobin gene or heme-binding fragments or variants thereof.
  • the heme-containing protein is encoded by a bovine myoglobin gene having a nucleic acid sequence of SEQ ID NO: 1 , or a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1
  • a bovine myoglobin gene may encode an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2..
  • the myoglobin protein may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NO: 2-16 (as provided in Table A).
  • Table A List of heme-containing gene and protein sequences
  • compositions and methods described herein are substantially free of antibiotics, animal growth hormones, and animal meat.
  • substantially free refers to a level of any antibiotic, animal growth hormone, and animal meat tissue that is not detectable using routine detection methods.
  • a heme-containing protein composition or a plantbased meat, poultry, or seafood analog composition as described herein, comprises a yeast cell or population of yeast cells from the genus Pichia, such as Pichia pastoris cells, genetically modified by insertion of a nucleic acid sequence encoding the heterologous heme protein into the cell chromosome.
  • a population of such yeast cells makes up a biomass, comprising a population of microbial cells genetically modified to express a heme-containing protein heterologous to the microbial cells.
  • the titer of the heme-containing protein in the biomass is at least about 15 grams/Liter.
  • the biomass is combined with a plant-based meat, poultry, or seafood analog dough, to form a plant-based meat, poultry, or seafood analog composition.
  • the biomass may also be added to other food products including but not restricted to gravies, sauces, purees, broths, soups, pastes and spreads.
  • the plant-based meat, poultry, or seafood analog dough composition comprises at least one plant-derived protein and one or more components selected from a fat, oil (including vegetable and plant oil), a dietary fiber, a carbohydrate, amino acids, a stabilizer, a thickener, texturizers, a plant based connective tissue analog, a vitamin, and a dietary mineral.
  • a fat, oil including vegetable and plant oil
  • a dietary fiber including a dietary fiber
  • carbohydrate including amino acids, a stabilizer, a thickener, texturizers
  • a plant based connective tissue analog a vitamin, and a dietary mineral.
  • Food compositions in which biomass compositions may be included are final edible products ready to be consumed by human beings and/or animals. They may comprise various components or ingredients, each imparting a desired feature or characteristic to the products, such as nutrition, flavor, appearance, taste, and texture.
  • Food compositions contemplated herein include meat, poultry, or seafood analogs comprising as a component, a biomass of microbial cells, or a biomass of lysed microbial cells which are genetically modified to express a heme-containing protein.
  • meat analog compositions include compositions mimicking ground meat, meatloaf mix, steaks, pinwheels, sausages, salami, jerky, bacon, pork boneless rib meat, chicken cutlets, tenders, drumsticks, or hams, soups or stews.
  • Non-limiting examples of poultry analog compositions include vegan chicken, mock chicken, vegan turkey, and compositions mimicking nuggets, cutlets, breasts, slices or strips sourced from chicken, quail, duck, ostrich, turkey, bantam, or geese.
  • Non-limiting examples of seafood analog include fish, clams, oysters, mussels, lobsters, shrimp, crab, and echinoderms analogs.
  • the food compositions described herein may be formulated to mimic any real meat, poultry, or seafood product, such as ground meat, ground meat patties, ground meat meatballs, meat steaks, meat sausage, meat jerky strips, ground chicken, poultry slices, fish fillets, seafood cutlets, seafood pies, salmon burgers, fish sticks, crab cakes, fish burgers, fish cakes, sushi, chowder, bisques, rolls and seafood stews or any combination thereof.
  • the food compositions described herein may be formed as any such product formed from real beef, poultry, or seafood.
  • plant-based food compositions in the form of plant-based ground beef, which may take the form of a ground beef patty or slider, a ground beef meatball, a beef sausage or hot dog, a cut of beef, corned beef, or a dried beef strip.
  • the meat alternative formulation described herein may alternatively be prepared in the form taken by other real meat products such as meat (beef, chicken, or turkey) nuggets or strips, meat loaf or meat cake forms, canned seasoned meat, sliced meat, sausage of any size, or processed meats such as salami, bologna, luncheon meat and the like.
  • the meat alternative formulation after cooking, may provide the color, the flavor, and the texture of cooked meat which is pleasurable and palatable to the consumer.
  • Meat analog compositions may comprise a plant-based meat-like base combined with microbial cells containing heme-containing protein to impart the color and flavor of a real animal, poultry, meat or seafood to the meat analog compositions.
  • the plant-based meat, poultry, or seafoodlike base is a base material or composition of plant origin that may have a nutritional profile and/or taste and/or texture similar to real animal, bird, or seafood meat.
