Structured high protein meat substitute compositions with microbial heme flavors

Abstract

To provide alternative protein-rich food products that are not derived from animals, but are free of unhealthy meat-related components such as saturated fatty acids and cholesterol, are free of the deleterious environmental effects of animal husbandry, and provide similar texture and flavor characteristics and functional properties as animal meat.SOLUTION: Structured food compositions, such as meat substitute compositions, comprising microbial-derived protein products, such as microbial-derived protein hydrolysates, are provided. Methods of making such products are also provided. Also provided are flavor potentiator compositions comprising heme-containing polypeptides, such as microbial-derived flavohemoglobin, and food compositions, such as meat substitute compositions, comprising the heme-containing flavor potentiator compositions. In one aspect, the flavor potentiator comprises a heme-containing polypeptide from a hydrogen-oxidizing microorganism, wherein (i) the hydrogen-oxidizing microorganism is a Cupriavidus microorganism or (ii) the hydrogen-oxidizing microorganism grows under microaerobic conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 001,215, filed March 27, 2020, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to structured food compositions comprising microbially-derived protein products, such as structured meat substitute compositions that are suitable for human or animal consumption, closely mimic the properties of meat, and function as meat substitute products, particularly structured meat substitute compositions comprising microbially-produced heme-containing polypeptides that, upon heating, catalyze the production of a meat-like flavor or aroma. [Background technology]

[0003] Eating meat derived from animal sources is a part of daily life for many people. The adverse effects of a meat-based diet on human health and the environment are well documented. There is growing consumer demand for alternative protein-rich foods that are not derived from animals but that offer similar texture and flavor characteristics as animal meat, as well as similar functional properties, without the unhealthy components associated with meat, such as saturated fatty acids and cholesterol, and without the harmful environmental impacts of animal agriculture. Summary of the Invention

[0004] Disclosed herein are structured food products, such as structured meat products, eg, structured meat substitute products, and methods of producing such products.

[0005] In one aspect, a microbially derived heme-containing protein product (e.g., a single-cell protein, a protein hydrolysate, a cell lysate, a protein isolate, a protein extract, a peptide, an oligopeptide, or an isolated heme-containing protein) is provided. When heated, the microbially derived heme-containing protein product releases heme that reacts with organic compounds, such as sugars and / or amino acids, to produce a desired flavor and / or aroma, such as a flavor and / or aroma that mimics the flavor and / or aroma of meat, for example, in a structured meat product. In some embodiments, the heme-containing protein product can be derived from a hydrogen-oxidizing microorganism, such as, but not limited to, a Cupriavidus microorganism. In some embodiments, the hydrogen-oxidizing microorganism is grown under O2-limited conditions.

[0006] In one aspect, a heme-containing protein product is provided having a heme content within the heme-containing protein product, i.e., on a weight basis, from about 1% (w / w) to about 15% (w / w), from about 1% (w / w) to about 10% (w / w), from about 1% (w / w) to about 9% (w / w), from about 1% (w / w) to about 8% (w / w), from about 1% (w / w) to about 7% (w / w), from about 1% (w / w) to about 6% (w / w), from about 1% (w / w) to about 5% (w / w), from about 2% (w / w) to about 10% (w / w), from about 3% (w / w) to about 10% (w / w), from about 4% (w / w) to about 10% (w / w), or from about 5% (w / w) to about 10% (w / w). In some embodiments, the heme content in the heme-containing protein product is at least about 0.5% (w / w), at least about 1.0% (w / w), at least about 2.0% (w / w), at least about 3% (w / w), at least about 4% (w / w), at least about 5% (w / w), at least about 6% (w / w), at least about 7% (w / w), at least about 8% (w / w), at least about 9% (w / w), at least about 10% (w / w), or at least about 15% (w / w). In some embodiments, such heme-containing protein products are used as ingredients to form structured food products according to the methods and compositions described herein. In some embodiments, the resulting structured food product has a saturation of about 0.1% (w / w) to about 3% (w / w), about 0.1% (w / w) to about 2% (w / w), about 0.1% (w / w) to about 1.5% (w / w), about 0.1% (w / w) to about 1% (w / w), about 0.1% (w / w) to about 0.9% (w / w), about 0.1% (w / w) to about 0.8% (w / w), about 0.1% (w / w) to about 0.7% (w / w), about 0.1% (w / w) to about 0. 6% (w / w), about 0.1% (w / w) to about 0.5% (w / w), about 0.2% (w / w) to about 1.5% (w / w), about 0.3% (w / w) to about 1.5% (w / w), about 0.4% (w / w) to about 1.5% (w / w), about 0.5% (w / w) to about 1.5% (w / w), about 0.5% (w / w) to about 3% (w / w), about 1% (w / w) to about 2% (w / w), or about 2% (w / w) to about 3% (w / w).

[0007] In one aspect, a structured food product is provided that includes a protein product derived from a microorganism. In some embodiments, the microorganism comprises or consists of one or more chemoautotrophically grown microorganisms. In one embodiment, the microorganism, for example, the chemoautotrophically grown microorganism, comprises or consists of a Cupriavidus microorganism.

[0008] In some embodiments, the structured food comprises a protein product from a first microorganism grown on a nutrient source that comprises a protein product from a second microorganism. In one embodiment, the second microorganism comprises or consists of a Cupriavidus microorganism. For example, the first microorganism may be a Generally Recognized As Safe (GRAS) microorganism, and in some cases, the second microorganism may be a non-GRAS microorganism.

[0009] In some embodiments, the structured food comprises protein products derived from microbial organisms that are non-GMO. In some embodiments, the structured food does not comprise animal-derived biomolecules such as animal-derived proteins, lipids, and / or carbohydrates.

[0010] In some embodiments, the structured food comprises a protein product comprising one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof derived from one or more microorganisms. In one embodiment, the protein product comprises or consists of protein hydrolysates derived from one or more microorganisms. In some embodiments, the structured food comprises at least about 5% to about 50%, or about 10% to about 50%, or about 20% to about 50%, or about 30% to about 50% protein by weight from the microbial protein product. In some embodiments, the protein product comprises one or more heme-containing proteins.

[0011] In some embodiments, the structured food comprises a non-animal protein ingredient that comprises or consists of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof, where such protein ingredients impart one or more beneficial functional properties to the structured food.

[0012] In some embodiments, the structured food product is a structured meat substitute product, such as, but not limited to, structured beef, poultry, pork, fish, lamb, or seafood (e.g., crab, shrimp, lobster) substitute products. For example, the structured meat substitute product can replicate the texture and / or organoleptic properties of natural meat. In some embodiments, the meat substitute product mimics the structure of ground or muscle meat. In certain embodiments, the structured meat substitute product comprises one or more flavorings, such as a flavohemoprotein flavoring. In one embodiment, the structured meat substitute comprises a flavohemoprotein flavoring produced by a microorganism. In some embodiments, the structured meat substitute product comprises a protein product derived from chemoautotrophically grown microorganisms or from microorganisms grown in a medium comprising a protein product derived from chemoautotrophically grown microorganisms. In one embodiment, the chemoautotrophically grown microorganisms comprise or consist of Cupriavidus microorganisms.

[0013] In some embodiments, the structured food product, such as but not limited to, a structured meat substitute product, has one or more added substances, such as vitamins, nutrients, or substances with beneficial functional properties. For example, the added substance(s) may be amino acids, lipids, oils, fatty acids, vitamin B, or the like. 12 or may include one or more of other vitamins, biotin, antioxidants, minerals, surfactants, and emulsifiers.

[0014] In another aspect, a dough composition is provided for producing a structured food product as described herein, e.g., a structured food product comprising a microbially derived protein product. In some embodiments, the microorganism comprises or consists of one or more chemoautotrophically grown microorganisms. In one embodiment, the microorganism, e.g., the chemoautotrophically grown microorganism, comprises or consists of a Cupriavidus microorganism.

[0015] In some embodiments, the dough composition comprises a protein product from a first microorganism grown on a nutrient source that comprises a protein product from a second microorganism. In one embodiment, the second microorganism comprises or consists of a Cupriavidus microorganism. For example, the first microorganism can be a Generally Recognized As Safe (GRAS) microorganism, and in some cases, the second microorganism can be a non-GRAS microorganism.

[0016] In some embodiments, the dough composition comprises a protein product derived from a non-GMO microorganism. In some embodiments, the dough composition does not comprise animal-derived biomolecules, such as animal-derived proteins, lipids, and / or carbohydrates.

[0017] In some embodiments, the dough composition comprises a protein product comprising one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof derived from one or more microorganisms. In one embodiment, the protein product comprises or consists of protein hydrolysates derived from one or more microorganisms. For example, the average molecular weight of the proteins in the hydrolysates may be from about 5 kD to about 10 kD. In certain embodiments, the dough composition comprises a protein hydrolysate produced under conditions that preserve intact globular proteins, such as gentle cell lysis and physical separation of soluble material (e.g., protein) from solid material (e.g., cell debris).

[0018] In some embodiments, a dough composition comprising, for example, one or more partially or fully hydrolyzed protein products derived from microorganisms is added to a second protein product to promote structure and / or fiber formation. Non-limiting examples of second protein products include one or more of wheat gluten, soy, pea, wheat, dairy, algae, and other non-animal proteins.

[0019] In some embodiments, the moisture content of the dough composition is from about 40% (w / w) to about 80% (w / w). In some embodiments, the shear strength of the dough is at least about 1000 psig.

[0020] In another aspect, a method for producing a structured food product, e.g., a structured meat substitute product, is provided, comprising: (a) culturing a microorganism in the presence of a carbon source, thereby producing a protein-containing biomass; (b) converting the biomass to a protein product; and (c) processing the protein product into a structured food composition.

[0021] In some embodiments, step (a) comprises chemoautotrophic culture conditions. For example, the chemoautotrophic culture conditions can include a gaseous C1 molecule, such as CO2, as a carbon source. In some embodiments, the microorganisms grown under chemoautotrophic culture conditions comprise or consist of Cupriavidus microorganisms.

[0022] In some embodiments, the protein product produced in step (b) comprises one or more of single cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof.

[0023] In some embodiments, step (c) comprises processing the protein product into a dough composition. For example, a thermochemical process such as extrusion can be employed. In some embodiments, step (c) can comprise a spinning process, which can result in the production of fibers. In some embodiments, step (c) can comprise the formation of a structured hydrocolloid.

[0024] In some embodiments, the structured food product produced in step (c) is a structured meat substitute product, such as a structured beef, poultry, pork, fish, or seafood (e.g., crab, shrimp, lobster) substitute product. For example, the structured meat substitute product may replicate the texture and / or organoleptic properties of natural meat. In some embodiments, the meat substitute product mimics the structure of ground or muscle meat. In certain embodiments, the structured meat substitute product comprises one or more flavorings, such as a flavohemoprotein flavoring. In one embodiment, the structured meat substitute comprises a flavohemoprotein flavoring produced by a microorganism. In some embodiments, the structured meat substitute product comprises a protein product derived from chemoautotrophically grown microorganisms or from microorganisms grown in a medium comprising a protein product derived from chemoautotrophically grown microorganisms. In one embodiment, the chemoautotrophically grown microorganisms comprise or consist of Cupriavidus microorganisms.

[0025] In another aspect, a thermochemical process is provided for processing a dough composition as described herein into a structured meat substitute composition. The thermochemical process comprises extruding a dough composition containing one or more microorganism-derived protein products to produce aligned fibers as described herein. In some embodiments, the protein product comprises or consists of a protein hydrolysate. In some embodiments, the microorganism from which the protein product is derived is a chemoautotrophically grown microorganism or a microorganism grown in a medium containing a protein product derived from a chemoautotrophically grown microorganism. For example, the chemoautotrophically grown microorganism may comprise or consist of a Cupriavidus microorganism. [Brief explanation of the drawings]

[0026] [Figure 1] Figure 1 shows a representative, non-limiting workflow for one embodiment of the production of structured foods as described herein. WCB = total cell biomass [Figure 2] FIG. 2 shows emulsion capacity for whey protein isolate, as described in Example 2. [Figure 3] FIG. 3 shows emulsion volume relative to whole cell biomass (WCB), as described in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0027] Provided herein are structured food compositions and methods for producing the same. The structured food compositions described herein, including structured meat substitute compositions, comprise protein products (e.g., protein products that comprise or consist of protein hydrolysates) produced by microorganisms. For example, the microorganisms from which the protein products are derived may grow chemoautotrophically on, for example, gaseous C1 substrates as a carbon source.

[0028] definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley & Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods, systems, and compositions described herein.

[0029] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are fully explained in the literature, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Current Protocols in Molecular Biology (F.M. Usubel et al., eds., 1994); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transfer and Expression: A Laboratory Manual (Kriegler, 1990).

[0030] Numerical ranges provided herein are inclusive of the numbers defining the range.

[0031] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0032] The terms "a," "an," and "the" include plural references unless the context clearly indicates otherwise, and thus the indefinite articles "a," "an," and "the" as used in the specification and claims shall be understood to mean "at least one" unless clearly indicated to the contrary.

[0033] The term "about," as used herein, when referring to a measurable value such as an amount, temporal duration, etc., is meant to encompass variations of ±5%, ±1%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods or in connection with the disclosed compositions.

[0034] "Acetogen" refers to a microorganism that produces acetate and / or other short-chain organic acids of up to C4 chain length as a product of anaerobic respiration.

[0035] "Acidophilic microorganisms" refer to a type of extremophilic microorganism that thrives under strongly acidic conditions (usually pH 2.0 or below).

[0036] The term "amino acid" refers to a molecule containing both an amine group and a carboxyl group attached to a carbon referred to as the alpha carbon. Suitable amino acids include, but are not limited to, both D- and L-isomers of natural amino acids and unnatural amino acids prepared by organic synthesis or other metabolic pathways. In some embodiments, a single "amino acid" may have multiple side chain moieties available per extended aliphatic or aromatic backbone. Unless the context specifically dictates otherwise, as used herein, the term amino acid is intended to include amino acid analogs.

[0037] As used in this specification and the claims, the phrase "and / or" shall be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present in some cases jointly and in other cases disjunctively. In some cases, other elements other than those specifically identified by the "and / or" clause may be present, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," can refer to, in one embodiment, A without B (optionally including elements other than B); in another embodiment, B without A (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so forth.

[0038] The term "biomass" refers to the material produced by the growth and / or reproduction of cells, such as microbial cells. Biomass can include cells and / or intracellular contents as well as extracellular material, including, but not limited to, compounds secreted by the cells.

[0039] The term "bioreactor" or "fermentor" refers to a sealed or partially sealed vessel in which cells, such as microbial cells, are grown and maintained. The cells may, but need not, be maintained in liquid suspension. In some embodiments, rather than being maintained in liquid suspension, the cells may instead be grown and / or maintained in contact with, on, or within another non-liquid substrate, including, but not limited to, a solid growth support material.

[0040] The term "carbon fixation" process, reaction, or pathway refers to an enzymatic reaction or metabolic pathway that converts forms of carbon that are gaseous under ambient conditions, including, but not limited to, CO, CO, and CH, into carbon-based biochemicals that are liquid or solid under ambient conditions, dissolved in aqueous solution, or held in suspension in aqueous solution.

[0041] "Carbon source" refers to the type of molecule from which a microorganism obtains the carbon needed for organic biosynthesis.

[0042] "Carboxydotroph" refers to a microorganism that can tolerate or oxidize carbon monoxide. In preferred embodiments, a carboxydotrophic microorganism can utilize CO as a carbon source and / or as a source of reduced electrons for biosynthesis and / or respiration.

[0043] "Chemoautotroph" refers to an organism that obtains energy from the oxidation of chemical electron donors by chemical electron acceptors and synthesizes from carbon dioxide all of the organic compounds it needs to survive and grow.

[0044] "Chemolithoautotroph" refers to an organism that derives energy from the oxidation of inorganic compounds and uses carbon dioxide as its sole carbon source for growth.

[0045] In the claims and the specification, all transitional phrases such as "comprising," "including," "holding," "having," "containing," "involving," "holding," etc. shall be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0046] As used herein, a "consortium" refers to two or more different species or strains of microorganisms and / or multicellular organisms grown together, for example, grown in co-culture in the same growth medium.

[0047] The term "culturing" refers to growing a population of cells, such as microbial cells, in a liquid or solid medium under conditions suitable for growth.

[0048] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," and "made from," and generally indicates that a particular substance originates from, or has characteristics that can be described with reference to, another particular substance.

[0049] The term "dough" as used herein refers to a blend of dry ingredients ("dry mix"; e.g., proteins, carbohydrates, and lipids, including liquid oils) and liquid ingredients ("liquid mix"; e.g., water or juice, such as a liquid-based extract from a non-animal source, such as a plant or any part of a plant). The dough may also include one or more additional protein products with structural and / or functional properties that impart or enhance the structuring qualities of the dough, e.g., during the shearing process.

[0050] "Energy source" refers to either an electron donor that is oxidized by oxygen in aerobic respiration or a combination of an electron donor that is oxidized and an electron acceptor that is reduced in anaerobic respiration.

[0051] "Extremophile" refers to a microorganism that thrives in extreme physical or geochemical conditions (e.g., high or low temperature, high or low pH, or high salinity) relative to the Earth's surface or ocean conditions typically tolerated by most life forms found at or near the Earth's surface.

[0052] The terms "functional properties," "functional characteristics," or "functionality" or similar descriptors refer to how food ingredients behave during preparation and cooking and how they affect the finished food product in terms of appearance, taste, texture, and handling. Functional properties may include water absorption, water solubility, oil absorption index, swelling index, bulk density, viscosity, binding, aeration, thickening, setting, color, dextrinization, caramelization, jelling, denaturation, coagulation, emulsion capacity, or emulsion stability.

