High-protein food composition

Abstract

A method for producing a food product that is not derived from animals, is free from unhealthy components associated with meat such as saturated fatty acids and cholesterol, does not have the harmful environmental impacts of animal farming, and provides a food product that has similar texture and flavor characteristics to animal meat. [Solution] A method comprising (a) processing microbial cells collected from a culture medium to produce a protein product, wherein the protein product contains one or more of the following: single-cell protein, cell lysate, protein concentrate, protein isolate, protein extract, protein hydrolysate, free amino acids, peptides, and oligopeptides; and (b) processing the protein product to produce a food product.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 956,110, filed on December 31, 2019, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to a novel protein composition that is suitable for human consumption, closely mimics the characteristics of meat, and functions as a meat substitute or artificial meat product.

Background Art

[0003] Eating meat from animal sources is part of the daily life of many people. The adverse effects of meat - based diets on human health and the environment have been well - reported. There is a growing consumer demand for foods rich in alternative proteins that are not of animal origin, do not contain unhealthy components associated with meat such as saturated fatty acids and cholesterol, and do not have the harmful environmental impacts of animal agriculture, and provide similar texture and flavor characteristics to animal meat.

Summary of the Invention

Problems to be Solved by the Invention

[0004] According to the present invention, chemolithoautotrophic microorganisms, such as hydrogen - oxidizing microorganisms, grow in a bioreactor, for example, in a culture growth medium within the bioreactor. In some embodiments, a gas composition is introduced into a bioreactor containing a carbon and / or energy source for the growth of chemolithoautotrophic microorganisms. In one embodiment, the gas composition contains hydrogen, carbon dioxide, and oxygen for the growth of hydrogen - oxidizing microorganisms. Alternatively, the biomass can be grown heterotrophically using an organic carbon source such as sugar molecules instead of a gas feedstock. In one embodiment, the microorganisms include Cupriavidus necator, such as DSM531 or DSM541, but are not limited thereto.

[0005] The biomass grown from this process can be harvested and processed into artificial meat products, e.g., meat-like products, or other food products. The biomass can first be processed into one or more protein products, such as single-cell proteins (e.g., whole-cell biomass), cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof. Such an initial process may include (1) releasing organic molecules (including at least a portion of the microbial proteins) from microbial cells via cell excretion, secretion, or cell lysis, and (2) processing the released organic molecules to break down the bonds between at least a portion of the amino acids of the protein to produce peptides, polypeptides, oligopeptides (peptides having 2 to 20 amino acids) or free amino acids with a desired chain length. These protein products can then be processed into food products such as artificial meat products.

[0006] According to one embodiment, the protein product contains any of the following free amino acids: about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%. According to one embodiment, the protein product contains any of the following oligopeptides: about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%. According to one embodiment, the protein product comprises a polypeptide containing 20 to 50 amino acids or 21 to 50 amino acids, in any of the following proportions: approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 98%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 98%.According to one embodiment, the protein product comprises a polypeptide containing 50 to 200 amino acids or 51 to 200 amino acids, in any of the following proportions: approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 98%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 98%. According to one embodiment, the protein product comprises a polypeptide containing 200 to 500 amino acids or 201 to 500 amino acids, in any of the following proportions: approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 98%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 98%. According to one embodiment, the protein product comprises a polypeptide containing less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids, comprising about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%.According to one embodiment, the protein product comprises a combination of free amino acids, oligopeptides, and polypeptides having 20 to 50 amino acids or 21 to 50 amino acids, with free amino acids to oligopeptides to 20 to 50 amino acids. The ratio of amino acids or polypeptides having 21 to 50 amino acids is approximately 1:1:1, or approximately 0-3:0-3:0-3, or approximately 3-6:0-3:0-3, or approximately 0-3:3-6:0-3, or approximately 0-3:0-3:0-3, or approximately 0-3:0-3:0-3:0-3, or approximately 0-3:0-3:0-3:0-3, or approximately 3-6:0-3:0-3, or approximately 0-3:0-3 The ratios are approximately 6:3-6, or 3-6:0-3:3-6, or 6-9:0-3:0-3, or 0-3:6-9:0-3, or 0-3:6-9:0-3, or 0-3:0-3:6-9, or 6-9:6-9:0-3, or 0-3:0-3:6-9, or 6-9:6-9:0-3, or 0-3:6-9:6-9, or 6-9:0-3:6-9. According to one embodiment, the protein product does not contain or essentially contains whole cells.