  • Nonlimiting examples of a plant-based meat base or composition may include plant proteins and/or fibers, such as proteins isolated from soy, rice, peas, chickpea, canola, pulses, beans, nuts, corn, wheat, gluten, and animal proteins such as milk and egg.
  • plant proteins and/or fibers such as proteins isolated from soy, rice, peas, chickpea, canola, pulses, beans, nuts, corn, wheat, gluten, and animal proteins such as milk and egg.
  • Food compositions and accompaniments contemplated herein further include for example, gravies, sauces, purees, broths, soups, pastes, spreads and similar foods that could benefit in flavor, texture or color by addition of hemecontaining biomass.
  • a plant-based meat, poultry, or seafood analog composition comprises a biomass composition comprising heme protein-containing microbial cells as disclosed herein, a plant-based protein, a hydrocolloid base, a plantbased fiber, an additional plant-based protein, and a second additional plant-based protein.
  • a plant-based meat, poultry, or seafood analog composition comprises a biomass composition comprising heme protein-containing microbial cells as disclosed herein, a plant-based protein, a hydrocolloid base, a plant-based fiber, and an additional plant-based protein.
  • a plant-based meat, poultry, or seafood analog composition that comprises a biomass composition comprising heme protein-containing microbial cells as disclosed herein, a plant-based protein, an additional plant-based protein, a hydrocolloid base, and a plantbased fiber.
  • a plant-based meat, poultry, or seafood analog composition comprises a biomass composition comprising heme protein-containing microbial cells as disclosed herein, a plant-based protein, a hydrocolloid base, a plantbased fiber, an additional plant-based protein, a second additional plant-based protein, fat and/ or oil.
  • a plant-based meat, poultry, or seafood composition comprises a biomass composition comprising heme protein-containing microbial cells as disclosed herein, a plant-based protein, a hydrocolloid base, a plant-based fiber, an additional plant-based protein, a second additional plant-based protein, a fat and/or oil, and a binder.
  • heme protein is used interchangeably with “heme-containing protein”, to refer to a protein which comprises or is configured to bind to a heme prosthetic group.
  • Heme prosthetic groups typically comprise one or more highly conjugated rings complexed to an iron ion.
  • a heme prosthetic group (which may be referred to interchangeably as ‘heme’ or ‘heme moiety’) may denote iron (e.g., Fe+ 2 , Fe+ 3 , or Fe+ 4 ) bound to a porphyrin ring.
  • heme moieties include, but are not limited to, heme a, heme b, heme c, heme d, heme d1 , heme I, heme s, heme o, heme m, and siroheme.
  • a heme moiety comprises a porphyrin, porphyrinogen, corrin, corrinoid, chlorin, bacteriochlorophyll, corphin, chlorophyllin, bacteriochlorin, or an isobacteriochlorin moiety complexed to an iron ion.
  • a heme protein may possess one or several iron porphyrins.
  • the heme protein may be expressed in a microbe (e.g., a bacterial or fungal expression system).
  • the heme protein is expressed in Pichia pastoris engineered to express the heme-containing protein.
  • the heme-containing protein may comprise one or more of a vertebrate (e.g., bovine) myoglobin and/or hemoglobin produced in a yeast fermentation system.
  • a plant-based meat, poultry, or seafood composition as contemplated herein may comprise the combination of a microbial paste, such as but not limited to a yeast paste comprising a biomass of microbial cells (e.g., yeast cells) or cell lysate which have been engineered to express the heme-containing protein as described herein at titers sufficient to produce results as described herein.
  • a microbial paste such as but not limited to a yeast paste comprising a biomass of microbial cells (e.g., yeast cells) or cell lysate which have been engineered to express the heme-containing protein as described herein at titers sufficient to produce results as described herein.
  • microbial paste such as but not limited to a yeast paste comprising a biomass of microbial cells (e.g., yeast cells) or cell lysate which have been engineered to express the heme-containing protein as described herein at titers sufficient to produce results as described herein.
  • the microbial e.g
  • yeast paste is combined with a plant-based meat-like, or poultry-like or seafood-like base dough, thereby forming a plant-based substitute 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 a heme-containing microbial paste as provided herein are 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, 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®, 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 Bee
  • the amount of heme-containing biomass added to the plant-based meat-like base can vary depending on the application and the desired flavor, color, and texture profile. In some aspects, the amount of a heme-containing microbial paste combined with a plant-based meat-like base is, in percentage from about 1 % to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25% of the total weight.
  • the biomass may comprise about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25% or the total composition by weight.
  • the present disclosure encompasses use of a heme-containing protein composition to prepare a plant-based meat, poultry, or seafood composition.