[0053] The term "gasification" refers to a general high-temperature process that converts carbonaceous materials into a mixture of gases containing hydrogen, carbon monoxide, and carbon dioxide, called synthesis gas, syngas, or producer gas. The process generally involves partial combustion and / or the application of externally generated heat, along with the controlled addition of oxygen and / or steam so that insufficient oxygen is present for complete combustion of the carbonaceous material.

[0054] "Halophiles" refer to a type of extremophile microorganism that thrives in environments with very high concentrations of salt.

[0055] A "heterotroph" is an organism that cannot synthesize all of the organic compounds necessary for its survival and growth from carbon dioxide and must utilize these compounds for growth. Heterotrophs cannot produce their own food; that is, rather than fixing carbon from inorganic sources such as carbon dioxide, they instead obtain food and energy by ingesting and metabolizing organic matter, such as plants or animals.

[0056] "Hydrogen-oxidizing bacteria" refers to microorganisms that utilize reduced H2 for the production of intracellular reducing equivalents and / or as an electron donor in respiration.

[0057] "Hyperthermophiles" refer to a type of extremophile microorganism that thrives in extremely hot environments for life, typically above about 60°C (140°F).

[0058] The term "detonation gas" refers to a mixture of molecular hydrogen and oxygen gases. "Hydrogen-oxidizing microorganisms" are microorganisms that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration for the production of intracellular energy carriers such as adenosine-5'-triphosphate (ATP). The terms "oxyhydrogen" and "oxyhydrogen microorganisms" can be used synonymously with "detonation gas" and "hydrogen-oxidizing microorganisms," respectively. Hydrogen-oxidizing microorganisms generally use molecular hydrogen through hydrogenases, and some of the electrons donated from H are converted to NAD. +(and / or other intracellular reducing equivalents), and some of the electrons from H are used in aerobic respiration. Hydrogen-oxidizing microorganisms generally fix CO autotrophically through pathways including, but not limited to, the Calvin cycle or the reverse citric acid cycle ["Thermophilic bacteria," Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0059] The term "lysate" refers to a liquid and / or solution of cellular contents, including a mixture resulting from cell lysis, such as microbial cell lysis. In some embodiments, the methods described herein involve purification of a chemical or mixture of chemicals in the cell lysate. In some embodiments, the methods involve purification of amino acids and / or proteins in the cell lysate.

[0060] The term "lysis" refers to the disruption of the plasma membrane, and, if present, the cell wall, of cells, such as microbial cells, such that a significant amount of intracellular material escapes into the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal, or viral means. In some embodiments, the methods described herein comprise performing lysis of cells or microorganisms as described herein to separate a chemical or mixture of chemicals from the contents of a bioreactor. In some embodiments, the methods comprise performing lysis of cells or microorganisms as described herein to separate an amino acid or a mixture of amino acids and / or proteins from the contents of a bioreactor or cell growth medium.

[0061] The term "meat substitute" or "meat substitute" or "fake meat" or "meat substitute" as used herein refers to a food product that is not derived from an animal or contains a significant amount of non-animal protein sources, but has structure, texture, aesthetic qualities, and / or other properties comparable to or similar to animal meat. The term refers to uncooked, undercooked, and cooked meat-like foods.

[0062] "Methanogen" refers to a microorganism that produces methane as a product of anaerobic respiration.

[0063] "Methylotrophic bacteria" refers to microorganisms that can use reduced one-carbon compounds, such as, but not limited to, methanol or methane, as a carbon source and / or as an electron donor for their growth.

[0064] The terms "microorganism" and "microbe" refer to microscopic, single-celled organisms such as bacterial and fungal microorganisms.

[0065] The term "molecule" means any distinct or identifiable structural unit of matter comprising one or more atoms, and includes, for example, carbohydrates, lipids, polypeptides, and polynucleotides.

[0066] An "oligopeptide" refers to a peptide that includes a relatively small number of amino acid residues, for example, from about 2 to about 20 amino acids.

[0067] As used herein and in the claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including more than one and not just the inclusion of at least one of a number or list of elements, and possibly further including unlisted items. Conversely, only explicitly stated terms such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or," as used herein, shall only be interpreted as indicating exclusive alternatives (i.e., "either one of, but not both") when preceded by exclusive terms such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0068] The term "organic compound" refers to any gaseous, liquid, or solid compound containing carbon atoms, with the following exceptions, which are considered inorganic: carbides, carbonates, simple oxides of carbon, cyanides, and allotropes of pure carbon, such as diamond and graphite.

[0069] "Peptide" refers to a compound (polypeptide) consisting of two or more amino acids joined in a chain in which the carboxyl group of each acid is linked to the amino group of the next by a bond of the type R-OC-NH-R', e.g., from about 2 amino acids to about 50 amino acids, or from 21 amino acids to about 50 amino acids.

[0070] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length and any three-dimensional structure, single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), containing deoxyribonucleotides, ribonucleotides, and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides containing modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides encoding specific amino acid sequences. Any type of modified nucleotide or nucleotide analog can be used, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, phosphorothioate, etc.), so long as the polynucleotide retains the desired function under the conditions of use. Labels, such as radioactive or non-radioactive labels or anchors, such as biotin, may also be incorporated for detection or capture purposes. The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides can be natural or non-natural. The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably herein. A polynucleotide may comprise RNA, DNA, or both, and / or modified forms and / or analogs thereof. The sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl, optionally containing an ether (-O-) linkage. Not all linkages within a polynucleotide need be identical. Polynucleotides can be linear or circular, or contain a combination of linear and circular portions.

[0071] As used herein, "polypeptide" refers to a composition composed of amino acids and recognized by those skilled in the art as a protein. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are altered naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0072] The term "precursor to" or "precursor of" refers to an intermediate toward the production of one or more components of an end product.

[0073] "Producer gas" refers to a gas mixture containing varying proportions of H2, CO, and CO2, typically having a heating value per unit volume ranging from one-half to one-tenth that of natural gas under standard conditions. Producer gas can be produced in a variety of ways from a wide variety of feedstocks, including gasification, steam reforming, or auto-reforming of carbonaceous feedstocks. In addition to H2, CO, and CO2, producer gas can contain other components, including but not limited to methane, hydrogen sulfide, liquefied gases, tar, and ash, depending on the production process and feedstock. The percentage of N2 in the mixture can be higher or lower depending on whether air is used as an oxidant in the reactor and whether the heat for the reaction is provided by direct combustion or through indirect heat exchange.

[0074] The term "producing" includes both intracellular and extracellular production of a compound, including secretion of the compound from the cell.

[0075] "Psychrophiles" refer to a type of extremophile microorganism that can grow and reproduce at low temperatures, typically below about 10°C.

[0076] The terms "recovered," "isolated," "purified," and "separated" as used herein refer to material (e.g., proteins, nucleic acids, or cells) that is removed from at least one component with which it is naturally associated. For example, these terms can refer to material that is substantially or essentially free from components that normally accompany it as found in its native state, such as in an intact biological system.

[0077] The terms "structured meat product" or "structured meat substitute product" or "meat-structured protein product" as used herein refer to a product comprising a protein fiber network and / or aligned protein fibers that result in a meat-like texture, with optional post-processing after the fiber and / or aligned structure has been generated and fixed (e.g., hydration, soaking, drying, coloring). Methods for measuring the degree of protein fiber network formation and / or protein fiber alignment are known in the art and include, for example, visual determination based on photographs and microscopic images, as exemplified in U.S. Patent Application Publication No. 2015 / 029683, which is incorporated herein by reference.

[0078] With respect to any given component, the phrase "substantially free" or "essentially free" means that such component, if present, is present only in a functionally insignificant amount, i.e., has no significant negative impact on the intended performance or function of any process or product. Typically, substantially free means less than about 1%, including less than about 0.5%, including less than about 0.1%, and also including zero percent, of such component by weight. The term "substantially free" or "essentially free" refers to less than 1% of the component.

[0079] "Sulfur-oxidizing bacteria" refers to microorganisms that utilize reduced sulfur-containing compounds, including but not limited to H2S, for the production of intracellular reducing equivalents and / or as electron donors in respiration.

[0080] "Syngas" or "synthesis gas" refers to a type of gas mixture that, like producer gas, contains H2 and CO, but is more specifically tailored in terms of H2 and CO content and ratios and impurity levels for the synthesis of specific types of chemical products, such as, but not limited to, methanol or Fischer-Tropsch diesel. Syngas generally contains H2, CO, and CO2 as its primary components and can be produced through established methods, including methane steam reforming; or through the gasification of any organic, combustible carbonaceous material, including, but not limited to, biomass, organic matter, or peat. The hydrogen content of syngas can be increased through the reaction of CO2 with steam in a water-gas shift reaction, with a concomitant increase in CO2 in the syngas mixture.

[0081] "Thermophiles" refer to a type of extremophile microorganism that thrives at relatively high temperatures for life, typically from about 45°C to about 122°C.

[0082] "Wild-type" refers to a microorganism as it occurs in nature.

[0083] "Yield" refers to the amount of product produced from a feed material relative to the total amount of material that would be produced if all of the feed material were converted to the product. For example, product yield can be expressed as the percentage of product produced relative to the theoretical yield if 100% of the feed material were converted to the product.

[0084] Heme flavoring ingredients Provided herein are food flavor enhancers that are rich in heme. In some embodiments, the flavor enhancers provide a flavor and / or aroma that mimics the flavor and / or aroma of meat when heated. For example, upon denaturation during cooking or pelleting, heme-containing polypeptides denature, releasing iron-containing heme that reacts with other organic molecules, such as sugars, amino acids, and lipids (e.g., organic molecules in microbial protein products), to produce flavor- and / or aroma-imparting molecules.

[0085] The food flavor enhancer can be in the form of a microbially derived protein product (e.g., single-cell protein, protein hydrolysate, cell lysate, protein isolate, protein extract, peptide, oligopeptide, or isolated or purified heme-containing protein, or a combination thereof) that has a high hemoglobin content, such as flavohemoglobin (FHb).

[0086] In some embodiments, the flavor enhancer is derived from a chemoautotrophic microorganism, e.g., a microorganism that grows on CO2 and H2. In some embodiments, the flavor enhancer is derived from a hydrogen-oxidizing microorganism, such as, but not limited to, a Cupriavidus microorganism, such as Cupriavidus necator, or any of the hydrogen-oxidizing microorganisms described herein. In some embodiments, the microorganism is engineered to express or overexpress FHb and / or produce or overproduce FHb or another hemoglobin-type protein under the culture conditions in which it is grown. For example, a strong promoter or an O2-regulated promoter can be used to express FHb to enhance production. In some embodiments, a heterologous polynucleotide, such as a heterologous promoter sequence, e.g., a strong promoter or an O2-regulated promoter to enhance FHb production, is introduced into the microorganism to enhance production of the native FHb sequence. In other embodiments, the microorganism is engineered to express a heterologous FHb (e.g., from a different species, e.g., a different microorganism). In another embodiment, a promoter from the same species of microorganism, such as a strong promoter or an O2-regulated promoter to enhance production of FHb, is introduced to enhance expression of the native FHb sequence.

[0087] Cupriavidus produces soluble membrane-associated proteins containing heme cofactors that are expressed under aerobic, microaerobic, and / or anaerobic fermentation conditions. The types of heme produced by Cupriavidus include a-, b-, c-, and d-type hemes, in which the iron ion is coordinated to a porphyrin acting as a tetradentate ligand and one or two axial ligands contributed by the protein.

[0088] Flavohemoglobin is naturally expressed by hydrogen-oxidizing bacteria under microaerobic conditions and performs O scavenging or O buffering functions at low O concentrations. Expression levels can be controlled using operating parameters for bacterial culture, such as low O partial pressure for high FHb expression. In some embodiments, microaerobic culture conditions are disrupted. For example, dissolved oxygen may be present at levels below the detection limit of a standard dissolved oxygen probe, e.g., below 1 ppm, which is lower than the level found in air or air-saturated solutions.

[0089] Soluble FHb from Cupriavidus is a three-domain monomer protein encoded by the bacterial genome. In vivo, FHb catalyzes the flavin-mediated oxidation of NADH, which in turn reduces b-type heme, which binds O2. The enzyme contains an NAD(H)-binding domain, a flavodoxin reductase-type domain, and a globin domain containing a b-type heme. The heme domain can be expressed separately. For example, a truncated form of the enzyme containing the heme, i.e., the heme domain, can be expressed in a microorganism and recovered.

[0090] A heme-containing food flavor enhancer as described herein can be included in any food composition, such as the meat substitute compositions described herein. The flavor enhancer produces a meat-like flavor and / or aroma during cooking (i.e., when heated).

[0091] Heme-containing food flavor enhancers (e.g., heme-containing polypeptides) as described herein may be processed from a microorganism as a single-cell protein, protein hydrolysate, cell lysate, protein isolate, protein extract, peptide, or oligopeptide, or a combination thereof, derived from the microorganism, and / or may be isolated or purified from the microorganism and provided in a purified form to the food composition. In one embodiment, the heme domain polypeptide is processed from a microorganism as a single-cell protein, protein hydrolysate, cell lysate, protein isolate, protein extract, peptide, or oligopeptide, or a combination thereof, derived from the microorganism, and / or isolated or purified from the microorganism and provided in a purified form to the food composition.

[0092] structured food Structured foods are provided herein. The structured foods include microbially derived protein products. The protein products are derived from microorganisms, such as bacterial or fungal microorganisms. Structured foods encompass food structures that are formed when food ingredients are mixed together and processed to produce a food, i.e., an edible product for human or animal consumption.

[0093] In some embodiments, the microorganism is a chemoautotrophically grown microorganism, such as a microorganism grown on a gaseous C1 carbon source such as CO2, CO, or CH4, or a microorganism grown in a medium containing a protein product derived from a chemoautotrophically grown microorganism. The chemoautotrophic microorganism can be any of the chemoautotrophic microorganisms described later herein. In certain non-limiting embodiments, the chemoautotrophic microorganism is a Cupriavidus microorganism, such as Cupriavidus necator or Cupriavidus metallidurans.

[0094] In some embodiments, the microorganism is a GRAS microorganism, such as any of the GRAS microorganisms described later herein, such as lactic acid bacteria (LAB). In certain embodiments, the GRAS microorganism is grown in a medium containing a protein product derived from a non-GRAS microorganism. The non-GRAS microorganism can be, for example, a chemoautotrophically grown microorganism, such as a Cupriavidus microorganism, for example, Cupriavidus necator or Cupriavidus metallidurans.

[0095] The microbially-derived protein product in the structured food may comprise one or more of single-cell protein, cell lysate, protein isolate, protein extract, protein hydrolysate, free amino acid, peptide, and oligopeptide. The protein product, such as any protein product described herein, may be present in the structured food composition in an amount of at least 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 35% (w / w), 40% (w / w), 45% (w / w), 50% (w / w), 55% (w / w), 60% (w / w), 65% (w / w), or 70% (w / w). In some embodiments, the microbially derived protein content of the structured food product is from about 5% (w / w) to about 50% (w / w), from about 5% (w / w) to about 10% (w / w), from about 10% (w / w) to about 15% (w / w), from about 15% (w / w) to about 20% (w / w), from about 20% (w / w) to about 25% (w / w), from about 25% (w / w) to about 30% (w / w), from about 30% (w / w) to about 35% (w / w), from about 35% (w / w) to about 40% (w / w), from about 40% (w / w) to about 45% (w / w), or from about 45% (w / w) to about 50% (w / w). / w), about 5% (w / w) to about 15% (w / w), about 10% (w / w) to about 20% (w / w), 15% (w / w) to about 25% (w / w), about 20% (w / w) to about 30% (w / w), about 25% (w / w) to about 35% (w / w), about 30% (w / w) to about 40% (w / w), about 35% (w / w) to about 40% (w / w), about 40 (w / w) to about 50% (w / (w / w)w), about 5% (w / w) to about 25% (w / w), about 20% (w / w) to about 40% (w / w), or about 25% to about 50% (w / w).Protein products include at least about 5% (w / w) to about 50% (w / w) single cell protein, at least about 5% (w / w) to about 50% (w / w) cell lysate, at least about 5% (w / w) to about 50% (w / w) protein extract, at least about 5% (w / w) to about 50% (w / w) protein isolate, at least about 5% (w / w) to about 50% (w / w) protein hydrolysate, at least about 5% (w / w) to about 50% (w / w) free amino acids. , at least about 5% (w / w) to about 50% (w / w) peptides, at least about 5% (w / w) to about 50% (w / w) oligopeptides, at least about 25% (w / w) to about 80% (w / w) single cell proteins, at least about 25% (w / w) to about 80% (w / w) cell lysates, at least about 25% (w / w) to about 80% (w / w) protein extracts, at least about 25% (w / w) to about 80% (w / w) protein isolates, at least about 25% (w / w) to about 80% (w / w) at least about 25% (w / w) to about 80% (w / w) protein hydrolysate, at least about 25% (w / w) to about 80% (w / w) free amino acids, at least about 25% (w / w) to about 80% (w / w) peptides, at least about 25% (w / w) to about 80% (w / w) oligopeptides, at least about 75% (w / w) to about 100% (w / w) single cell protein, at least about 75% (w / w) to about 100% (w / w) cell lysate, at least about 75% (w / w) to about 80% (w / w) It may comprise about 100% (w / w) protein extract, at least about 75% (w / w) to about 100% (w / w) protein isolate, at least about 75% (w / w) to about 100% (w / w) protein hydrolysate, at least about 75% (w / w) to about 100% (w / w) free amino acids, at least about 75% (w / w) to about 100% (w / w) peptides and / or at least about 75% (w / w) to about 100% (w / w) oligopeptides.