  • the heme-containing protein composition comprises a biomass comprising a population of microbial cells genetically modified to express a hemecontaining protein heterologous to the microbial cells, wherein the titer of the heme- containing protein in the biomass is at least about 15 grams/Liter.
  • the microbial cells may comprise genetically modified yeast, bacteria, or fungi.
  • the microbial cells comprise a yeast cell from the genus Pichia, such as Pichia pastoris cells.
  • Plant-based meat, poultry, or seafood compositions are prepared according to known methods and using other known and readily commercially available components in addition to the biomass compositions described herein.
  • biomass is generated by fermentation using optimized conditions for culturing the microbial cell engineered to produce the heme protein.
  • a microbial cell paste is then obtained from the culture by one or more methods known in the art including but not restricted to centrifugation, membrane filtration, decantation, gravity separation, sedimentation or drying.
  • the microbial biomass obtained may be washed and added directly to one or more ingredients that go into a food composition.
  • the washed microbial paste may be further processed before adding to the one or more ingredients that go into a food composition.
  • the further processing may include one or more of freezing, refrigeration, vacuum sealing, dehydration, lyophilization, freeze-drying, spray drying or hydration.
  • the paste can then be combined with the ingredients and processed using known methods in the art including combining, homogenization, grinding, centrifugation, hydrating, formulation, casting, forming, dehydrating, heating, cooling, packaging, and storing.
  • a centrifuge uses rotation around a fixed axis to generate centripetal acceleration resulting in the separation of materials based on density. Separation using centrifugation can be accomplished in a batch or continuous process. Typically, a continuous process is used for large volumes. In one embodiment a disc stack centrifuge is used. In another embodiment, a decanter centrifuge is used. Disc stack and decanter centrifuges are well known in the art and commercially available from a number of manufacturers. Centrifugation may be applied to untreated material or used in combination with additional dewatering processes such as filtration. Membrane Filtration
  • membrane filtration is a type of filtration physical process where a fluid is passed through a special pore-sized membrane to separate microorganisms and suspended particles from process liquid.
  • the membrane size used in the current disclosure is suitable to retain microbial cell while allowing the culture medium to pass through.
  • the membrane filtration may be batch filtration process.
  • Freeze drying also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure by applying vacuum, then removing the ice by sublimation. This is in contrast to dehydration by most conventional methods that evaporate water using heat.
  • the sample to be freeze dried may have undergone a pretreatment process prior to freezing including but not restricted to washing, addition of other ingredients and formulation for example antibiotics, anti-microbials, color stabilizers, odor reductants, or flavorings.
  • a freeze-dried culture in the form of a powder is physically significant different from a frozen culture among others due to that a freeze-dried powder comprises significantly less water as compared to a frozen culture.
  • Spray drying is a means of converting a liquid or slurry to a powder using hot gas. All spray dryers use some type of atomizer or spray nozzle to disperse the liquid or slurry into a controlled drop size spray. The most common of these are rotary disk and single-fluid high pressure swirl nozzles. Depending on the process requirements, drop sizes from 10 to 500 pm can be achieved with the appropriate choices. The most common applications are in the 100 to 200 pm diameter range. In some aspects the drop size is between 50-100 pm, or 100-150 pm, or 150-200 pm, or 200-250 pm, or 250-300 pm, or 300-350 pm, or 350-400 pm, or 400-450 pm, or 450- 500 pm. The dry powder is often free flowing.
  • any commercially available spray dryer may be used for example a Buchi, Aveka, , Vettertec dryer of suitable scale for the application.
  • the inlet and outlet temperatures and feed rates for the dryer may vary depending on the desired product, and the make of the spray dryer. In some aspects, the inlet and outlet temperatures range from about 130°C to about 250°C. In some aspects the feed rate is between 1-50mL/min, or between 5-25 mL/min or between 6-10mL/min.
  • a “substantially homogenous mixture” is described herein above.
  • the ingredients/components that form the substantially homogenous mixture are preferably in solid and dry form, such as powders, lyophilized powders, particles, flours, sheets, cubes, and blocks.
  • Combining the ingredients may be achieved through any commonly used means such as blending, stirring, whisking, rotating, breaking, pounding, grinding, milling, rolling, chopping, cutting, pulverizing, or any other physical means or maneuvers to allow the even distribution of ingredients in the mixture.
  • the tools or instrumentations used in the combining may include, but not limited to scales to measure the ingredients, mixing bowls for holding and mixing the ingredients, and stir bars, whisk wires or mixers to facilitate the combining to form the substantially homogenous mixture.