[0096] Structured foods as described herein can be analogs of natural foods and can include the rheological and structural (geometric and surface) attributes of foods, including attributes perceptible by mechanical, tactile and / or visual receptors. Food structure attributes include, for example: adhesiveness, springiness, brittleness, chewiness, chewiness, adhesion, coating, cohesion, creaminess, crispness, flaking, firmness, denseness, doughiness, dryness, springiness, oiliness, hardness, flaking, fleshy, fuzziness, foaminess, brittleness, richness, stickyness, graininess, coarseness, gummyness, hardness, heavyness, heterogeneity, juiciness, leanness, lightness, suppleness, graininess, moisture, mouth-coating, slushiness, oiliness, pastyness, plasticity, porosity, mealiness, expandability, pulpy, richness, roughness, rubberiness, flowability, sandiness, grittyness, etc.

[0097] In some embodiments, the structured food product is a structure that resembles the texture or appearance of a meat substitute product, such as a natural meat product derived from an animal.For example, the meat substitute product can be a substitute for beef, poultry (e.g., chicken, turkey, duck), pork, fish, or seafood.The meat substitute product can be in the form of a natural meat product, such as a burger, nuggets, etc., and can replicate the texture and / or organoleptic (i.e., one or more organoleptic) characteristics of the natural meat product.

[0098] The substitute meat product can mimic the structure of natural ground meat or muscle meat. Muscle meat is naturally structured by individual muscle fibers assembled into anisotropic fibers. The fibrous material process using microbial protein products as described herein can result in a three-dimensional imitation of natural meat structure. Structuring methods include, but are not limited to, self-organization, forced organization, or a combination, and induced self-organization.

[0099] In some embodiments, the protein fibers are substantially aligned. While the protein fiber network and / or protein fiber arrangement can impart cohesiveness and firmness, the open spaces in the protein fiber network and / or protein fiber arrangement can tenderize the meat structured protein product, provide pockets for trapping water, carbohydrates, salt, lipids, flavors, and other materials that are gradually released during mastication, facilitate the shearing process, and impart other meat-like sensory characteristics.

[0100] Structured foods, such as structured meat substitute products, can include one or more flavorings. In certain embodiments, structured foods, such as structured meat products, can include a flavohemoprotein flavoring. In one embodiment, the flavohemoprotein is produced by the microorganism from which the protein product is derived. In one embodiment, the flavohemoprotein and protein product are produced by a Cupriavidus microorganism, such as Cupriavidus necator or Cupriavidus metallidurans.

[0101] In some embodiments, the structured food, e.g., structured meat substitute product, comprises one or more additional vitamins, nutrients, or functional substances (i.e., can be added to the formulation (e.g., dough) for production of the structured food, or to the growth medium for the microorganism from which the protein product is derived, or can be produced by the microorganism from which the protein product is derived. Non-limiting examples of such additional substances include amino acids (e.g., essential amino acids), lipids, oils, fatty acids, vitamins (e.g., vitamin B 12 , biotin, and other essential vitamins), antioxidants, minerals, surfactants, and emulsifiers.

[0102] In some embodiments, the microbial derived protein products incorporated into the structured food compositions, eg, structured meat substitute compositions, are non-GMO.

[0103] In some embodiments, the structured food composition, e.g., structured meat substitute composition, does not include animal-derived materials or substances, such as animal-derived biomolecules or biochemicals. In some embodiments, e.g., in meat substitute or meat substitute products, hydrogels, lipogels, and / or emulsions are included in the composition, e.g., as drug release systems (e.g., for the release of colorants, flavorants, fatty acids, leavening agents, and / or gelling agents (e.g., bicarbonate (e.g., potassium bicarbonate), calcium hydroxide, and / or alginate (e.g., sodium or potassium alginate) where the drug(s) can be released during cooking of the food to mimic animal meat).

[0104] In some embodiments, the structured food product comprises one or more plant protein sources, such as, but not limited to, bean, rice, glutinous rice, wheat, gluten, soy, hemp, canola, insects, algae, and / or buckwheat, in combination with a protein product produced by a microorganism as described herein (e.g., one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof), wherein the protein product imparts a flavor, e.g., a meat-like flavor, to the food composition.

[0105] In some embodiments, the food product, e.g., a meat substitute or meat substitute product, includes a heme compound, such as a heme-containing polypeptide. In one embodiment, the food product includes heme (e.g., a heme-containing polypeptide) from the microorganism from which the protein product is derived. In some embodiments, the heme-containing polypeptide is in the form of a protein product (e.g., one or more of a single-cell protein, a protein hydrolysate, a cell lysate, a protein isolate, a protein extract, a peptide, an oligopeptide, and an isolated heme-containing protein) from a hydrogen-oxidizing microorganism, such as, for example, Cupriavidus or any other hydrogen-oxidizing microorganism described herein. In some embodiments, the hydrogen-oxidizing microorganism grows under O2-limited conditions, such as low O2 partial pressure conditions, e.g., microaerobic conditions.

[0106] In some embodiments, the structured meat substitute product (e.g., a beef, poultry, pork, fish, or seafood substitute product) includes a microbially produced protein product (e.g., one or more of single-cell protein, cell lysate, protein isolate, protein extract, protein hydrolysate, free amino acids, peptides, oligopeptides, or combinations thereof) as described herein. In some embodiments, the meat substitute product is a vegan product that does not contain any ingredients derived from animal sources. In some embodiments, enhanced meat products comprising animal protein (e.g., beef, poultry, pork, fish, seafood, or egg products, a portion of which is a microbially produced protein product feedstock (e.g., one or more of single-cell protein, cell lysate, protein isolate, protein extract, protein hydrolysate, free amino acids, peptides, oligopeptides, or combinations thereof) as described herein are provided. For example, the protein product can be included as a bulking agent in an enhanced meat or meat substitute product, e.g., the protein product replaces at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the meat or artificial or fake meat ingredient (e.g., a plant-based artificial or fake meat ingredient) to produce the enhanced meat product or meat substitute / fake product, respectively. In some embodiments, the microorganism is a CO2-grown or air-grown microorganism, e.g., an oxygen-hydrogen microorganism. Non-limiting examples of meat substitute products are provided in U.S. Patent Nos. 10,327,464, 10,314,325, 10,287,568, 10,273,492, 10,172,380, 10,172,381, 10,093,913, 10,087,434, 10,039,306, 9,943,096, 9,938,327, 9,833,768, 9,826,772, 9,808,029, 9,737,875, 9,700,067, and 9,011,949, which are incorporated by reference herein in their entireties.

[0107] In some embodiments, at least a portion or all of the protein products in the structured food products described herein, including but not limited to meat substitute products, comprise a protein product (e.g., one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) derived from Cupriavidus, such as, but not limited to, Cupriavidus necator, e.g., DSM 531 or DSM 541.

[0108] In some embodiments, at least some or all of the protein products in the food products described herein, including but not limited to meat substitute products, comprise protein products (e.g., one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) derived from lactic acid bacteria, such as, but not limited to, bacteria of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacteria are GRAS bacteria.

[0109] In some embodiments, at least some or all of the protein product in the food products described herein, including but not limited to meat substitute products, comprises a protein product (e.g., one or more of single-cell protein, cell lysate, protein isolate, protein extract, protein hydrolysate, free amino acids, peptides, oligopeptides, or combinations thereof) derived from a fungal microorganism of the genus Fusarium or Rhizopus, such as, but not limited to, Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae. In some embodiments, the fungal microorganism is a GRAS microorganism.

[0110] meat substitute products Provided are meat substitute products that resemble animal meat. The meat substitute products comprise protein products (e.g., one or more of single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) derived from one or more microorganisms as described herein, and mimic the texture and / or physical characteristics of animal meat, such as, for example, flavor, aroma, texture, and appearance.

[0111] In some embodiments, the meat substitute product comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% by weight of a microbial protein product as described herein, optionally bound together with one or more binders to produce a food product having one or more similar texture and / or functional characteristics compared to animal meat. In some embodiments, the meat substitute product resembles animal meat, such as ground animal meat (e.g., ground beef, ground pork, ground turkey). In some embodiments, the meat substitute product is primarily, substantially, or entirely composed of ingredients derived from non-animal sources. In alternative embodiments, the meat substitute product is composed partially of ingredients derived from animal sources, but with the addition of ingredients derived from non-animal sources. In some embodiments, the meat substitute product further comprises one or more drug release systems and / or other ingredients. In various embodiments, the meat substitute products herein may be sliced, cut, minced, shredded, grated, or otherwise processed, or may remain unprocessed. Examples of sliced ​​forms include, but are not limited to, dried meat, cured meat, and sliced ​​lunch meat. In some embodiments, the meat substitute foods provided herein are shredded and then combined together, chunked and formed, ground and formed, or shredded and formed, thus producing a product similar in appearance and texture to, for example, animal jerky.

[0112] In some embodiments, the meat substitute product is vegan. In some embodiments, the meat substitute product does not contain GMO ingredients. In some embodiments, the meat substitute product does not contain ingredients derived from nuts. In some embodiments, the meat substitute product contains less than about 0.6% or less than about 0.5% sodium by weight. In some embodiments, the meat-like food product is gluten-free. In some embodiments, the meat-like food product is soy-free.

[0113] In some embodiments, the meat substitute foods provided herein contain about 5% to about 30% fat by weight. In some embodiments, the meat substitute products contain about 0.5% to about 10% total carbohydrates by weight. In some embodiments, the meat substitute products contain about 0.5% to about 5% edible fiber by weight.

[0114] The meat substitute products provided herein have a moisture content (MC) of at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight. In some embodiments, the meat substitute products comprise a MC similar to that of animal meat.

[0115] In some embodiments, the meat substitute product contains one or more colorants. In some embodiments, the meat substitute product contains one or more color enhancers. In some embodiments, the meat-like food product contains a mixture of two or more colorants, color stabilizers, and / or color enhancers. Non-limiting examples of such mixtures include beet extract and annatto, beet extract and turmeric, beet extract and saffron, beet extract and purple carrot, beet extract and grapeseed extract, beet extract and tomato extract, beet extract and lycopene, beet extract and beta-carotene, beet extract and anthocyanins, beet extract and anthocyanins and annatto, beet extract and annatto and lycopene, beet extract and ascorbic acid, anthocyanins and annatto, beet extract and annatto and ascorbic acid, beet extract and annatto and beta-carotene, beet extract and turmeric and ascorbic acid, and anthocyanins, lycopene, and annatto. In some such embodiments, the colorants, color stabilizers, and / or color enhancers are present in equal weight ratios. In other such embodiments, the colorants, color stabilizers, and / or color enhancers are present in unequal weight ratios (e.g., 55:45, 60:40, 65:35, 2:1, 70:30, 75:25, 80:20, 5:1, 85:15, 90:10, 20:1, 95:5, 99:1). In some embodiments, the meat substitute product comprises a browning agent, such as, but not limited to, a pentose (e.g., ribose, arabinose, xylose), a hexose (e.g., glucose, fructose, mannose, galactose), a dextrin, and a commercially available browning agent (e.g., Red Arrow dextrose, wood-derived agents).

[0116] In some embodiments, the meat substitute products referred to herein comprise one or more plant protein sources, such as, but not limited to, bean, rice, glutinous rice, wheat, gluten, soy, hemp, canola, insects, algae, and / or buckwheat, in combination with a microbially produced protein product as described herein, wherein the protein product imparts a meat-like flavor to the composition.

[0117] In some embodiments, the meat substitute product herein comprises a heme compound, such as a heme-containing polypeptide. For example, the heme compound (e.g., heme-containing polypeptide) can be derived from the microorganism from which the protein product is derived. In some embodiments, the heme-containing polypeptide is in the form of a protein product (e.g., one or more of a single-cell protein, a protein hydrolysate, a cell lysate, a protein isolate, a protein extract, a peptide, an oligopeptide, and an isolated heme-containing protein) derived from a hydrogen-oxidizing microorganism, such as Cupriavidus, or any other hydrogen-oxidizing microorganism described herein. In some embodiments, the hydrogen-oxidizing microorganism grows under O2-limited conditions, such as low O2 partial pressure conditions, e.g., microaerobic conditions.

[0118] Dough composition As described herein, a dough composition for producing a structured food product, such as a structured meat substitute product, is provided. The composition of the dough mixture includes one or more protein products (e.g., single-cell protein, cell lysate, protein isolate, protein extract, protein hydrolysate, free amino acid, peptide, and / or oligopeptide) derived from one or more microorganisms as described herein, and may include, for example, binders, salts, texturing agents, etc. The mixture may also be supplemented with one or more flavoring materials and / or flavor enhancers, such as, for example, flavohemoglobulin, one or more vitamins, and / or one or more nutritional additives, as described herein above. Non-limiting examples of dough compositions are described, for example, in U.S. Patent No. 9,526,267, the entire contents of which are incorporated herein by reference.

[0119] In some embodiments, the dough contains one or more heme-containing polypeptides derived from a microorganism. For example, the included polypeptides can be in the form of a protein product (e.g., one or more of single-cell protein, protein hydrolysate, cell lysate, protein isolate, protein extract, peptide, oligopeptide, and isolated heme-containing protein) derived from a hydrogen-oxidizing microorganism such as Cupriavidus, or any other hydrogen-oxidizing microorganism described herein. In some embodiments, the hydrogen-oxidizing microorganism grows under O2-limited conditions, such as low O2 partial pressure conditions, e.g., microaerobic conditions.

[0120] In some embodiments, the dough composition comprises a protein hydrolysate, wherein the average molecular weight of the proteins in the hydrolysate is from about 5 kD to about 10 kD, or any of about 5 kD, 6 kD, 7 kD, 8 kD, 9 kD, or 10 kD, or any of about 5 kD to about 7 kD, about 6 kD to about 8 kD, about 7 kD to about 9 kD, 8 kD to about 10 kD, about 5 kD to about 8 kD, or about 7 kD to about 10 kD.

[0121] In some embodiments, the protein products in the dough composition are produced under conditions that preserve globular, native proteins. For example, such conditions can include gentle cell lysis followed by separation of soluble components (e.g., proteins) from insoluble components (e.g., cell debris).

[0122] In some embodiments, the dough comprises, in addition to the microbial protein products as described herein, proteins from one or more additional sources, non-limiting examples of which include plant sources (e.g., wheat gluten, soy, peas, wheat), algae, other non-animal proteins, or milk.

[0123] In some embodiments, the dough may be about 40% (w / w), about 45% (w / w), about 50% (w / w), about 55% (w / w), about 60% (w / w), about 65% (w / w), about 70% (w / w), about 75% (w / w), about 80% (w / w), about 40% (w / w) to about 80% (w / w), about 40% (w / w) to about 50% (w / w), about 45% (w / w) to about 55% (w / w), about 50 ...50% (w / w), about 45% (w / w) to about 55% (w / w), about 50% (w / w), about 60% (w / w), about 60% (w / w), about 65% (w / w), about 70% (w / w), about 75% (w / w), about 80% (w / w), about 40% (w / w) to about 50% (w / w), about 45% (w / w) to about 55% (w / w), about 50% (w / w), about 6 The moisture content of the composition may be from about 40% (w / w) to about 60% (w / w), from about 55% (w / w) to about 65% (w / w), from about 60% (w / w) to about 70% (w / w), from about 65% (w / w) to about 75% (w / w), from about 70% (w / w) to about 80% (w / w), from about 40% (w / w) to about 60% (w / w), from about 50% (w / w) to about 70% (w / w), or from about 60% (w / w) to about 80% (w / w).

[0124] In some embodiments, the shear strength of the dough composition is greater than about 1000 psig.

[0125] In some embodiments, the dough is an emulsion, for example, an emulsion of one or more proteins and one or more oils as described herein.

[0126] Methods for Producing Structured Food Compositions This specification describes a method for producing a structured food composition, such as a structured meat substitute composition. Protein products (e.g., single-cell proteins, cell lysates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, and / or oligopeptides) derived from microorganisms are processed into structured food compositions. One non-limiting embodiment of the workflow for producing structured food is shown in Figure 1.

[0127] The physical processes of shearing, mixing, and / or spinning can be used to deform and create fiber arrays from protein products, such as protein hydrolysate products. Fiber creation requires phase separation of the material, followed by alignment of the internal structure and subsequent solidification. The type of fiber array and properties of the resulting structured material depend on the composition and rheological properties of the pre-processed dough material, its water-holding capacity, and other factors that impart structure and food functionality, including water absorbency, water solubility, oil absorption index, swelling index, bulk density, and viscosity.

[0128] As described hereinabove, the dough composition can be converted into a structured food composition. Thermochemical processes, such as extrusion, can be used to structure proteins. For example, extrusion can involve: (i) pre-treating the material outside the extruder; (ii) mixing / cooking inside the extruder barrel; and (iii) cooling in the die. Physical factors that affect the formation of structure by extrusion include, for example, temperature, screw speed, and extruder design. Factors that impart structure and food functionality include the water absorption, water solubility, oil absorption index, expansion index, bulk density, and viscosity of the dough. In some embodiments, extrusion of the dough composition as described herein produces aligned fibers, i.e., a protein fiber network and / or aligned protein fibers, which produce structured foods, such as products with a meat-like texture. For example, the dough composition is processed in a temperature-controlled extruder designed to apply mechanical shear to the isotropically mixed dough composition in a cone-in-cone shape to form a macroscopic anisotropic mixture with a substantially aligned fiber structure. Non-limiting examples of this are described, for example, in US Pat. No. 9,526,267, which is incorporated herein by reference in its entirety.