  • Hydrating generally refers to the process of introducing an aqueous liquid to a dry phase. Hydrating the substantially homogeneous mixture may be achieved by introducing to the mixture a hydration agent, such as water in any form and at any temperature, another aqueous solvent, a gelling agent, or any combination thereof. Hydration is achieved when a viscous, sticky composition, /.e., a gel is produced.
  • the hydration agent may be a water, an ionized water, a buffered water, a non-water solvent, a gelling agent, or any combination thereof.
  • the water used may be a tap water, a distilled water, and a filtered water, such as those from Millipore filtration.
  • the water can be cold water, hot water, or introduced to the mixture as steam.
  • the gelling agent may be an aqueous or a non-aqueous solution or liquid, comprising an inorganic ion, an organic ion, a crosslinking agent, an enzyme, a sugar, a salt, an acid, or a base, or anything that may facilitate the formation of a gel.
  • the hydration agent may be treated to reach a desired temperature, such as heating to a temperature above room temperature, boiling to a steam, or chilled to below room temperature.
  • hydrating the substantially homogenous mixture may be achieved by adding the hydration agent, mixing, stirring, heating, cooling, setting, any combinations thereof, or any other means or maneuvers to allow dispersing of the substantially homogenous mixture to the hydration agent and gelling of the mixture.
  • the tools and instrumentations in hydrating may comprise volumetric flasks to measure out the hydration agent, stir bar, wire whisk or mixers to facilitate mixing and hydrating, an oven/heater to heat up the hydration agent, or refrigerator/freezer to cool down the hydration agent. It will be understood that the selection of the hydration agent and amount used in the hydrating step will vary with the nature and the amount of the various ingredients in the substantially homogenous mixture.
  • the method of making may also comprise a step of safely packaging and storing the microbial biomass alone or food product obtained through the above steps.
  • the biomass alone or meat, poultry, or seafood analog comprising it may be frozen or refrigerated.
  • the biomass alone or meat, poultry, or seafood analog comprising it may be packaged using a vacuum seal.
  • the biomass alone or meat, poultry, or seafood analog comprising it may be packaged using routine procedures in a container or a bag suitable for holding food and facilitating its stability.
  • the container or bag may have a setup to prevent air or water diffusion into the microbial biomass.
  • the container or bag used may also possess a setup to prevent microorganisms, such as bacteria from entering the container or bag.
  • the container or bag suitable for holding food may be one of disposable, airtight, zippered, sealable, or with vacuum sealing.
  • the packaged biomass alone or meat, poultry, or seafood analog comprising it may be stored at room temperature, in a refrigerator, or in a freezer.
  • the packaged and stored microbial biomass alone or meat, poultry, or seafood analog comprising it may be ready to use at any time.
  • a “genetically modified” cell refers to a cell in which the nuclear, organellar or extrachromosomal nucleic acid sequences of a cell has been modified, i.e., the cell contains at least one nucleic acid sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and/or a substitution of at least one nucleotide.
  • the terms “genome modification” and “genome editing” refer to processes by which a specific nucleic acid sequence in a genome is changed such that the nucleic acid sequence is modified.
  • the nucleic acid sequence may be modified to comprise an insertion of at least one nucleotide, a deletion of at least one nucleotide, and/or a substitution of at least one nucleotide.
  • the modified nucleic acid sequence is inactivated such that no product is made.
  • the nucleic acid sequence may be modified such that an altered product is made.
  • heterologous refers to an entity that is not native to the cell or species of interest.
  • nucleic acid and polynucleotide refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms may encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties. In general, an analog of a particular nucleotide has the same basepairing specificity, i.e., an analog of A will base-pair with T.
  • the nucleotides of a nucleic acid or polynucleotide may be linked by phosphodiester, phosphothioate, phosphoram idite, phosphorodiamidate bonds, or combinations thereof.
  • nucleotide refers to deoxyribonucleotides or ribonucleotides.
  • the nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs.
  • a nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety.
  • a nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a synthetic nucleotide.
  • Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7 -deaza purines).
  • Nucleotide analogs also include dideoxy nucleotides, 2’-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • amino acid sequence that is “derived from” an amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more amino acids compared to the reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein.
  • derivative when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide.
  • a part or fragment of a polypeptide may correspond to at least 1 %, at least 2%, at least 3 %, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% of the length of a polypeptide, such as a polypeptide having an amino acid sequence identified by a specific SEQ ID NO., or having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length (in amino acids) of the polypeptide.
  • the “variants” and “fragments” are capable of binding heme.
  • target site refers to a nucleic acid sequence that defines a portion of a nucleic acid sequence to be modified or edited and to which a homologous recombination composition is engineered to target.
  • upstream and downstream refer to locations in a nucleic acid sequence relative to a fixed position. Upstream refers to the region that is 5' (/.e., near the 5' end of the strand) to the position, and downstream refers to the region that is 3' (7.e. , near the 3' end of the strand) to the position.
  • nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences may also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) may be compared by determining their percent identity.
  • the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482- 489 (1981 ). This algorithm may be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
  • Percent identities between sequences can be at least 70-75%, or at least 85-80%, or at least 80-85%, or at least 85-90%, or at least 90-95% or at least 95-100% or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100% sequence identity.
  • Pichia pastoris is considered as a universal cell factory or production system for industry, agriculture and biomaterial applications including chemicals, enzymes and antimicrobials.
  • One aspect of the current disclosure provides compositions and methods to improve the ease of use of synthetically expressed, recombinant heme protein-containing compositions.
  • the current disclosure provides method of making and using yeast cells for example Pichia pastoris (referred herein as yeast) cells comprising heme protein directly in food products with minimal downstream processing (DSP).
  • yeast Pichia pastoris
  • DSP minimal downstream processing
  • Pichia pastoris provided in the current disclosure is an engineered methylotrophic yeast adapted to express high concentrations of heme protein.
  • a yeast culture expressing heme protein was obtained through M3 fermentation. Approximately 20 g/L of heme in broth was present in the culture. A raw yeast paste was obtained by centrifugation at 15900 X g for 10 min. About 46 mg heme/g wet cell weight (WCW) was present in the resulting wet paste. The yeast paste was washed with de-ionized water. The paste was divided into samples at different storage conditions and evaluated for color and taste. Ten grams of processed heme protein was used for comparison. A summary of the evaluation is provided in Table 1 and FIG 1 provides photographs of the yeast paste samples used in this evaluation.
  • Results suggest that the color from the yeast paste could be sufficient to be used directly in burger patties.
  • the taste of the paste was sour, and the pH varied between samples. Further tests were conducted to address desirable taste and pH requirements.
  • yeast cells may be used as raw yeast paste, spray dried yeast paste or freeze-dried yeast paste.
  • Table 2 Exemplary batch of yeast culture
  • the yeast culture was diluted with two times the volume of culture with buffer A (50m M NaPC , 10mM Na ascorbate, 100mM NaCI). The resulting sample was centrifuged at 15900 X g for 10 minutes. The pellet was scooped and resuspended in 8 L of buffer A and centrifuged again. This process was repeated until the supernatant was clear (about 6 times). 1.1 kg of cell paste was recovered and stored at 4°C.
  • buffer A 50m M NaPC , 10mM Na ascorbate, 100mM NaCI.
  • a Buchi B290 Mini spray dryer was used to obtain a spray dried yeast paste.
  • Raw yeast paste was obtained as detailed above.
  • 257g of washed yeast paste was resuspended in 2.3kg of water to form a 11 % wet solids feed.
  • the resuspended paste was passed through the spray drier at a feed rate of 6-9mL/min.
  • the inlet temperature was maintained at 210°C and the outlet temperature was 114°C.
  • Raw yeast paste was similarly obtained. 180g of washed yeast paste was placed in a lyophilizer. The sample was frozen at -50°C for 10 min before starting the vacuum and lyophilized at 500mTorr for 24 hours and 30 minutes. 71.6g of the lyophilized product was recovered and vacuum sealed within 1 hr. [0095] Table 3 provides a summary of the heme content in the samples obtained by the three processes and FIG. 2 provides photographs showing characteristics of each form used.
  • Results further support the potential to use yeast paste to achieve the color and flavor of downstream processed heme protein. All three forms preserve the color of the product. Drying yeast (spray dried or freeze dried) can further enhance shelf-life.
  • yeast paste (wet or dried) can be used in food products
  • the three forms of yeast pastes were used in plant-based burger formulations.
  • the amount of paste needed in the patty was determined.
  • Table 4 provides the amount of paste needed in each formulation and test condition.
  • Table 4 Yeast paste (at 15g/L) needed for each formulation and test condition
  • Heme yeast cells HYCP - paste, or in dried form
  • processed heme protein was added to the following burger formulation as provided (see Table 5). Briefly, water and HYCP were first combined to form an homogenous mixture. Texturized vegetable protein (TVP) was added to the mix and hydrated for 30 minutes. Separately, methylcellulose, soy protein, salt, pepper, and potato starches were combined and set aside (dry powder). Sunflower lecithin, sunflower oil and coconut oil were also combined separately and melted. The TVP mixture, dry powders and oil were mixed together for 2 minutes in an homogenizer for 2 minutes on low speed. Forty (40) gram patties were made from the mixture using ring molds. The patties were frozen overnight, and vacuum sealed the next day and stored frozen.