[0129] High moisture extrusion can result in protein denaturation and cross-linking, reducing the solubility of the extruded material. In some embodiments, the extrusion conditions can cause previously denatured protein molecules to break down into subunits. The subunits can then reassemble into a product that exhibits the characteristic texture and microstructure of assembled proteins. This reassembly can occur primarily through intermolecular peptide bonds, although hydrophobic interactions plus hydrogen and disulfide bonds can also play a role, to a lesser extent.

[0130] In some embodiments, a spinning process is used to process microbial protein products into structured food compositions. For example, a solution containing a protein product can be extruded through a spinneret and then immersed in a bath containing a non-solvent for the protein product. The exchange of the solvent and non-solvent can result in the precipitation and solidification of the extruded protein phase, forming elongated filaments (e.g., filaments having a thickness of about 20 μm). (Dekkers, et al. (2018) Trends Food Sci Technol 81:25-36)

[0131] In some embodiments, converting a protein product into a structured food product involves forming a structured hydrocolloid. The structured hydrocolloid can be formed from a mixture of a microbially derived protein product, such as a protein hydrolysate, as described herein, and a hydrocolloid, which is precipitated with metal cations, for example, at elevated temperatures, to form a fibrous product. Non-limiting examples of hydrocolloids include pectin, for example, in the form of an alginate or gum.

[0132] Solidifying the structured food compositions produced by the methods as described herein can involve heating, cooling, drying, and / or solidifying.

[0133] Protein hydrolysate In some embodiments, at least some or all of the protein product is produced by hydrolyzing proteins (e.g., single-cell proteins, cell lysates, protein isolates, and / or protein extracts) from at least one microorganism described herein. For example, hydrolysis of cellular proteins can produce peptides, oligopeptides, and / or free amino acids.

[0134] The hydrolysis of microbial protein can be carried out by acid, base and / or enzymatic treatment.Methods for hydrolyzing protein are well known in the art.Non-limiting examples of microbial protein hydrolysis methods and hydrolyzed compositions can be found in PCT Application No. US20 / 50902, filed on September 15, 2020, which is incorporated herein by reference in its entirety.

[0135] In some embodiments, the hydrolysis method can include increasing or decreasing the pH of a proteinaceous suspension, e.g., a suspension of microbial biomass, thereby producing an alkaline or acidic suspension, respectively. The starting biomass suspension can include a suitable amount of biomass in a liquid, e.g., a microbial biomass in a growth medium. In some embodiments, the amount of biomass, dry weight / reaction volume, is at least about 0.01%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 2%, or at least about 3%, or about 0.1% to about 8%, e.g., about 3% to about 5%, including about 0.2% to about 8%, about 0.5% to about 6%, about 1% to about 6%, or about 2% to about 6%.

[0136] In some embodiments, cells within the biomass are subjected to lysis at the beginning of the process, eg, before increasing or decreasing the pH, to facilitate recovery of proteins from the biomass into a suspension composition.

[0137] In certain embodiments, the alkaline or acidic suspension can be subjected to heating for an appropriate period of time to produce a protein hydrolysate composition. The suspension can be concentrated, dried (e.g., lyophilized), or utilized directly as a liquid suspension. In certain embodiments, the alkaline or acidic suspension is subjected to heat and high pressure to produce a protein hydrolysate composition, for example, by autoclaving the alkaline or acidic suspension. In some embodiments, the suspension is neutralized with a buffer to lower or raise the pH after the heating or heat / pressure treatment. In certain embodiments, the pH is lowered (in the case of an alkaline suspension) or raised (in the case of an acidic suspension) sufficiently to allow subsequent enzymatic treatment of the suspension with a hydrolytic enzyme, such as a protease (e.g., an alkaline protease, an acid protease, or a metalloprotease). After enzymatic hydrolysis, a protein hydrolysate composition is produced. In other embodiments, the biomass suspension is hydrolyzed with a proteolytic enzyme, such as a protease (e.g., an alkaline protease, an acid protease, or a metalloprotease), without prior alkaline or acid treatment.

[0138] In certain embodiments, the hydrolyzed protein in the protein hydrolysate is mainly in the soluble fraction of the suspension.The resulting suspension can be clarified, for example, by centrifugation, to obtain a supernatant fraction containing hydrolyzed protein.In some embodiments, the hydrolysis treatment (for example, alkaline or acid hydrolysis, optionally including enzymatic (for example, protease) treatment, or enzymatic hydrolysis alone) is followed by clarification of the suspension (hydrolysate) to remove undissolved substances in the suspension, for example, to separate the soluble and insoluble fractions.The suspension can be clarified using any suitable method, such as centrifugation, filtration, etc.In some embodiments, after the suspension is clarified, for example, by centrifugation, the supernatant can be separated from the pellet.

[0139] In some embodiments, a clarified liquid composition comprising hydrolyzed protein (e.g., a soluble fraction such as the supernatant of a separated suspension) is dried, e.g., lyophilized, to produce a dry or substantially dry composition. In some embodiments, the lyophilized composition has a moisture content of about 10% or less, e.g., about 3% or less, about 8% or less, about 6% or less, or about 5% or less. In some embodiments, the lyophilized protein hydrolysate composition has a moisture content of about 1% to about 10%, e.g., about 1% to about 8%, or about 1% to about 6%, including about 2% to about 5%.

[0140] In some embodiments, the clarified liquid composition (e.g., a soluble fraction such as the supernatant of a separated suspension) is dehydrated or concentrated to reduce its water content. In some embodiments, the concentrated composition has a water content of about 80% or less, e.g., about 75% or less, about 50% or less, about 40% or less, including about 30% or less; and in some embodiments, each of the above water content ranges can be at least about 20%, at least about 25%, at least about 30%, at least about 40%, or at least about 50% (to the extent that such a range exceeds such lower limit). In some embodiments, the dehydrated product is dried using heating and / or evaporation, for example, using one or more methods such as, but not limited to, spray drying; drum drying; oven drying; vacuum drying; vacuum oven drying; drying under an inert gas such as N2; and solar evaporation. In some embodiments, the clarified product is first dehydrated using a rotary evaporator, for example, to remove about 50% to about 65% of the water. In some embodiments, further dehydration is achieved, for example, by lyophilization, such that the lyophilized protein hydrolysate composition has a moisture content of about 1% to about 10%, e.g., about 1% to about 8%, about 1% to about 6%, including about 2% to about 5%.

[0141] In some embodiments, at least some or all of the protein from which the protein hydrolysate is produced (e.g., single-cell protein, cell lysate, protein isolate, and / or protein extract) is derived from a Cupriavidus microorganism, such as, but not limited to, Cupriavidus necator, e.g., DSM 531 or DSM 541. In some embodiments, the protein hydrolysate composition (e.g., comprising peptides, oligopeptides, and / or free amino acids) is derived from protein from a Cupriavidus microorganism, such as, but not limited to, Cupriavidus necator, e.g., DSM 531 or DSM 541.

[0142] In some embodiments, at least some or all of the proteins from which the protein hydrolysate is produced (e.g., single-cell proteins, cell lysates, protein isolates, and / or protein extracts) are derived from lactic acid bacteria, such as, but not limited to, bacteria of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the protein hydrolysate composition (e.g., comprising peptides, oligopeptides, and / or free amino acids) is derived from proteins from lactic acid bacteria, such as, but not limited to, bacteria of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacteria are GRAS bacteria.

[0143] In some embodiments, at least some or all of the protein (e.g., single-cell protein, cell lysate, protein isolate, and / or protein extract) from which the protein hydrolysate is produced is derived from a fungal microorganism of the genus Fusarium or Rhizopus, such as, but not limited to, Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae. In some embodiments, the protein hydrolysate composition (e.g., comprising peptides, oligopeptides, and / or free amino acids) is derived from proteins from a fungal microorganism of the genus Fusarium or Rhizopus, such as, but not limited to, Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae.

[0144] In some embodiments, the protein hydrolysate herein comprises peptides that comprise or consist of peptides in a size range that is typically non-allergenic, for example, non-allergenic to humans.In some embodiments, the protein hydrolysate incorporated into the food composition as described herein comprises peptides and free amino acids, and the peptides are typically in a size range that is non-allergenic.In some embodiments, the non-allergenic peptides are in a size range with an average molecular weight distribution of about 800 to about 1500 Da.For example, the peptides obtained by protein hydrolysis as described herein may have an average molecular weight less than about 1500, 1400, 1300, 1200, 1100, 1000, 900, or 800 Da.

[0145] In some embodiments, salts (e.g., acid or alkali salts used for hydrolysis) are removed from the protein hydrolysate before incorporating the hydrolysate into a food composition as described herein. For example, the protein hydrolysate can be purified by filtration (e.g., ultrafiltration) or dialysis to remove salts and / or other impurities.

[0146] microorganisms The proteinaceous material (protein product) used in the methods and incorporated into the compositions described herein is derived from one or more microorganisms. The microorganisms from which the single-cell proteins, cell lysates, protein isolates, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof are derived can be photoautotrophic, heterotrophic, methanotrophic, methylotrophic, carboxydotrophic, or chemoautotrophic organisms. In some embodiments, the microorganisms include oxyhydrogen microorganisms. The microorganisms can be wild-type, genetically modified (e.g., recombinant), or a combination thereof.

[0147] Microbial biomass can be harvested from the culture of one or more suitable microorganisms, for example, in a fermenter or bioreactor. The biomass can be harvested using any suitable method, such as centrifugation, to separate the cell mass from the culture medium. In some embodiments, the harvested biomass can be used to produce a protein hydrolysate composition. In some embodiments, the harvested biomass can be spray-dried or freeze-dried to produce a dry biomass, which can then be used as a feedstock for the production of food compositions as described herein or to produce a protein hydrolysate composition. In some embodiments, a protein product (e.g., single-cell protein, cell lysate, protein extract, protein-containing extract, protein isolate, protein hydrolysate, free amino acids, peptides, oligopeptides, or a combination thereof) is produced from the harvested biomass.

[0148] In some embodiments, the microorganism or its protein product includes a strain within the genus Cupriavidus, or Ralstonia, or Hydrogenobacter. In some embodiments, the microorganism includes the species Cupriavidus necator or Cupriavidus metallidurans. In some embodiments, the microorganism includes a strain of the species Cupriavidus necator DSM531 or DSM541. In some embodiments, the microorganism includes the species Cupriavidus metallidurans. In some embodiments, the microorganism includes a strain of the species Cupriavidus metallidurans DSM2839.

[0149] In some embodiments, the microorganism or protein product thereof comprises a strain within the genus Xanthobacter. In some embodiments, the microorganism comprises the species Xanthobacter autotrophicus. In some embodiments, the microorganism comprises a strain of the species Xanthobacter autotrophicus DSM432.

[0150] In some embodiments, the microorganism or protein product thereof includes a microorganism of the genus Rhodococcus or Gordonia. In some embodiments, the microorganism includes Rhodococcus opacus. In some embodiments, the microorganism includes Rhodococcus opacus (DSM 43205) or a Rhodococcus species (DSM 3346). In some embodiments, the microorganism includes Rhodococcus opacus, Hydrogenovibrio marinus, Rhodopseudomonas capsulata, Hydrogenobacter thermophilus, or Rhodobacter sphaeroides. In some embodiments, the microorganism includes a strain in the family Burkholderiaceae.

[0151] In some embodiments, the microorganism or protein product thereof includes a lactic acid bacterium, such as, but not limited to, a bacterium of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacterium is a GRAS bacterium.

[0152] In some embodiments, the microorganism or protein product thereof includes a fungal microorganism of the genus Fusarium or Rhizopus, such as, but not limited to, Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae. In some embodiments, the fungal microorganism is a GRAS microorganism.

[0153] In some embodiments, a consortium of microorganisms (i.e., two or more microorganisms grown together) is used as a protein product source in the methods and compositions described herein. The consortium can include one or more of any of the microbial species or strains described herein, or one or more microorganisms having one or more microbial traits described herein. In some embodiments, the consortium includes two or more of any of the microbial species or strains or microorganisms described herein, or two or more microorganisms having one or more microbial traits described herein.

[0154] In some embodiments, a microorganism as described herein is capable of accumulating protein to about 50% or more of the total cell mass by weight. In some embodiments, a microorganism as described herein is capable of accumulating protein to about 60% or more of the total cell mass by weight. In some embodiments, a microorganism is capable of accumulating protein to about 70% or more of the total cell mass by weight. In some embodiments, a microorganism is capable of accumulating protein to about 80% or more of the total cell mass by weight. In some non-limiting embodiments, a microorganism exhibiting these traits is Cupriavidus necator, such as Cupriavidus necator DSM531 or DSM541.

[0155] In some embodiments, a microorganism as described herein can grow naturally on H2 / CO2 and / or syngas and / or producer gas. In some embodiments, the microorganism can naturally accumulate polyhydroxyalkanoic acid (PHA) (e.g., polyhydroxybutyric acid (PHB)) to about 50% or more of its cellular biomass by weight. In some embodiments, the microorganism has the inherent ability to direct a high flux of carbon through acetyl-CoA metabolic intermediates and can direct it to numerous other synthetic pathways, e.g., PHA synthesis, such as PHB, and / or fatty acid biosynthesis along with amino acid biosynthesis. In some embodiments, a microorganism exhibiting these traits is Cupriavidus necator, e.g., Cupriavidus necator DSM531 or DSM541. In some embodiments, the microorganism does not produce and / or accumulate PHA (e.g., PHB).

[0156] In some non-limiting embodiments, the microorganism or protein product thereof includes Corynebacterium autotrophicum. In some non-limiting embodiments, the microorganism includes Corynebacterium autotrophicum and / or Corynebacterium glutamicum. In some embodiments, the microorganism includes Hydrogenovibrio marinus. In some embodiments, the microorganism includes Rhodopseudomonas capsulata, Rhodopseudomonas palustris, or Rhodobacter sphaeroides.

[0157] In some embodiments, the microorganism or protein product thereof includes one or more of the following genera: Cupriavidus, Rhodococcus, Hydrogenovibrio, Rhodopseudomonas, Hydrogenobacter, Gordonia, Arthrobacter, Streptomycetes, Rhodobacter, and / or Xanthobacter.

[0158] In some embodiments, the microorganism or its protein product comprises a microorganism of the class Actinobacteria. In some embodiments, the microorganism comprises a microorganism of the suborder Corynebacterineae (family Corynebacterium, Gordoniaceae, Mycobacteriaceae, and Nocardiaceae). In some embodiments, the microorganism comprises a microorganism of the family Nocardiaceae. In some embodiments, the microorganism comprises a microorganism extracted from one or more of the following genera: Corynebacterium, Gordonia, Rhodococcus, Mycobacterium, and Tsukamurella. In some embodiments, the microorganism includes Rhodococcus opacus, Rhodococcus aurantiacus; Rhodococcus baikonurensis; Rhodococcus boritolerans; Rhodococcus equi; Rhodococcus coprophilus; Rhodococcus corynebacterioides; Nocardia corynebacterioides (synonym: Nocardia corynebacterioides); Rhodococcus erythropolis; erythropolis); Rhodococcus fascians; Rhodococcus globerulus; Rhodococcus gordoniae;Rhodococcus jostii; Rhodococcus koreensis; Rhodococcus kroppenstedtii; Rhodococcus maanshanensis; Rhodococcus marinonascens; Rhodococcus opacus; Rhodococcus percolatus; Rhodococcus phenolicus; Rhodococcus polyvorum; Rhodococcus pyridinivorans; Rhodococcus rhodochrocus rhodochrous; Rhodococcus rhodnii; (synonym: Nocardia rhodnii); Rhodococcus ruber (synonym: Streptothrix rubra); Rhodococcus species RHA1; Rhodococcus triatomae; Rhodococcus tukisamuensis; Rhodococcus wratislaviensis (synonym: Tsukamurella wratislaviensis); Rhodococcus yunnanensis;or Rhodococcus zopfii. In some embodiments, the microorganism includes Rhodococcus opacus strain DSM 43205 or DSM 43206. In some embodiments, the microorganism includes Rhodococcus species strain DSM 3346.

[0159] In some embodiments, the microorganism or protein product thereof includes a microorganism (e.g., a microorganism of any microbial genus or species described herein) that can naturally grow on H2 / CO2 and / or syngas and / or producer gas and naturally accumulate lipids to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more of its cellular biomass by weight. In some embodiments, the microorganism includes a microorganism (e.g., a microorganism of any microbial genus or species described herein) that has the natural ability to direct a high flux of carbon into the fatty acid biosynthetic pathway. In some embodiments, the microorganism exhibiting these traits is Rhodococcus opacus (e.g., Rhodococcus opacus DSM 43205 or DSM 43206 or DSM 44193) or Cupriavidus necator (e.g., Cupriavidus necator DSM 531 or DSM 541).