  • TVP Texturized vegetable protein
  • Table 5 Plant-based burger formulation comprising heme yeast cells (as HYCP)
  • FIG. 3 and FIG. 4 provide a series of photographs of raw and cooked versions of the patties after day 1 and day 14 of the initial freezing.
  • the remaining two sets of stored patties kept frozen 14 days and after the frozen storage were refrigerated for 7 days and 14 days respectively and subsequently cooked.
  • FIG. 5 shows a series of photographs of patties cooked after refrigeration for 7 days after freezing for 14 days.
  • FIG. 6 shows a series of photographs of patties cooked after refrigeration for 14 days after freezing for 14 days.
  • Each of the patties was further characterized for color values (L- value, a-value and b-value).
  • the L-value is a measure of the observed color along a black-to-white axis.
  • the “a-value” is a measure of the observed color along a “redness” axis, and the “b-value” is a measure of the observed color along a yellow to blue axis.
  • FIG. 7A, FIG 7B and FIG. 7C provide graphical representation of the L-value, a-value and b-value respectively for raw patties.
  • FIG. 8A, FIG 8B and FIG. 8C provide bar graphs of the color values (L-value, a-value and b-value) respectively for the cooked patties. Both L-value and a-value were largely consistent across the sets and storage conditions. B-value was lower for heme protein (C279018) with storage but was largely consistent on storage for yeast paste.
  • FIG. 9 shows bar graphs characterizing the pH of each cooked set. The pH was found to decrease for wet paste and freeze-dried paste but remained consistent for spray dried paste.
  • FIG. 10 provides an estimate of the moisture loss seen for each set under different storage conditions. Surprisingly, burger samples with yeast paste exhibited greater moisture retention on refrigeration than the sample containing heme protein.
  • Example 4 The effect of increasing HYCP concentration in vegetable broth
  • HYCP Increasing concentrations of HYCP were tested in vegetable stock.
  • HYCP was added at a range of concentrations 0-5% (specifically, 0%, 1 %, 2.5% and 5%) with or without 0.5% salt, and the resulting formulations were tested for their flavor profile. Results are described in FIG. 11. Specifically, it was found that lower concentrations of HYCP in the vegetable broth without salt produced very mild iron and umami notes, while adding salt increased savory flavor and umami notes. Approximately 2.5% HYCP and approximately 0.5% salt was found to have stronger umami, meaty flavor, and better mouth feel, with taste like a beef bouillon, while the higher concentration of HYCP (e.g., 5%) produced a metallic after taste and a salty taste (FIG. 11).
  • a plant-based faux gras was prepared with HYCP, according to the proportions in the Table 7. Tofu and mushrooms were roasted in olive oil at 350 0 F for 15 minutes. Garlic and shallots were slowly reduced in sherry and marsala wines, until liquid was reduced to a syrup. All the ingredients were pureed in a blender until smooth. The Faux Gras was spread and served on crackers. The plant-based faux gras was tasted and found to share an iron rich flavor profile with real liver meat.
  • a rich, deep flavored, iron fortified plant-based alternative to shredded beef was prepared using HYCP (Table 8 below). It was found that HYCP easily solubilizes to create concentrated aromatics and flavors which simultaneously imparting a reddish-brown color to mimic beef. Salt was found to enhance and amplify the flavor profile.
  • the gluten matrix was developed using temperature and shear to create a fibrous network that sets during cooking under high pressure.
  • HYCP was used with other synergizing compounds like salts, IMP and GMP as well as with enzymatic treatments (cellulases, etc) to modify and enhance vegetable texture.
  • Table 8 Composition of Plant-based Shredded Beef Mimic with HYCP
  • tofu was pressed firmly to remove any excess moisture, and placed in a Vitamix blender. Salt was added and the tofu was blended on high for 2-3 min to produce a mayonnaise-like texture. The tofu mixture was then placed in a vacuum seal bag and vacuum-seal fully and steamed for 60 min. The mixture was removed from heat and let to cool until just warm to the touch. The slightly warm tofu were mixed with wet ingredients in the bowl of a stand mixer. HCYP and 9.4g distilled water were combined and mixed separately. The proteins were then added into wet ingredients and mixed to pre-hydrate. The remaining ingredients were added to a stand mixer bowl with a paddle attachment, and mixed for 5 minutes on low to moderate speed until smooth.
  • the dough thus formed was allowed to rest for 30 min at room temperature covered with plastic wrap.
  • the dough was further kneaded in a Thermomix multi-cooker with a kneading function for 9 min on speed 3 in reverse.
  • the dough was shaped into a log about 2 inches around, wrapped tightly first in parchment paper brushed with a neutral oil, then in foil, and sealed with tape.
  • the dough was cooked in a pressure cooker set on high for one hour and was chilled once cooled. The outside layer was peeled with a vegetable peeler and discarded. The cooked dough was then shredded using a peeler.
  • the shreds can be cooked by heating and/or browning in a preheated pan with a drizzle of oil, for approximately 2- 4 minutes.
  • a preheated pan with a drizzle of oil, for approximately 2- 4 minutes.
  • both beef products were served with BBQ sauce and the flavor profile examined.
  • the HYCP supplemented product was judged to have an enhanced meatier, more savory and generally more desirable flavor.
  • Example 7 Exemplary Food Product Made with HCYP - Tofu
  • a tofu-based meat substitute product was prepared using HCYP.
  • Example 6 imitation beef product
  • the high solubility of HYCP lead to more concentrated aromatics and flavors while simultaneously imparting a reddish-brown color. Salt enhanced the flavor profile.
  • the tofu matrix was developed using temperature, shear and transglutamase to create an extra dense meat-like texture that cues real meat.
  • the iron- rich beefy flavor profile allowed HYCP to bring rich flavor quality to the tofu space.
  • Table 9A Compositions of HYCP and Tofu Products
  • the meaty tofu described above was prepared as follows. The tofu was pressed firmly to remove excess moisture and placed in a Vitamix blender. Salt was added and tofu was blended on high for 2-3 min to produce a mayonnaise-like texture. The blended tofu mixture was placed in a vacuum seal bag and vacuum-sealed fully. The mixture was then steamed for 60 min and let cooled until just warm to the touch. The tofu mixture was then removed from the steam pouch and mixed using an immersion blender until smooth. The mixture was cooled to just below 45 °C and HYCP solution comprising HYCP, water, and flavor was added to the mixture and blended until smooth and homogeneous.
  • HYCP gave umami-like flavors to otherwise flavorless vegetables like zucchini and squash. Grilling the vegetables was hypothesized to enhance the meat-like flavor from HYCP.
  • the ingredients in the marinade are shown in Table 10. Approximately 0.5% heme was present in the marinade.
  • vegetables marinaded without HYCP was used. Briefly, carrots, onions, zucchini and potatoes were cut in unform size. The infusion liquid was prepared by gently heating water and adding HYCP and salt. The infusion liquid was stirred until the added HYCP and salt were dissolved. The cut vegetables were then placed in a cryovac bag with the infusion liquid, vacuum sealed, and let sit for at least 10 minutes. The vegetables were then drained from the infusion liquid and then cooked either by roasting in an oven or a grill. Table 10: Composition of a Savory Infusion
  • HYCP easy water solubility, savory flavor profile, iron-rich nutrient composition and protein thickening potential elevated plant-based broth taste and texture.
  • HYCP HYCP
  • HYCP could be leveraged as an iron supplement for manufacturers looking for highly bioavai lable iron.
  • HYCP could be used with other synergizing compounds (salts, IMP, GMP, etc) to modify and enhance vegetable flavor.
  • the composition of an exemplary vegan meaty broth is shown in Table 12.
  • the vegan meaty broth was prepared by combining a commercially available vegan broth, HYCP and salt in a small pot and heating over medium heat until the salt was dissolved. Taste testing was performed on hot broth, but the broth could be served hot or cold. The broth had approximately 0.3% by weight of the HYCP.
  • Table 13 shows the composition of an exemplary broth booster.
  • the broth booster was prepared by combining all the ingredients shown in Table 13 in powder form, and mixing to evenly disperse. Five grams of the dry broth booster mix was then added to 150g of water in a small pot and heated over medium heat until boiling. The broth was then removed from heat and served hot. The broth booster had approximately 0.5% by weight of HYCP.
  • Table 14 shows the composition of a soup comprising HYCP.
  • the soup was prepared by adding HYCP to a commercially available canned vegetable soup and heating over medium heat until the HYCP was dissolved, and served hot.
  • the soup comprised approximately 0.3% by weight of HYCP.
  • Ramen noodles comprising HYCP were prepared.
  • the beef flavor powder packet in a package of commercially available Maruchan Ramen noodles was combined with 3% by weight HYCP.
  • the powder mix was then added to water and brought to boil, until HCYP dissolved.