[0160] In some embodiments, the microorganism or protein product thereof includes an oxyhydrogen or hydrogen oxidizing strain. In some embodiments, the microorganism includes the following hydrogen-oxidizing microorganisms: Aquifex pyrophilus, Aquifex aeolicus, or other Aquifex species; Cupriavidus necator or Cupriavidus metallidurans or other Cupriavidus species; Corynebacterium autotrophicum or other Corynebacterium species; Gordonia desulfuricans, Gordonia polyisoprenivorans, Gordonia rubripertincta, Gordonia hydrophobica, or other hydrogen-oxidizing microorganisms. hydrophobica, Gordonia westfalica, or other Gordonia species; Nocardia autotrophica, Nocardia opaca, or other Nocardia species;Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophila purple non-sulfur photosynthetic bacteria, including, but not limited to, Rhodobacter species, Rhodospirillum rubrum, or other Rhodospirillum species; Rhodococcus opacus or other Rhodococcus species; Rhizobium japonicum or other Rhizobium species; Thiocapsa roseopersicina or other Thiocapsa species;Pseudomonas facilis, Pseudomonas flava, Pseudomonas putida, Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, Pseudomonas palleronii, Pseudomonas pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophile, or other Pseudomonas species; Hydrogenomonas pantotropha, Hydrogenomonas eutropha eutropha, Hydrogenomonas facilis, or other Hydrogenomonas species; Hydrogenobacter thermophiles, Hydrogenobacter halophilus, Hydrogenobacter hydrogenophilus, or other Hydrogenobacter species; Hydrogenophilus islandicus or other Hydrogenophilus species; Hydrogenovibrio marinus or other Hydrogenovibrio species; Hydrogenothermus marinus marinus) or other Hydrogenothermus species;Helicobacter pylori or other Helicobacter species; Xanthobacter autotrophicus, Xanthobacter flavus, or other Xanthobacter species; Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava, or other Hydrogenophaga species; Bradyrhizobium japonicum or other Bradyrhizobium species; Ralstonia eutropha eutropha or other Ralstonia species; Alcaligenes eutrophus, Alcaligenes facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Alcaligenes paradoxus, Alcaligenes ruhlandii, or other Alcaligenes species; Amycolata species; Aquaspirillum autotrophicum or other Aquaspirillum species; Arthrobacter strain 11 / X, Arthrobacter methylotrophus methylotrophus, or other Arthrobacter species; Azospirillum lipoferum, or other Azospirillum species;Variovorax paradoxus or other Variovorax species; Acidovorax facilis or other Acidovorax species; Bacillus schlegelii, Bacillus tusciae, or other Bacillus species; Calderobacterium hydrogenophilum or other Calderobacterium species; Derxia gummosa or other Derxia species; Flavobacterium autothermophilum autothermophilum or other Flavobacterium species; Microcyclus aquaticus or other Microcyclus species; Mycobacterium gordoniae or other Mycobacterium species; Paracoccus denitrificans or other Paracoccus species; Persephonella marina, Persephonella guaymasensis, or other Persephonella species; Renobacter vacuolatum or other Renobacter species;Seliberia carboxydohydrogena or other Seliberia species, Streptomycetes coelicoflavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetes thermocarboxydus, and other Streptomycetes species; Thermocrinis ruber or other Thermocrinis species; Wautersia species; Anabaena oscillarioides, Anabaena spiroides, Anabaena cylindrica cylindrica or other Anabaena species, and cyanobacteria, including but not limited to Arthrospira platensis, Arthrospira maxima, or other Arthrospira species; Scenedesmus obliquus or other Scenedesmus species, Chlamydomonas reinhardii or other Chlamydomonas species, Ankistrodesmus species, and green algae, including but not limited to Rhaphidium polymorphium or other Rhaphidium species. In some embodiments, a microbial consortium comprising an oxyhydrogen microorganism, such as any of the above, is used to produce a protein product as described herein.

[0161] In some embodiments, the microorganism or protein product thereof includes one or more of the following genera: Cupriavidus; Xanthobacter; Dietzia; Gordonia; Mycobacterium; Nocardia; Pseudonocardia; Arthrobacter; Alcanivorax; Rhodococcus; Streptomyces; Rhodopseudomonas; Rhodobacter; and Acinetobacter; or a consortium of microorganisms comprising one or more of these genera of microorganisms.

[0162] In some embodiments, the microorganism or protein product thereof includes any of the following: Arthrobacter methylotrophus DSM 14008; Rhodococcus opacus DSM 44304; Rhodococcus opacus DSM 44311; Xanthobacter autotrophicus DSM 431; Rhodococcus opacus DSM 44236; Rhodococcus ruber DSM 43338; ​​Rhodococcus opacus DSM 44315; Cupriavidus metallidurans metallidurans DSM 2839; Cupriavidus necator DSM 531; Cupriavidus necator DSM 541; Rhodococcus aetherivorans DSM 44752; Gordonia desulfuricans DSM 44462; Gordonia polyisoprenivorans DSM 44266; Gordonia polyisoprenivorans DSM 44439; Gordonia rubripertincta DSM 46039; Rhodococcus percolatus percolatus DSM44240; Rhodococcus opacus DSM43206; Gordonia hydrophobica DSM44015; Rhodococcus zopfii DSM44189;Gordonia westfalica DSM44215, Xanthobacter autotrophicus DSM1618; Xanthobacter autotrophicus DSM2267; Xanthobacter autotrophicus DSM3874; Streptomyces coelicoflavus DSM41471; Streptomyces griseus DSM40236; Streptomyces species DSM40434; Streptomyces xanthochromogenes In some embodiments, the microorganism or protein product thereof includes one or more of the following: Streptomyces xanthochromogenes DSM 40111; Streptomyces thermocarboxydus DSM 44293; Rhodobacter sphaeroides DSM 158. In some embodiments, the microorganism or protein product thereof includes one or more of these microbial strains, or a consortium of microorganisms comprising one or more of any of the microbial genera or species disclosed herein.

[0163] Many different microorganisms capable of growth using carbon monoxide as an electron donor and / or carbon source (i.e., carboxydotrophs) have been characterized. In some cases, carboxydotrophs can also use H2 as an electron donor and / or grow mixotrophically. In some cases, carboxydotrophs are facultative chemolithoautotrophs (Biology of the Prokaryotes, edited by J. Lengeler, G. Drews, H. Schlegel, John Wiley & Sons, July 10, 2009, incorporated herein by reference in its entirety).In some embodiments, the microorganism or protein product thereof includes the following carboxydotrophic microorganisms: Acinetobacter spp.; Alcaligenes carboxydus or other Alcaligenes spp.; Arthrobacter spp.; Azomonas spp.; Azotobacter spp.; Bacillus schlegelii or other Bacillus spp.; Hydrogenophaga pseudoflava or other Hydrogenophaga spp.; Pseudomonas carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas complansoris, compransoris, Pseudomonas gazotropha, Pseudomonas thermocarboxydovorans, or other Pseudomonas species; Rhizobium japonicum or other Rhizobium species; and Streptomyces G26, Streptomyces thermoautotrophicus, or other Streptomyces species. In some embodiments, the microorganism or protein product thereof comprises a microbial consortium comprising a carboxydotrophic microorganism, such as one or more of the carboxydotrophic microorganisms described above. In certain embodiments, a carboxydotrophic microorganism capable of chemoautotrophy is used. In certain embodiments, a carboxydotrophic microorganism capable of utilizing H as an electron donor in respiration and / or biosynthesis is used.

[0164] In some embodiments, the microorganism or protein product thereof includes any of the following: Acetoanaerobium spp.; Acetobacterium spp.; Acetogenium spp.; Achromobacter spp.; Acidianus spp.; Acinetobacter spp.; Actinomadura spp.; Aeromonas spp.; Alcaligenes spp. s species; Alcaliqenes species; Aquaspirillum species; Arcobacter species; Aureobacterium species; Bacillus species; Beggiatoa species; Butyribacterium species; Carboxydothermus species; Clostridium species; Comamonas species; Cupriavidus riavidus species; Dehalobacter species; Dehalococcoide species; Dehalospirillum species; Desulfobacterium species; Desulfomonile species; Desulfotomaculum species; Desulfovibrio species; Desulfurosarcina species; Ectothiorhodospira orhodospira species; Enterobacter species; Eubacterium species; Ferroplasma species; Halothibacillus species; Hydrogenobacter species; Hydrogenomonas species; Leptospirillum species; Metallosphaera species; Methanobacterium species;Methanobrevibacter spp.; Methanococcus spp.; Methanococcoides spp.; Methanogenium spp.; Methanolobus spp.; Methanomicrobium spp.; Methanoplanus spp.; Methanosarcina spp.; Methanospirillum spp.; Methanothermus spp. nothermus species; Methanothrix species; Micrococcus species; Nitrobacter species; Nitrobacteraceae species, Nitrococcus species, Nitrosococcus species; Nitrospina species, Nitrospira species, Nitrosolobus species; Nitrosomonas species; Nitrosospira Nitrosospira species; Nitrosovibrio species; Nitrospina species; Oleomonas species; Paracoccus species; Peptostreptococcus species; Planctomycetes species; Pseudomonas species; Ralstonia species; Rhodobacter species; Rhodococcus species; Rhodococcus species; Rhodocyclus species Rhodocyclus species; Rhodomicrobium species; Rhodopseudomonas species; Rhodospirillum species; Shewanella species; Siderococcus species; Streptomyces species; Sulfobacillus species; Sulfolobus species; Thermothrix species, Thiobacillus species;The microorganisms include obligate and / or facultative chemoautotrophic microorganisms such as Thiomicrospira species; Thioploca species; Thiosphaera species; Thiothrix species; Thiovulum species; sulfur-oxidizing bacteria; hydrogen-oxidizing bacteria; iron-oxidizing bacteria; acetogens; and methanogens; a microbial consortium including chemoautotrophs; chemoautotrophs indigenous to at least one of hydrothermal vents, geothermal vents, hot springs, cold springs, underground aquifers, salt lakes, saline formations, and soil; and one or more extremophiles selected from one or more of thermophiles, hyperthermophiles, acidophiles, halophiles, and psychrophiles. In some embodiments, the microorganism, or its protein product, includes a microbial consortium including chemoautotrophic microorganisms such as one or more of the above chemoautotrophic microorganisms.

[0165] In some embodiments, the microorganism or protein product thereof includes an extremophile microorganism that can withstand extremes in various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and / or chemicals. Such microorganisms include hyperthermophiles such as Pyrolobus fumarii; thermophiles such as Synechococcus lividis; mesophiles and psychrophiles such as Psychrobacter and / or extremely thermophilic sulfur-metabolizing bacteria such as Thermoproteus species, Pyrodictium species, Sulfolobus species, and Acidianus species; radioresistant organisms such as Deinococcus radiodurans; pressure-tolerant microorganisms, including piezophiles or barophiles; desiccation-tolerant and anhydrobiotic microorganisms, including xerozoans such as Artemia salina; microorganisms and fungi; Halobacteriacea and Dunaliella salina. salina; pH-tolerant microorganisms, including alkaliphiles such as Natronobacterium, Bacillus firmus OF4, and Spirulina species, and acidophiles such as Cyanidium caldarium and Ferroplasma species; gas-tolerant microorganisms, including Cyanidium caldarium, that can tolerate, for example, pure CO2; and metal-tolerant microorganisms (metal-resistant bacteria), such as Ferroplasma acidarmanus and Ralstonia species.

[0166] In certain embodiments, the microorganism, or protein product thereof, includes a cell line selected from eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophiles, yeast, fungi, proteobacteria, recombinant organisms thereof, and synthetic organisms. In certain embodiments, the genus Spirulina is utilized.

[0167] In certain embodiments, the microorganism or protein product thereof includes green non-sulfur bacteria, including, but not limited to, the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.

[0168] In certain embodiments, the microorganism or protein product thereof includes green sulfur bacteria, including but not limited to the following genera: Chlorobium, Clathrochloris, and Prosthecochloris.

[0169] In certain embodiments, the microorganism or protein product thereof includes purple sulfur bacteria, including, but not limited to, the following genera: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis.

[0170] In certain embodiments, the microorganism or protein product thereof comprises purple non-sulfur bacteria, including, but not limited to, the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rhodovibrio, and Roseospira.

[0171] In some embodiments, the microorganism or its protein product includes a methanotroph and / or a methylotroph. In some embodiments, the microorganism is of the genus Methylococcus. In some embodiments, the microorganism is Methylococcus capsulatus. In some embodiments, the microorganism is a methylotroph. In some embodiments, the microorganism is of the genus Methylobacterium. In some embodiments, the microorganism includes one or more of the following species: Methylobacterium zatmanii; Methylobacterium extorquens; Methylobacterium chloromethanicum.

[0172] In some embodiments, the microorganism or protein product thereof includes a hydrogen-oxidizing chemoautotrophic microorganism and / or a carboxydotrophic bacterium and / or a methylotrophic bacterium and / or a methanotroph.

[0173] In certain embodiments, the microorganism or protein product thereof includes a microorganism capable of heterotrophic growth, utilizing multi-carbon organic molecules as a carbon source, such as, but not limited to, sugars, including, but not limited to, glucose and / or fructose. In some embodiments, the microorganism can grow on unprocessed crude glycerol and / or glucose and / or methanol and / or acetate as the sole electron donor(s) and carbon source(s). In some embodiments, the microorganism can grow mixotrophically, e.g., on an organic carbon source and an inorganic energy source (e.g., an inorganic electron donor).

[0174] In certain embodiments, the microorganism or protein product thereof includes one or more of eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophiles, archaea, yeast, fungi, proteobacteria, recombinant organisms thereof, and synthetic organisms.

[0175] In some embodiments, the microorganism comprises or consists of gram-positive bacteria. In other embodiments, the microorganism comprises or consists of gram-negative bacteria.

[0176] In certain embodiments, the microorganisms or protein products thereof include naturally occurring and / or non-genetically modified (non-GMO) microorganisms and / or those that are non-pathogenic and / or that grow in specific environmental conditions provided by biological processes that are not present in the natural surrounding environment.

[0177] In certain embodiments, a microorganism or consortium of microorganisms is isolated from an environmental sample and grown in the presence of target electron donors, including but not limited to, one or more of H, CO, syngas, and / or methane, and / or electron acceptors, including but not limited to, one or more of O, nitrate, ferric iron, and / or CO, and / or environmental conditions (e.g., temperature, pH, pressure, dissolved oxygen (DO), salinity, presence of various impurities and contaminants, etc.), to enrich for the desired microorganisms using methods known in the art of microbiology.

[0178] In certain embodiments, the microorganism or consortium of microorganisms includes a probiotic microorganism. In certain embodiments, the microorganism or consortium of microorganisms includes a "Generally Recognized as Safe" (GRAS) microorganism, such as a bacterial and / or fungal GRAS microorganism. In certain embodiments, the microorganism or consortium of microorganisms includes a yeast, such as, but not limited to, one or more of the following: Candida humilis; Candida milleri; Debaryomyces hansenii; Kazachstania exigua (Saccharomyces exiguous); Saccharomyces cerevisiae; Saccharomyces florentinus; Torulaspora delbrueckii; Trichosporon beigelli; and / or Aspergillus oryzae. oryzae; Aspergillus sojae; Fusarium venenatum A3 / 5; Neurospora intermedia var. oncomensis; Rhizopus oligosporus; Rhizopus oryzae; Aspergillus luchuensis; and / or fungi such as, but not limited to, one or more of the following: Bacillus amyloliquefaciens; Bacillus subtilis; Bifidobacterium animalis (lactis);Bifidobacterium bifidum; Bifidobacterium breve; Bifidobacterium longum; Lactobacillus acidophilus; Lactobacillus brevis; Lactobacillus casei; Lactobacillus delbrueckii subsp. bulgaricus; Lactobacillus fermentum; Lactobacillus helveticus; Lactobacillus kefiranofaciens; Lactobacillus lactis lactis; Lactobacillus plantarum; Lactobacillus rhamnosus; Lactobacillus reuteri; Lactobacillus sakei; Lactobacillus sanfranciscensis; Lactococcus lactis (Streptococcus lactis, Streptococcus lactis subsp. diacetylactis); Leuconostoc; Leuconostoc carnosum; Leuconostoc cremoris; Leuconostoc mesenteroides mesenteroides); Pediococcus spp.;Bacteria include one or more of Propionibacterium freudenreichii; Arthrospira (Spirulina) platensis; Streptococcus faecalis; and Streptococcus thermophilus.

[0179] The protein-containing biomass from which the protein product is derived may be produced by a consortium of different species of microorganisms. The consortium may, in some cases, include multicellular organisms. In some embodiments, the consortium includes one or more of: oxyhydrogen microorganisms; carboxydotrophs; methanotrophs; methylotrophs; chemoautotrophs; photoautotrophs; and heterotrophs.

[0180] In some embodiments, the protein product also includes one or more vitamins produced by the microorganism from which the protein product is derived. In some non-limiting embodiments, the microorganism includes Cupriavidus necator (e.g., Cupriavidus necator DSM 531 or Cupriavidus necator DSM 541). In some non-limiting embodiments, the vitamin is a B vitamin, including but not limited to vitamin B1, B2, and / or B12. In a non-limiting example, B vitamins (e.g., B1, B2, and / or B12) can be produced by Cupriavidus necator (e.g., Cupriavidus necator DSM 531 or Cupriavidus necator DSM 541).

[0181] microbial culture Microorganisms can be cultured using any suitable method. Microorganisms can be grown under any suitable conditions in an environment suitable for growth and biomass production. In some embodiments, microorganisms can be grown under autotrophic, heterotrophic, or a combination of autotrophic and heterotrophic culture conditions. Heterotrophic cultures can include suitable carbon and energy sources, such as one or more sugars (e.g., glucose, fructose, etc.). Autotrophic cultures can include C1 chemicals, such as carbon monoxide, carbon dioxide, methane, methanol, formate, and / or formic acid, and / or mixtures containing C1 chemicals, including, but not limited to, various syngas or producer gas compositions produced from low-value carbon and energy sources, such as, but not limited to, lignocellulosic energy crops, crop residues, bagasse, sawdust, forestry waste, or food, through gasification, partial oxidation, pyrolysis, or steam reforming of the low-value carbon source, and which can use oxyhydrogen microorganisms or hydrogen-oxidizing or carbon monoxide-oxidizing microorganisms as carbon and energy sources. Suitable methods for culturing microorganisms and generating biomass for use in the present methods are described, for example, in PCT Application Nos. PCT / US2010 / 001402, PCT / US2011 / 034218, PCT / US2013 / 032362, PCT / US2014 / 029916, PCT / US2017 / 023110, PCT / US2018 / 016779, and U.S. Patent No. 9,157,058, each of which is incorporated by reference in its entirety. In some embodiments, the organisms can be grown photosynthetically in bioreactors, hydroponic systems, greenhouses, or cultivated fields, or can be recovered from waste or natural sources.

[0182] The liquid cultures used to grow the microbial cells described herein can be contained in culture vessels known and used in the art. In some embodiments, large-scale production in bioreactor vessels can be used to produce large quantities of desired molecules and / or biomass.

[0183] In certain embodiments, a bioreactor vessel is used to contain, isolate, and / or protect the culture environment. Culture vessels include those known to those skilled in the art of large-scale microbial culture. Such culture vessels include, but are not limited to, one or more of the following: airlift reactors; biological scrubber columns; bubble columns; stirred tank reactors; continuous stirred tank reactors; countercurrent, upflow, and expanded bed reactors; digesters and particularly digester systems, such as those known in the bioremediation art; filters, including but not limited to trickling filters, rotating biological contactor filters, rotating disks, and soil filters; fluidized bed reactors; gas lift fermenters; immobilized cell reactors; loop reactors; membrane biofilm reactors; Pachuca tanks; packed bed reactors; plug flow reactors; static mixers; trickle bed reactors; and / or vertical axis bioreactors.

[0184] Microbial cultures for the commercial production of biomass and / or organic compounds, such as protein products as described herein, particularly single-cell proteins, cell lysates, protein extracts, protein-containing extracts, protein isolates, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof, and / or other nutrients, such as, but not limited to, vitamins (e.g., B vitamins, e.g., B1, B2, and / or B12), can be carried out on a large scale in bioreactors (e.g., bioreactor volumes of 500 L, 1,000 L, 5,000 L, 10,000 L, 50,000 L, 100,000 L, 1,000,000 L, or more).

[0185] In certain embodiments, chemoautotrophic and / or heterotrophic and / or carboxydotrophic and / or methanotrophic and / or methylotrophic microorganisms are grown in liquid media inside a bioreactor using the methods described herein.

[0186] In some embodiments, the bioreactor containing the microorganisms is constructed of opaque materials that maintain the culture in near or complete darkness. Bioreactors constructed from opaque materials, such as steel and / or other metal alloys and / or reinforced concrete and / or fiberglass and / or various high-strength plastic materials, can be designed to have large working volumes. In some embodiments, fermenters constructed from steel or other metal alloys with volumes of 50,000 liters or more are utilized. In some embodiments, bioreactors capable of containing positive headspace pressure above ambient pressure are utilized. In some embodiments, egg-shaped or cylindrical digesters or vertical-axis bioreactors with volumes of 3,000,000 liters or more are utilized. In some embodiments, the bioreactor containing the microorganisms does not allow light to penetrate some, most, or all of the contained liquid volume. In certain non-limiting embodiments, the microorganisms used in the CO2 fixation step are not photosynthetic. In certain non-limiting embodiments, the bioreactor design does not confine the culture to a thin layer, as is typically required for photosynthesis, or have transparent walls to allow light to be available in all areas. In some embodiments, the microorganisms are cultured with little or no exposure to light. In certain such embodiments, net CO consumption still occurs as a result of chemoautotrophic metabolism and conditions even in the absence of light. In certain embodiments, conversion of electricity to artificial light is not required in biological systems for CO capture and conversion.

[0187] In certain embodiments, the lack of light dependency facilitates continuous CO2 capture operation day and night, year-round, under all weather conditions, without the need for artificial lighting.

[0188] In some embodiments, microorganisms are grown and maintained in the absence of light in a medium containing a gaseous carbon source, such as, but not limited to, syngas, producer gas, or a H and CO gas mixture; such growth is known as chemoautotrophic growth.

[0189] In some embodiments, for example, syngas produced from the gasification of organic matter is utilized by the microorganisms for chemoautotrophic growth. The organic matter can be, for example, from agricultural sources (e.g., corn stover, bagasse).

[0190] In some embodiments, food-grade CO and / or air passing through the direct air capture unit is utilized by the microorganisms for chemoautotrophic growth. Non-limiting examples of direct air capture can be found in U.S. Patent Application Publication No. 2017 / 0106330 and Keith, D., et al. (2018) Joule 2(8):1573-1594, which are incorporated by reference in their entireties. In some embodiments, CO is provided from an industrial source and can optionally be concentrated via a gas separation procedure, resulting in highly concentrated food-grade CO.

[0191] In certain embodiments, increases in system capacity are met not only by horizontal scaling but also by vertical scaling. This contrasts with phototrophic approaches that use algae, cyanobacteria, or higher plants for CO2 capture. While various vertical cultivation schemes have been proposed for photosynthetic systems, practically and economically, phototrophic systems must be expanded horizontally, e.g., in the case of algae, in shallow ponds or photobioreactors. This results in a large geographic footprint with potentially numerous adverse environmental impacts.

[0192] Algae or higher plant systems grown under artificial lighting are challenged by inefficient light energy utilization and inefficient conversion of electrical energy to light energy. In certain embodiments, comparable algae or higher plant cultures grown under artificial lighting require more power for CO capture and / or biomass production than the CO capture and / or biomass production systems described herein. In certain embodiments, comparable algae or higher plant cultures grown under artificial lighting require at least 10 times more power per unit of CO capture and / or biomass production than the CO capture and / or biomass production systems described herein. For algae or higher plants grown under artificial lighting, the heat rejection requirements are approximately directly proportional to the electrical input. In certain embodiments of the methods described herein, the heat rejection requirements are lower than comparable algae or higher plant systems grown under artificial lighting for CO capture and / or biomass production. In certain embodiments, the heat rejection requirements are at least 10 times lower than comparable algal or higher plant systems for CO2 capture and / or biomass production when grown under artificial lighting.

[0193] In a representative, but non-limiting, embodiment, a bioreactor containing a nutrient medium is inoculated with production cells. Generally, following a lag phase, cells begin to double. After the lag phase, the cell doubling time shortens, and the culture enters the logarithmic phase. Following the logarithmic phase, the doubling time eventually increases, which, without intending to be bound by theory, is believed to be the result of either mass transfer limitations, depletion of nutrients, including nitrogen or mineral sources, or increased concentrations of inhibitory chemicals, or quorum sensing by the microorganisms. Growth slows and then ceases as the culture enters the stationary phase. In certain embodiments, the stationary phase is preceded by an arithmetic growth phase. In certain embodiments, the culture is harvested in the logarithmic and / or arithmetic and / or stationary phase to harvest the cell mass.

[0194] Bioreactors or fermenters are used to cultivate cells through various phases of their physiological cycle. Bioreactors can be used to cultivate cells and maintain them at specific phases of their growth curves. The use of bioreactors is advantageous in many methods for culturing chemoautotrophic growth. For certain embodiments, protein-rich cell masses used to produce proteins or protein hydrolysates are grown at high density in liquid suspension. Generally, control of growth conditions, including control of dissolved carbon dioxide, oxygen, and other gases such as hydrogen, as well as other dissolved nutrients, trace elements, temperature, and pH, is facilitated in bioreactors. For certain embodiments, protein-rich cell masses used to produce amino acids, peptides, proteins, hydrolysates, extracts, or whole cell products are grown at high density and / or with high productivity in liquid suspension in bioreactors.

[0195] Nutrient medium and gases can be added to the bioreactor by batch addition, or periodically, or in response to detected depletion or a programmed set point, or continuously over the period the culture is grown and / or maintained. For certain embodiments, the bioreactor at inoculation is filled with an initial batch of nutrient medium and / or one or more gases at the beginning of growth, and no additional nutrient medium and / or one or more gases are added after inoculation. For certain embodiments, nutrient medium and / or one or more gases are added periodically after inoculation. For certain embodiments, nutrient medium and / or one or more gases are added after inoculation in response to detected depletion of nutrients and / or gases. For certain embodiments, nutrient medium and / or one or more gases are added continuously after inoculation.

[0196] For certain embodiments, the supplemented nutrient medium does not contain any organic compounds.

[0197] In certain embodiments, a small number of microbial cells (i.e., an inoculum) is added to a volume of culture medium; the culture is then incubated; and the cell mass passes through lag, log, slowdown, and stationary phases of growth.

[0198] In batch culture systems, the conditions under which microorganisms are cultured (e.g., nutrient concentration, pH, etc.) generally change continuously throughout the growth period. In certain non-limiting embodiments, to avoid the fluctuating conditions inherent in batch culture and improve the overall productivity of the culture system, microorganisms used for the production of proteins and / or vitamins and / or other nutrients are grown in a continuous culture system called a chemostat. In such systems, the culture can be maintained in a perpetual logarithmic phase of growth by supplying fresh medium at a constant rate [F] while simultaneously maintaining a constant culture volume [V]. In certain embodiments, continuous culture systems ensure that cells are cultured under environmental conditions that remain nearly constant. In certain embodiments, cells are maintained in a perpetual logarithmic phase through the use of a chemostat system. In such cases, the dilution rate (D) of the culture is equal to the growth rate of the microorganism, given by D = F / V. The growth rate of the microorganism in continuous culture can be changed by altering the dilution rate. In certain embodiments, the growth rate of the microorganism is changed by altering the dilution rate. In certain non-limiting embodiments, the cells are incubated in a chemostat for about 0.2 h. -1 It grows at a dilution rate of .

[0199] In certain embodiments, inoculation of a bioreactor with a culture is carried out by methods including, but not limited to, transferring the culture from an existing culture residing in another bioreactor or by incubation from an incubator-grown seed stock. In certain embodiments, the seed stock of the strain can be transported and stored in a form including, but not limited to, powder, liquid, frozen, or lyophilized form, and any other suitable form readily recognized by one of skill in the art. In certain non-limiting embodiments, a backup bacterial culture is maintained in a metabolically inactive, lyophilized state until needed for resumption. In certain embodiments, when establishing a culture in a very large reactor, the culture is grown and established in progressively larger intermediate-scale vessels before inoculating the full-scale vessel.

[0200] For certain embodiments, the bioreactor has mechanisms that allow for mixing of the nutrient medium, including, but not limited to, one or more of the following: a spinning agitator bar, blades, impellers, or turbine; a spinning, rocking, or turning vessel; gas lift, sparging; recirculation of the broth from the bottom to the top of the vessel via a recirculation conduit, flowing the broth through a loop and / or static mixer. The culture medium can be mixed continuously or intermittently.

[0201] In certain embodiments, the microorganism-containing nutrient medium can be partially or completely removed from the bioreactor, periodically or continuously, and in certain embodiments, replaced with fresh cell-free medium to maintain the cell culture in exponential growth phase and / or to replenish depleted nutrients in the growth medium and / or to remove inhibitory waste products.

[0202] Ports, which are standard in bioreactors, can be used to deliver or withdraw gases, liquids, solids, and / or slurries to and / or from the bioreactor vessel containing the microorganisms. Many bioreactors have multiple ports for different purposes (e.g., ports for medium addition, gas addition, probes for pH and DO (dissolved oxygen), and sampling), and a given port can be used for various purposes during the course of a fermentation run. As an example, a port may be used to add nutrient medium to the bioreactor at one point and for sampling at another point. Preferably, multiple uses of a sampling port can be performed without introducing contaminants or invasive species into the growth environment. Valves or other actuators that allow for sample flow control or continuous sampling can be provided on the sampling port. For certain embodiments, the bioreactor includes at least one port suitable for inoculation of a culture and may additionally serve other uses, including medium or gas addition. Bioreactor ports allow for control of gas composition and flow rate to the culture environment. For example, a port can be used as a gas inlet into the bioreactor, through which gas is pumped.

[0203] For some embodiments, gases that can be pumped into the bioreactor include, but are not limited to, one or more of the following: syngas, producer gas, hydrogen gas, CO, CO, O, air, air / CO mixtures, natural gas, methane, ammonia, nitrogen, noble gases such as argon, and other gases. In some embodiments, the CO pumped into the system can be derived from sources including, but not limited to, CO from gasification of organic matter; CO from sintering limestone, CaCO to produce quicklime, CaO; CO from methane steam reforming, such as ammonia, methanol, or a CO by-product from hydrogen production; CO from combustion, incineration, or flaring; CO by-product of anaerobic or aerobic fermentation of sugars; CO by-product of methanotrophic biological processes; geologically or geothermally produced or released CO; and CO removed from acid gas or natural gas. In certain non-limiting embodiments, the CO is removed from industrial flue gases or intercepted from geological sources where it is otherwise naturally released into the atmosphere. In certain embodiments, the carbon source is CO and / or bicarbonates and / or carbonates dissolved in seawater or other surface or groundwater. In certain such embodiments, inorganic carbon can be introduced into the bioreactor dissolved in liquid water and / or as a solid. In certain embodiments, the carbon source is CO captured from the atmosphere. In certain non-limiting embodiments, CO is captured from a closed cabin as part of a closed-circuit life support system using a device such as, but not limited to, the CO Removal Assembly (CDRA) utilized on the International Space Station (ISS).

[0204] In certain non-limiting embodiments, a high concentration of an energy source (e.g., H, HS, CO gas) and / or a carbon source (e.g., CO, HCO - , CO3 2- Geological features such as geothermal and / or hydrothermal vents that release nitrates and / or other dissolved minerals can be utilized as a nutrient source for the microorganisms herein.

[0205] In certain embodiments, in addition to or in place of carbon dioxide as an alternative carbon source, one or more gases, including but not limited to, gaseous electron donors and / or carbon sources (e.g., hydrogen and / or CO and / or methane gas), are dissolved in solution and fed to the culture broth and / or dissolved directly in the culture broth. In certain embodiments, the input gases may include, but are not limited to, other gas components and impurities of syngas (e.g., hydrocarbons); ammonia; hydrogen sulfide; and / or other acid gases; and / or O; and / or other electron donors and / or electron acceptors and / or carbon sources and / or mineral nutrients, such as mineral-containing particles and ash.

[0206] In certain embodiments, one or more gases, including but not limited to: a gaseous electron donor such as one or more of hydrogen, carbon monoxide, methane, hydrogen sulfide, or other acidic gases; a gaseous carbon source such as one or more of CO, CO, or CH; and an electron acceptor such as, but not limited to, oxygen in air (e.g., 20.9% oxygen) or as pure O or an O-rich gas, are dissolved in the culture broth. In some embodiments, dissolution of these and other gases into solution is achieved using a system of compressors, flow meters, and flow valves known to those skilled in the art of fermentation engineering, and is fed into one or more of the following widely used systems for distributing gas into solution: sparging devices; spargers, including but not limited to dome, tubular, disk, or donut-shaped devices; coarse or fine bubble aerators; and Venturi devices. In certain embodiments, surface aeration and / or gas mass transfer can also be performed using paddle aerators, etc. In certain embodiments, gas dissolution is enhanced by mechanical mixing with an impeller or turbine and a hydraulic shearing device to reduce bubble size. After passing through a reactor system that holds the gas-uptake microorganisms, in certain embodiments, the residual gas can be either recycled to the bioreactor, combusted for process heat, flared, injected underground, or released to the atmosphere. In certain embodiments utilizing H2 as the electron donor herein, H2 can be supplied to the culture vessel by bubbling it through the culture medium or by diffusing it through a hydrogen-permeable, water-impermeable membrane known in the art that contacts the liquid culture medium.

[0207] In certain embodiments, microorganisms grow and multiply on H and CO and other dissolved nutrients under microaerobic conditions. In certain embodiments, mixtures containing C chemicals, including but not limited to carbon monoxide, methane, methanol, formate, or formic acid, and / or various syngas compositions produced from various gasification, pyrolysis, or steam reforming fixed carbon feedstocks, are biochemically converted to long-chain organic chemicals (i.e., C or higher, in some embodiments, C or higher carbon chain molecules) under one or more of the following conditions: aerobic, microaerobic, anaerobic, anaerobic, and / or facultative conditions.

[0208] A controlled amount of oxygen can also be maintained in the culture broth in some embodiments, and in certain embodiments, oxygen is actively dissolved in a solution fed to the culture broth and / or dissolved directly in the culture broth. In certain aerobic or microaerobic embodiments that require pumping air or oxygen into the culture broth to maintain a target DO level, oxygen bubbles can be injected into the broth at a diameter optimal for mixing and oxygen transfer.

[0209] In some embodiments, the culture is maintained under low O2 partial pressure to promote high FHb expression. In some embodiments, microaerobic culture conditions are developed. For example, dissolved oxygen may be present at a level below the detection limit of a standard dissolved oxygen probe, e.g., below 1 ppm, which is lower than the level found in air or air-saturated solutions.