  • the noodles, such as the commercially available ramen noodles were added to the boiling water and cooked al dente and served hot or cooled.
  • the noodles comprise approximately 0.3% by weight of HYCP.
  • flavor bombs comprising HYCP were prepared by adding powdered yeast powder (HYCP, 3% HYCP, 0.75% Heme) to water (10 grams powder to 90g hot water).
  • HYCP powdered yeast powder
  • HYCP 3% HYCP
  • Heme powdered yeast powder
  • the flavor bomb mix comprising HYCP was found to have richer umami and meaty taste, than the control (Hemami) flavor bomb.
  • a plant-based “butter” enhanced with HYCP was prepared. This product was then used to prepare seafood plates. It was found that HYCP’s easy water solubility, savory flavor profile, iron-rich nutrient composition, and protein thickening potential elevated the plant-based butter taste in taste testing. Further, adding protein and iron to a plant-based butter can boost the nutritional profile. It was also found that lacing butter with HYCP enhanced an iron rich flavor found in lobster, making HYCP- enhanced real dairy butter or HYCP-enhanced plant-based butter ideal accompaniments for real seafood and shellfish, or plant-based seafood and shellfish analogs.
  • Table 16 shows the ingredients in a plant-based butter enhanced with HYCP.
  • the plant-based butter enhanced with HYCP was prepared by heating a commercially available plant-based butter (e.g., Earth Balance Original) together with HYCP, until the composition was melted. The melted mix was stirred to obtain a homogenous mixture, and taste tested by drizzling over other food.
  • a commercially available plant-based butter e.g., Earth Balance Original
  • Table 16 Composition of enhanced plant-based butter Example 11 - Exemplary Pet Food Product Made with HCYP
  • a pet food product was prepared using HCYP. As described in earlier examples, HYCP easily solubilizes into broths which enhances dry dog food aromatically and flavorfully without adding sodium. The nutritional iron content also can combat anemia.
  • a wet broth was prepared as the delivery vehicle to deliver the HYCP product to dry dog food.
  • the wetness further augmented flavor and improved the texture of dry dog foods for aging dogs. It was found that adding heme paste to pet food gave a more robust, iron scent attractive to dogs which made the food more appealing.
  • the pet food was prepared by straining a broth mixture (for e.g., Canidae Sustain Bone Broth Toppers), followed by adding and mixing HYCP at about 3% (wt/wt). The strained solids were then added back into the broth mixture. The pet food was found to have approximately 0.3 wt% Heme.
  • a broth mixture for e.g., Canidae Sustain Bone Broth Toppers
  • Example 12 Chocolate Product Made with HYCP
  • composition of a sample milk and a sample dark chocolate product combined with HYCP are described in Table 18 below.
  • Commercially available chocolate products e.g., Accent milk chocolate; Barry Callebaut, Breda Dark chocolate; Barry Callebaut
  • HYCP powder was gradually stirred into the melted chocolate, until fully dispersed and smooth.
  • the chocolate was molded into a desired shape and cooled.
  • Table 18 Composition of milk and dark chocolate products comprising HYCP
  • a yeast culture expressing fish heme protein e.g., any of SEQ ID NO: 1
  • NOs: 12-16 will be obtained through M3 fermentation.
  • the level of heme in the broth in the culture will be measured and a raw yeast paste will be obtained by centrifugation at 15900 X g for 10 min.
  • the amount of heme per gram wet cell weight (WCW) will be measured in the resulting wet paste.
  • the yeast paste will be washed with de-ionized water.
  • the paste will be divided into samples at different storage conditions and evaluated for color and taste. Ten grams of processed heme protein will be used for comparison.
  • the yeast paste prepared with fish heme protein produced as described in Example 13 will be used as described for bovine heme yeast paste in Examples 5 to 11 to prepare associated savory and sweet food products.
  • the yeast paste prepared with fish heme protein will be used to prepare a flavor enhancer or other composition useful for flavoring a fish dish (e.g., as described in Example 10) or to prepare a fish-like product (e.g., fish fillet, seafood cutlets, seafood pies, salmon burgers, fish sticks, crab cakes, fish burgers, fish cakes, chowder, bisques, rolls, or seafood stews).

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Abstract

L'invention concerne des compositions de biomasse comprenant des cellules microbiennes telles que des cellules de levure modifiées pour exprimer une protéine contenant de l'hème, et leur utilisation dans la préparation de diverses compositions alimentaires.
PCT/US2023/073993 2022-09-12 2023-09-12 Biomasse contenant de l'hème dans une composition alimentaire à base de plantes WO2024059578A2 (fr)

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