[0210] In some embodiments, the microorganisms convert fuel gases, including but not limited to syngas, producer gas, CO, CO, H, natural gas, methane, and mixtures thereof. In some embodiments, the heat content of the fuel gas is at least 100 BTU per standard cubic feet (scf). In some embodiments, the bioreactor used to contain and grow the microorganisms is equipped with a fine bubble sparger and / or a high shear impeller for gas delivery.

[0211] Introducing and / or increasing the gas flow rate into the bioreactor enhances mixing of the culture and generates turbulence when the gas inlet is located below the surface of the liquid medium, causing the gas to bubble or be sparged through the medium. In certain embodiments, mixing is enhanced through turbulence provided by bubbling and / or sparging and / or gas plugging through the liquid medium. In some embodiments, the bioreactor includes a gas outlet port for venting gas and relieving pressure. In some embodiments, the gas inlet and outlet are preferably equipped with check valves to prevent backflow of gas.

[0212] In certain embodiments in which chemosynthetic reactions occur in a bioreactor, one or more types of electron donors and one or more types of electron acceptors are pumped or otherwise added in a bolus, or periodically or continuously, to a nutrient medium containing chemoautotrophic organisms in a reaction vessel. Driven by the transfer of electrons from electron donors to electron acceptors in cellular respiration, chemosynthetic reactions fix inorganic carbon dioxide and / or other dissolved carbonates and / or other carbon oxides into organic compounds and biomass.

[0213] In certain embodiments, nutrient media for culture growth and production are used that comprise aqueous solutions containing appropriate minerals, salts, vitamins, cofactors, buffers, and other components necessary for microbial growth, as known to those skilled in the art [Bailey and Ollis, Biochemical Engineering Fundamentals, 2nd ed.; pp. 383-384 and 620-622; McGraw-Hill: New York (1986)].

[0214] In certain embodiments, chemicals used in the maintenance and growth of microbial cultures as known in the art are included in the nutrient medium. In certain embodiments, these chemicals include: nitrogen sources such as ammonia, ammonium (e.g., ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4)), nitrates (e.g., potassium nitrate (KNO3)), urea, or organic nitrogen sources; phosphates (e.g., disodium phosphate (Na2HPO4), potassium phosphate (KH2PO4), phosphoric acid (H3PO4), potassium dithiophosphate (K3PS2O2), potassium orthophosphate (K3PO4), dipotassium phosphate (K2HPO4)); sulfates; yeast extract; chelated iron; potassium (e.g., potassium phosphate (KH2PO4), potassium nitrate (KNO3), potassium iodide (KI), potassium bromide (KBr)); and other inorganic salts, minerals, and micronutrients (e.g., sodium chloride (NaCl), magnesium sulfate (MgSO4 7H2O) or magnesium chloride (MgCl2), calcium chloride (CaCl2) or calcium carbonate (CaCO3), Manganese sulfate (MnSO4 7H2O) or manganese chloride (MnCl2), iron chloride (FeCl3), iron sulfate (FeSO4 7H2O) or iron chloride (FeCl2 4H2O), sodium bicarbonate (NaHCO3) or sodium carbonate (Na2CO3), zinc sulfate (ZnSO4) or zinc chloride (ZnCl2), ammonium molybdate (NH4MoO4) or sodium molybdate (Na2MoO4 2H2O), copper sulfate (CuSO4) or copper chloride (CuCl2 2 The additives may include, but are not limited to, one or more of: copper chloride (CuSeO4 5H2O), cobalt chloride (CoCl2 6H2O), aluminum chloride (AlCl3 6H2O), lithium chloride (LiCl), boric acid (H3BO3), nickel chloride (NiCl2 6H2O), tin chloride (SnCl2H2O), barium chloride (BaCl2 2H2O), copper selenate (CuSeO4 5H2O) or sodium selenate (Na2SeO3), sodium metavanadate (NaVO3), chromium salts).In certain embodiments, mineral salts medium (MSM) as formulated by Schlegel et al. can be used ["Thermophilic bacteria", Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0215] The microorganisms described herein can, in some embodiments, be cultured in any type of medium (rich or minimal), including fermentation media and any composition. As will be understood by those skilled in the art, routine optimization allows for the use of a wide variety of media types. The selected medium can be supplemented with various additional components. Some non-limiting examples of additional components include glucose, antibiotics, isopropyl-β-d-1-thiogalactopyranoside (IPTG) for gene induction, and American Type Culture Collection (ATCC) trace mineral supplements. Similarly, other aspects of the media and growth conditions of the microorganisms described herein can be optimized through routine experimentation. For example, pH and temperature are non-limiting examples of factors that can be optimized. In some embodiments, factors such as medium selection, media additives, and temperature can affect the production level of the desired molecule. In some embodiments, the concentration and amount of additional components can be optimized. In some embodiments, the frequency at which one or more additional components are added to the medium and the quantitative time the medium is cultured before harvesting the desired molecule are optimized.

[0216] In certain embodiments, concentrations of nutrient chemicals (e.g., electron donors, electron acceptors, carbon sources, and / or various mineral nutrients) are maintained within the bioreactor near or at their respective optimized levels for optimal carbon uptake and / or fixation and / or biomass and / or organic compound conversion and / or production, which will vary depending on the microorganism utilized but can be routinely determined and / or optimized by those skilled in the art of microbial culture.

[0217] In certain embodiments, one or more of the following parameters are monitored and / or controlled within the bioreactor: waste level; pH; temperature; salinity; dissolved oxygen; dissolved carbon dioxide gas; liquid flow rate; agitation rate; and gas pressure. In certain embodiments, operating parameters affecting chemoautotrophic growth are monitored with sensors (e.g., dissolved oxygen probes or redox probes to measure electron donor / acceptor concentrations) and / or controlled either manually or automatically based on feedback from sensors through the use of devices including, but not limited to, actuated valves, pumps, and agitators. In certain embodiments, the temperature of the incoming broth and incoming gas is regulated by systems such as, but not limited to, coolers, heaters, and / or heat exchangers.

[0218] In certain embodiments, microbial cultures and biological reactions are maintained at steady state using continuous inflow and removal of nutrient medium and / or biomass, targeting constant levels of cell population and environmental parameters (e.g., cell density, pH, DO, chemical concentrations) over time. In certain embodiments, the constant level is the optimal level for feedstock conversion and / or production of target organic compounds. In certain embodiments, cell density can be monitored by direct sampling, by correlation of optical density with cell density, and / or using a particle size analyzer. In certain embodiments, water retention time and biomass retention time can be separated, allowing for independent control of both broth chemistry and cell density. In certain embodiments, the dilution rate can be maintained high enough so that the water retention time is relatively short compared to the biomass retention time and the broth is highly replenished for cell growth and / or feedstock conversion and / or organic compound production. In certain embodiments, the dilution rate is set with the optimal techno-economic tradeoff between culture broth and nutrient replenishment and / or waste removal, and increased process costs from pumping, increased inputs, and other demands that increase with the dilution rate.

[0219] In certain embodiments, the pH of the microbial culture is controlled. In certain embodiments, the pH is controlled within an optimal range for microbial maintenance and / or growth and / or feedstock conversion and / or organic compound production and / or survival. To address a drop in pH, in certain embodiments, a neutralization step can be performed within the bioreactor environment prior to recycling the medium back into the culture vessel, either directly or through a recirculation loop. Neutralization of acids in the broth in certain embodiments can be achieved by the addition of a base, including, but not limited to, one or more of the following: limestone, lime, sodium hydroxide, ammonia, ammonium hydroxide, caustic potash, magnesium oxide, iron oxide, and alkaline ash.

[0220] In certain embodiments, an aqueous suspension of chemoautotrophic microorganisms converts one or more electron donors and CO2 into protoplasm. In certain embodiments, an aqueous suspension of hydrogen-oxidizing microorganisms can be used to convert hydrogen and carbon dioxide into microbial protoplasm. In certain embodiments, an aqueous suspension of carbon monoxide-oxidizing microorganisms can be used to convert carbon monoxide and hydrogen and / or water into protoplasm. In certain embodiments, an aqueous suspension of methane-oxidizing microorganisms can be used to convert methane into protoplasm. In certain embodiments, the microorganisms in the suspension are bacteria or archaea. In certain non-limiting embodiments, an aqueous suspension or biofilm of H2-oxidizing chemoautotrophic microorganisms converts H2 and CO2, along with some other dissolved mineral nutrients, into biochemicals and protoplasm. In certain embodiments, the other dissolved mineral nutrients include, but are not limited to, a nitrogen source, a phosphorus source, and a potassium source. In certain embodiments, the produced protoplasm has food value for humans and / or other animals and / or other heterotrophic organisms. In certain embodiments, certain biochemicals may be extracted from the protoplasm and / or extracellular broth and are valuable for nutritional value and / or a wide variety of organic chemical or fuel applications. In certain embodiments, the intracellular energy driving this production of protoplasm comes from the oxidation of an electron donor by an electron acceptor. In certain non-limiting embodiments, the electron donor includes, but is not limited to, one or more of the following: H; CO; CH. In certain non-limiting embodiments, the electron acceptor includes, but is not limited to, O and / or CO. In certain non-limiting embodiments, the product of the energy-producing reaction, or respiration, includes, but is not limited to, water. In certain embodiments, the intracellular energy derived from respiration used to drive this synthesis of biochemicals and protoplasm from CO is stored and transported in biochemical molecules, including, but not limited to, ATP. For hydrogen-oxidizing microorganisms used in certain embodiments herein, the electron acceptor is O and the product of respiration is water.

[0221] In some embodiments, protein production and / or the distribution of produced amino acid molecules are optimized through one or more of the following: controlling bioreactor conditions, controlling nutrient levels, and / or genetically modifying the cells. In certain embodiments, pathways to amino acids, or proteins, or other nutrients, or whole-cell products are controlled and optimized for chemical production by maintaining specific growth conditions (e.g., levels of trace micronutrients such as nitrogen, oxygen, phosphorus, sulfur, inorganic ions, and any regulatory molecules, if present, that may not generally be considered nutrients or energy sources). In certain embodiments, dissolved oxygen (DO) can be optimized by maintaining the broth in aerobic, microaerobic, anoxic, anaerobic, or facultative conditions, depending on the requirements of the microorganisms. A facultative environment is considered to be one with an aerobic upper layer and an anaerobic lower layer caused by stratification of the water column. Biosynthesis of amino acids, or proteins, or other nutrients, or whole cell products by the microorganisms disclosed herein can occur during logarithmic phase or during the subsequent stationary phase when cell doubling has ceased, provided there is an adequate supply of carbon and energy and other nutrient sources.

[0222] In some embodiments, the growth medium for the microorganisms described herein includes a protein and / or nutrient source from another microorganism (e.g., cell lysate, protein hydrolysate, peptides, oligopeptides, and / or amino acids, and / or organic molecules and / or other nutrients from a different microorganism). In some embodiments, the microorganism in the growth medium is a GRAS microorganism. In one embodiment, the culture medium for lactic acid bacteria, such as, but not limited to, bacteria of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus (e.g., GRAS lactic acid bacteria, such as bacteria of the genus Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus), includes, but is not limited to, Cupriavidus necator, e.g., Cupriavidus The compositions may include cell lysates, protein hydrolysates, peptides, oligopeptides, and / or amino acids, and / or organic molecules and / or other nutrients from different microorganisms, such as any of the microorganisms described herein, including, but not limited to, Cupriavidus microorganisms such as Saccharomyces necator DSM531 or DSM541.In another embodiment, a growth medium for a fungal microorganism, such as a Fusarium or Rhizopus fungal microorganism (e.g., a GRAS fungal microorganism such as a GRAS Fusarium or Rhizopus fungal microorganism), such as any microorganism described herein, including but not limited to Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae, may be used. These include cell lysates, protein hydrolysates, peptides, oligopeptides, and / or amino acids, and / or organic molecules and / or other nutrients from different microorganisms, such as, but not limited to, Cupriavidus microorganisms, such as Saccharomyces cerevisiae DSM 531 or DSM 541.

[0223] In some embodiments, a fungal microorganism capable of lysing bacterial cells and / or hydrolyzing bacterial proteins is cultured in the presence of such bacterial cells. For example, a bacterial biomass can be isolated and optionally dehydrated, and then the fungal microorganism can be inoculated onto the bacterial biomass, or the fungal microorganism can be cultured in the presence of the bacterial biomass in a culture medium such as described herein. In certain non-limiting embodiments, the fungal microorganism includes, but is not limited to, a Fusarium or Rhizopus microorganism, such as Fusarium venenatum, Rhizopus oligosporus, or Rhizopus oryzae.

[0224] The specific examples of bioreactors, culture conditions, heterotrophic and chemotrophic growth, maintenance, and amino acid, or protein, or other nutrient, or whole cell product production methods described herein can be combined in any suitable way to improve the efficiency of microbial growth and amino acid, or protein, or other nutrient, or whole cell product production.

[0225] Electron donors and acceptors In certain non-limiting embodiments, the microorganisms described herein are grown chemoautotrophically. For example, the grown microorganisms can utilize biosynthetic reduction of CO utilizing an O electron acceptor and / or an H electron donor. In certain embodiments, O and H are produced by water electrolysis. In certain non-limiting embodiments, a portion of the O and all of the H produced by water electrolysis are supplied to an aqueous suspension of microorganisms as described herein. In certain non-limiting embodiments, the molar ratio of H to O supplied to the aqueous suspension of microorganisms is greater than 2:1. In certain non-limiting embodiments in which the O electron acceptor and H electron donor are produced by water electrolysis, excess O remains after all of the microorganisms' metabolic requirements for H and O are satisfied. In certain such embodiments, the excess O can be supplied to a hydroponic system for human and / or other aerobic organisms and / or root aeration, and / or used in a gasification or partial oxidation or combustion process, and / or stored and sold as a chemical by-product.

[0226] In certain embodiments utilizing molecular hydrogen as the electron donor, there may be chemical by-products formed in the production of molecular hydrogen using renewable and / or CO2-emission-free energy inputs. In certain embodiments, the acid-hydrogen reaction used in respiration is enzymatically linked to oxidative phosphorylation. In certain embodiments, the ATP and / or other intracellular energy carriers thus formed are utilized in the anabolic synthesis of amino acids and / or proteins. In certain embodiments, to maintain optimal conditions for carbon fixation and organic compound production by hydrogen-oxidizing microorganisms, the excess oxygen produced by the water splitting required for respiration can be processed into a form suitable for sale through process steps and commercial oxygen gas production science known in the art.

[0227] Certain embodiments employ hydrogen-oxidizing and / or CO-oxidizing and / or CH-oxidizing microorganisms that use more electronegative electron acceptors than CO in energy-conserving reactions for ATP production (e.g., respiration), such as, but not limited to, O. For example, hydrogenotrophic acidohydrogen- or hydrogen-oxidizing microorganisms that couple the acidohydrogen reaction, 2H + O → 2H O, to ATP production can produce more ATP per H and / or other electron donor consumed for respiration than acetogens or methanogens that use CO as the electron acceptor in respiration. For example, hydrogen-oxidizing microorganisms can produce at least two ATP per H consumed in respiration [L. Bongers (1970) "Energy generation and utilization in hydrogen bacteria," Journal of bacteriology 104(1):145-151 (http: / / jb.asm.org / content / 104 / 1 / 145.abstract), incorporated herein by reference in its entirety], producing eight times more ATP per H consumed in respiration than can be produced in microorganisms that perform methanogenesis and acetogenesis using H as the electron donor and CO as the electron acceptor in respiration. For this reason, using microorganisms that can utilize more electronegative electron acceptors in respiration and ATP production, such as, but not limited to, hydrogen-oxidizing microorganisms for anabolic biosynthesis, such as, but not limited to, the biosynthesis of amino acids, proteins, or fatty acids from syngas or H, may be more efficient than using acetogens or methanogens, such as those currently used in biological GTC technology for the production of short-chain acids or alcohols (e.g., acetic acid or ethanol). In certain embodiments, the acid-hydrogen reaction used in respiration is enzymatically linked to oxidative phosphorylation. In certain embodiments, aerobic respiration is utilized by the microorganisms described herein for the production of ATP. In certain embodiments, the ATP and / or other intracellular energy carriers thus formed are utilized for the anabolic biosynthesis of amino acids and / or proteins.In some embodiments, hydrogen-oxidizing and / or carboxydotrophic and / or methanotrophic and / or heterotrophic microorganisms or compositions comprising these microorganisms are utilized, and the microorganisms express one or more enzymes capable of biosynthesis of useful carbon-based products of interest, including, but not limited to, chemicals, monomers, polymers, proteins, polysaccharides, vitamins, dietary supplements, antibiotics, or pharmaceuticals or intermediates thereof, from carbon-containing gas feedstocks, including, but not limited to, syngas, producer gas, natural gas, biogas, or CO2 in combination with renewable H2, CO2, or methane-containing gases. In some embodiments, these carbon-based products of interest can be heterotrophically biosynthesized from organic multi-carbon feedstocks, such as, but not limited to, glucose, fructose, and other sugars. In some non-limiting embodiments, microorganisms or compositions comprising microorganisms are utilized, and the microorganisms require fewer than four H2 or NADH to produce one ATP through respiration. In other non-limiting embodiments, microorganisms that produce one or more ATP per H2 or NADH consumed through respiration are utilized. In other non-limiting embodiments, a microorganism that produces at least 2 ATP per H2 or NADH consumed through respiration, or at least 2.5 ATP per H2 or NADH consumed through respiration, is utilized.

[0228] An additional feature of certain non-limiting embodiments relates to the source, production, or recycling of electron donors used by chemoautotrophic microorganisms to fix carbon dioxide and / or other C1 feedstocks into organic compounds. Electron donors used for carbon dioxide capture and carbon fixation can, in certain embodiments, be produced or recycled electrochemically or thermochemically using electricity from many different renewable and / or low-carbon emission energy technologies, including, but not limited to, photovoltaic technology, solar, wind, hydroelectric, nuclear, geothermal, enhanced geothermal, ocean thermal contrast, ocean wave power, and tidal power. Many reduced inorganic chemicals (e.g., H2, CO2, HS, ferrous iron, ammonium, Mn) on which chemoautotrophic organisms can grow are readily available. 2+) can be readily produced using electrochemical and / or thermochemical processes known in the art and chemical engineering that can be powered by a variety of zero or low carbon emissions and / or renewable electricity sources, including, but not limited to, photovoltaic technology, solar, wind, hydroelectric, nuclear, geothermal, enhanced geothermal, ocean thermal, ocean wave, or tidal power.

[0229] Hydrogen production from renewable energy sources is gradually replacing production from fossil fuel systems, and technological advances in the energy sector are expected to reduce the price of green hydrogen production in the near future. For example, electrical energy efficiencies of up to 73% have already been achieved by commercial and industrial-grade electrolyzers, and research into novel materials and electrolyzer configurations has shown that efficiencies as high as 96% are possible. Certain embodiments utilize commercially available electrolysis technologies with electrical energy efficiencies greater than 70% for the production of H electron donors and / or O electron acceptors. Certain embodiments use electrolysis technologies with energy efficiencies of 73% or greater and / or up to 96% or greater.

[0230] In certain embodiments using molecular hydrogen as the electron donor, H is generated by one or more of the following methods well known in the art and in chemical and process engineering, including but not limited to: through electrolysis of water, including but not limited to proton exchange membranes (PEMs), approaches using liquid electrolytes such as KOH, alkaline electrolysis, solid polymer electrolyte electrolysis, high pressure electrolysis, high temperature steam electrolysis (HTES); and / or through thermochemical decomposition of water, including but not limited to the iron oxide cycle, the cerium(IV) oxide-cerium(III) oxide cycle, the zinc zinc-oxide cycle, the sulfur-iodine cycle, the copper-chlorine cycle, the calcium-bromine-iron cycle, the hybrid sulfur cycle; and / or electrolysis of hydrogen sulfide; and / or thermochemical decomposition of hydrogen sulfide; and / or other electrochemical or thermochemical processes known to produce hydrogen with low or no carbon dioxide emissions, including but not limited to carbon capture and storage (CCS) that enable methane reforming. In certain embodiments, approaches to producing H2 include, but are not limited to, electrolysis powered by renewable electrical energy and / or electricity from low greenhouse gas (GHG) sources. In certain embodiments, electrolysis is powered by one or more of the following: solar, including but not limited to photovoltaic technology and / or solar thermal; wind; hydroelectric; nuclear; geothermal; enhanced geothermal; ocean thermal gradient; ocean wave power; and tidal power.

[0231] The world has vast wind energy resources, only a small percentage of which is utilized. Current underutilization is primarily due to the intermittent nature of wind resources, resulting in varying amounts of power generated over time and underutilized capacity to meet energy demand most of the time. The prevalent mismatch between wind power supply and grid demand is evident in examples from around the world, such as in Scotland, where wind farms pay to shut down turbines due to oversupply [http: / / www.mnn.com / earth-matters / energy / blogs / blown-away-wind-turbines-generate-enough-energy-to-power-every-home-in], and in parts of Texas, where wind power is abundant and demand on the grid is low, free electricity is provided at night [http: / / www.nytimes.com / 2015 / 11 / 09 / business / energy-environment / a-texas-utility-offers-a-nighttime-special-free-electricity.html?_r=2]. This problem may be solvable by utilizing wind power produced during off-peak demand hours to produce H2 feedstock for the process in certain embodiments herein.

[0232] Hydrogen is currently gaining attention as a possible energy storage system in the so-called "power-to-gas" approach. The inherent instability of renewable energy production (especially solar and wind energy) and excess grid electricity (off-peak energy) can be mitigated by producing hydrogen through water electrolysis. According to most current schemes, the produced hydrogen gas is then converted to electricity using fuel cells and / or gas turbines during peak demand periods. Alternatively, H2 can be supplied to the gas grid or converted to methane via methanation. Furthermore, hydrogen can be used as a feedstock in the chemical, petrochemical, metallurgical, and food industries. Certain embodiments offer novel options within the power-to-gas framework by enabling H2 to be used in a wider range of products, including biochemicals, particularly proteins, amino acids, fertilizers, and biostimulants. In certain embodiments, hydrogen produced using excess grid electricity and / or off-peak energy is used as an electron donor for one or more metabolic pathways occurring in hydrogen-utilizing microorganisms. In certain embodiments, the hydrogen and / or oxygen required for microbial biosynthesis by hydrogen-oxidizing bacteria and / or aerobic bacteria is produced by electrolysis of water using renewable energy, in particular off-peak electricity, i.e., electricity available when energy supply exceeds demand, which in the current situation is often wasted.

[0233] In certain embodiments, on-site storage of H and CO gases allows for the diversion of power from the grid only during periods when renewable power generation exceeds electrical demand. In certain embodiments, power can flow into the grid as usual during periods of high demand. In certain embodiments, the process does not interrupt the renewable power supply, but rather allows for fuller utilization of renewable power generation capacity, such as, but not limited to, wind and solar. Certain embodiments allow for continuous renewable operation and power generation, even during periods when power generation exceeds grid demand (e.g., off-peak wind or solar power).

[0234] In certain embodiments, the hydrogen electron donor is not necessarily produced with low or no carbon dioxide emissions. However, in certain such embodiments, hydrogen is produced from sustainable or low-value energy and / or carbon sources using methods known in the fields of chemical and process engineering. Such methods generally include, but are not limited to, gasification, pyrolysis, steam reforming, or autothermal reforming of feedstocks such as, but not limited to, one or more of the following: agricultural materials, wood, methane hydrate, straw, seaweed and kelp, and low-value, lignocellulosic biomass. In certain embodiments, syngas or producer gas containing H and / or CO and / or CO is utilized as the electron donor and / or carbon source. In certain embodiments, the H and / or CO and / or CO contained in the syngas or producer gas is supplemented with H produced using renewable and / or low-GHG energy sources and conversion processes such as one or more of those described herein.

[0235] In certain non-limiting embodiments, reduction of CO2 and / or synthesis of cellular material that can be utilized as a food or nutrient source occurs. In certain embodiments, the ratio of hydrogen to carbon monoxide in the syngas or producer gas can be adjusted through a water-gas shift reaction and / or carbon capture before the gas is delivered to the microbial culture. In certain embodiments, C1 compounds are produced by methane steam reforming of methane or natural gas, particularly stranded natural gas, or natural gas otherwise flared or released to the atmosphere, or biogas, or landfill gas, and provided to the microbial culture as a gas or liquid stream of syngas and / or producer gas or C1 compounds; in certain embodiments, the ratio of hydrogen to carbon monoxide in the syngas or producer gas can be adjusted through a water-gas shift reaction and / or carbon capture before the gas is delivered to the microbial culture.

[0236] The following examples are intended to illustrate, but not limit, the present invention.

[0237] Example Example 1. Emulsion Stability The ability of the emulsion to resist changes in its properties over time was examined.

[0238] Research Samples: Whey Protein Isolate (PI) (50% Protein) · Whole cell biomass (WCB) grown on sugar (12.86% N; 70.9% protein using a 5.5 Jones factor)

[0239] Procedure: A mixture of 5g of protein solution and 5g of oil was mixed thoroughly in a homogenizer. The mixture was incubated at room temperature. The volume of the aqueous phase was measured. The emulsion stability percentage was calculated using the following formula:

[0240]

number

[0241] It was calculated according to.

[0242] Emulsion stability of whey PI and WCB A protein solution was prepared in distilled water: 4.58% whey PI, pH 6.50; 4.33% WCB, pH 6.26. Five grams of protein solution was mixed with 5 grams of corn oil and homogenized for 5 minutes using an IKA Turrax T25 digital homogenizer. The emulsion was transferred to a measuring cylinder, and the volume of the aqueous phase was measured. The results are shown in Table 1.

[0243] [Table 1]

[0244] Example 2. Emulsion Volume The ability of the protein solution to emulsify oil was investigated.

[0245] Research Samples: Whey Protein Isolate (PI) (50% Protein) · Whole cell biomass (WCB) grown on sugar (12.86% N; 70.9% protein using a 5.5 Jones factor)

[0246] Procedure: Different mixtures were prepared using a constant weight of protein solution and different weights of oil. Emulsions were formed using a homogenizer. The conductivity of each emulsified sample was measured. The emulsion volume was calculated at the inversion point from oil-water (oil droplets in water) to water-oil (water droplets in oil) using the following formula:

[0247]

number

[0248] It was calculated according to.

[0249] For whey PI, the inversion point occurred at 2 g of protein solution (4.583% solids) and 4 g and 5 g of oil. The emulsion volume was 98.19 g of oil per gram of protein. The results are shown in Figure 2.

[0250] For the WCB, the inversion point occurred at 3 g of protein solution (4.327% solids) and 4 g and 5 g of oil. The emulsion volume was 46.26 g of oil per gram of protein. The results are shown in Figure 3.

[0251] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications can be made thereto without departing from the spirit and scope of the invention. Therefore, the specification should not be construed as limiting the scope of the invention.

[0252] All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference as such.

Claims

1. A flavor enhancer comprising a heme-containing polypeptide derived from a hydrogen-oxidizing microorganism, (i) the hydrogen-oxidizing microorganism is a Cupriavidus microorganism; or (ii) A flavor enhancer, wherein the hydrogen-oxidizing microorganisms grow under microaerobic conditions.

2. 10. The flavor enhancer of claim 1, wherein the heme-containing polypeptide releases heme when heated.

3. 10. The flavor enhancer of claim 1, wherein the heme-containing polypeptide imparts a meaty flavor and / or aroma.

4. 10. The flavor enhancer of claim 1, wherein the heme-containing polypeptide is processed from the hydrogen-oxidizing microorganism as a single-cell protein, a protein hydrolysate, a cell lysate, a protein isolate, a protein extract, a peptide, an oligopeptide, or a combination thereof.

5. 2. The flavor enhancer of claim 1, wherein the heme-containing polypeptide is a heme-containing domain of a heme-containing enzyme.

6. 2. The flavor enhancer of claim 1, wherein the Cupriavidus microorganism is Cupriavidus necator.

7. 2. The flavor enhancer of claim 1, wherein the hydrogen-oxidizing microorganism is engineered for overexpression of the heme-containing polypeptide.

8. 10. A structured food comprising the flavor enhancer of claim 1 and a first protein product, wherein the flavor enhancer and the first protein product are derived from the hydrogen-oxidizing microorganism or the first protein product is derived from a different microorganism.

9. 10. The structured food of claim 8, wherein the hydrogen-oxidizing microorganisms comprise chemoautotrophically grown microorganisms.

10. 10. The structured food of claim 9, wherein the chemoautotrophically grown microorganisms comprise Cupriavidus microorganisms.

11. 10. The structured food of claim 8, wherein the different microorganism is grown on a nutrient source comprising a first protein product from a different, second microorganism.

12. 12. The structured food of claim 11, wherein the additional second microorganism comprises the hydrogen-oxidizing microorganism.

13. 13. The structured food of claim 12, wherein the different microorganisms are generally recognized as safe (GRAS) microorganisms.

14. 9. The structured food of claim 8, wherein the hydrogen-oxidizing microorganism is non-genetically modified organism (non-GMO).

15. 10. The structured food of claim 8, wherein the structured food is free of animal-derived biomolecules.

16. 9. The structured food of claim 8, wherein the first protein product comprises one or more of a single cell protein, a cell lysate, a protein isolate, a protein extract, a protein hydrolysate, a free amino acid, a peptide, an oligopeptide, or a combination thereof.

17. 10. The structured food of claim 8, comprising from about 5% to about 50% protein by weight from said first protein product.

18. 10. The structured food product of claim 8, wherein the structured food product is a structured meat substitute product.

19. 20. The structured food product of claim 18, wherein the structured meat substitute product comprises a structured beef, poultry, pork, fish, or seafood substitute product.

20. 20. The structured food product of claim 18, wherein the structured meat substitute product replicates the texture and / or organoleptic properties of natural meat.

21. 20. The structured food product of claim 18, wherein the structured meat substitute product mimics the structure of ground or muscle meat.

22. 9. The structured food of claim 8, to which vitamins and / or nutrients have been added.

23. 23. The structured food of claim 22, wherein the vitamins and / or nutrients comprise amino acids, lipids, oils, fatty acids, vitamin B12, biotin, antioxidants, minerals, surfactants, emulsifiers, or any combination thereof.

24. 10. A dough composition for the production of a structured food product comprising the flavor enhancer of claim 1 and a first protein product, wherein the flavor enhancer and the first protein product are derived from the hydrogen-oxidizing microorganism or the first protein product is derived from a different microorganism.

25. 25. The dough composition of claim 24, wherein the hydrogen-oxidizing microorganisms comprise chemoautotrophically grown microorganisms.

26. 26. The dough composition of claim 25, wherein the chemoautotrophically grown microorganisms comprise Cupriavidus microorganisms.

27. 25. The dough composition of claim 24, wherein the first protein product comprises one or more of a single cell protein, a cell lysate, a protein isolate, a protein extract, a protein hydrolysate, a free amino acid, a peptide, an oligopeptide, or a combination thereof.

28. 30. The dough composition of claim 27, wherein the first protein product comprises a protein hydrolysate.

29. 30. The dough composition of claim 28, wherein the average molecular weight of the proteins in the hydrolysate is from about 5 kD to about 10 kD.

30. 30. The dough composition of claim 28, wherein the protein hydrolysate is produced under conditions that preserve intact globular proteins.

31. 31. The dough composition of claim 30, wherein the conditions that preserve native globular proteins include gentle cell lysis and physical separation of soluble components from cell debris.

32. 31. The dough composition of claim 30, wherein a partially or fully hydrolyzed first protein product is added to a second protein product to promote structure and / or fiber formation.

33. 33. The dough composition of claim 32, wherein the second protein product comprises wheat, gluten, soy, pea, wheat, dairy, algae, other non-animal proteins, or combinations thereof.

34. 25. The dough composition of claim 24, wherein the moisture content of the dough composition is from about 40% (w / w) to about 80% (w / w).

35. 25. The dough composition of claim 24, wherein the shear strength of the dough composition is greater than about 1000 psig.

36. (a) culturing a hydrogen-oxidizing microorganism in the presence of a carbon source, thereby producing a protein-containing biomass, the biomass comprising at least one heme-containing polypeptide; (b) converting the biomass into a first protein product; and (c) processing said first protein product into a structured food product.

1. A method for producing a structured food product, comprising:

37. (a) culturing a hydrogen-oxidizing microorganism in the presence of a carbon source, thereby producing a protein-containing biomass; (b) converting the biomass into a first protein product; and (c) processing said first protein product into a structured food product.

1. A method of producing a structured food product, comprising: The method, wherein step (b) or step (c) comprises adding a flavor enhancer, wherein the flavor enhancer comprises a heme-containing polypeptide, and wherein the flavor enhancer and the first protein product are derived from the hydrogen-oxidizing microorganism, or the first protein product is derived from a different microorganism.

38. 38. The method of claim 36 or 37, wherein step (a) comprises chemoautotrophic culture conditions.

39. 39. The method of claim 38, wherein the chemoautotrophic culture conditions comprise microaerobic culture conditions.

40. 38. The method of claim 36 or 37, wherein the carbon source comprises gaseous C1 molecules.

41. 41. The method of claim 40, wherein the gaseous C1 molecules comprise carbon dioxide.

42. 39. The method of claim 38, wherein the hydrogen-oxidizing microorganism comprises a Cupriavidus microorganism.

43. 38. The method of claim 36 or 37, wherein the first protein product comprises one or more of a single cell protein, a cell lysate, a protein isolate, a protein extract, a protein hydrolysate, a free amino acid, a peptide, an oligopeptide, or a combination thereof.

44. 44. The method of claim 43, wherein step (c) comprises processing the first protein product into a dough composition.

45. 45. The method of claim 44, further comprising thermochemical processing of the dough composition.

46. 46. ​​The method of claim 45, wherein the thermochemical processing comprises extrusion.

47. 45. The method of claim 44, wherein step (c) comprises a spinning process, said spinning process producing fibers.

48. 45. The method of claim 44, wherein step (c) comprises a process for forming a structured hydrocolloid.

49. 38. The method of claim 36 or 37, wherein the structured food product is a structured meat substitute product.

50. 50. The method of claim 49, wherein the structured meat substitute product is a structured beef, poultry, pork, fish, or seafood substitute product.

51. 50. The method of claim 49, wherein the structured meat substitute product replicates the texture and / or organoleptic properties of natural meat.

52. 50. The method of claim 49, wherein the structured meat substitute product mimics the structure of ground or muscle meat.

53. 25. A thermochemical process for processing the dough composition of claim 24 into a structured food product, comprising extruding the dough composition to produce aligned fibers.

54. 54. The thermochemical process of claim 53, wherein the first protein product comprises a protein hydrolysate.

55. 55. The thermochemical process of claim 54, wherein the hydrogen-oxidizing microorganisms comprise chemoautotrophically grown microorganisms.

56. 56. The thermochemical process of claim 55, wherein the chemoautotrophically grown microorganisms comprise Cupriavidus microorganisms.

Images