[0007] According to one embodiment, protein products are combined with other edible ingredients to form food products such as artificial meat products that mimic one or more physical characteristics and / or functional properties of meat, such as texture, flavor, aroma, and / or appearance. Such other ingredients include apple cider, apple cider vinegar, baking powder, baking soda, beans, beef, beet juice, beet powder, black pepper, brown sugar, butter, canola oil, caramel, carrot fiber, carrots, cashews, cheese, chicken, chocolate, citrus fruits, citrus extracts, coconut oil, condensed milk, dairy products, eggs, egg substitutes, fish, wheat flour, chickpeas, garlic powder, honey, smoke flavoring, maple syrup, margarine, and monosodium glutamate. The ingredients may be selected from mustard powder, oil, olive oil, onion powder, paprika, pork, potatoes, potato starch, rice flour, salt, sodium benzoate, soy (protein and / or oil), soy sauce, spices, spirulina, sugar, sunflower oil, tomato juice, tomato powder, tomato sauce, tomatoes, turmeric, vanilla, vinegar, vitamins and minerals, walnuts, water, wheat, wheat flour, wheat gluten, xanthan gum, yeast, and / or yeast extract.

[0008] In some embodiments, the protein product comprises at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% of any artificial meat product (on a dry weight basis, e.g., by weight %) on a dry weight basis. [Modes for carrying out the invention]

[0009] This invention provides food products, such as artificial meat products, that contain protein products derived from microorganisms.

[0010] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the present invention pertains. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide general dictionaries of many of the terms used herein. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the methods, systems, and compositions described herein.

[0011] The implementation of the present invention will utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the scope of the art, unless otherwise indicated. Such techniques are fully described in literature such as, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1994); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Gene Transfer and Expression: A Laboratory Manual (Kriegler, 1990).

[0012] The numerical ranges provided herein include the numbers that define the ranges.

[0013] Unless otherwise specified, nucleic acids are written from left to right in the 5' to 3' direction. Amino acid sequences are written from left to right, with the amino to carboxyl orientation.

[0014] definition Unless the context explicitly indicates otherwise, “A,” “an,” and “the” include plural references, and therefore, in this specification and in the claims, the indefinite articles “a,” “an,” and “the” as used herein should be understood to mean “at least one” unless explicitly indicated otherwise.

[0015] As used herein, the term "about" refers to a measurable value such as quantity or temporal duration, and means that such variation includes a variation of ±5%, ±1%, or ±0.1% from the specified value, such that it is appropriate for carrying out the disclosed method or in relation to the disclosed composition.

[0016] "Acetic acid production" refers to microorganisms that produce acetates and / or other short-chain organic acids up to C4 chain length as products of anaerobic respiration.

[0017] "Acidicophilic" refers to a type of extremophilic microorganism that grows under highly acidic conditions (usually pH 2.0 or lower).

[0018] The term "amino acid" refers to a molecule containing both an amine group and a carboxyl group bonded to a carbon called the alpha carbon. Preferred amino acids include, but are not limited to, D and L isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. In some embodiments, a single "amino acid" may have multiple side chain moieties, each available for extended aliphatic or aromatic scaffolding. Unless the context specifically indicates otherwise, the term amino acid as used herein is intended to include amino acid analogs.

[0019] In this specification and in the claims, the phrase “and / or” as used herein means “either or both” of the elements thus combined (i.e., elements that exist together in some cases and separately in others). Other elements other than those specifically identified by the “and / or” phrase may exist, whether related to the specifically identified elements or not, unless explicitly indicated otherwise. Thus, as a non-restrictive example, a reference to “A and / or B” when used in combination with open-ended language such as “comprising” may, in one embodiment, mean including A but not B (optionally including elements other than B); in another embodiment, include B but not A (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements), and so on.

[0020] As used herein, the terms “artificial meat,” “meat substitute,” “imitation meat,” or “meat look-alike” refer to food products that are not derived from animals or contain a substantial amount of non-animal protein sources, but have a structure, texture, aesthetic quality, and / or other characteristics equivalent to or similar to substitutes / look-alikes of animal meat, e.g., livestock (e.g., beef, pork), game meat (e.g., venison), poultry (e.g., chicken, turkey, duck), and / or fish or seafood. The terms refer to uncooked, cooked, and cooked meat-like food products.

[0021] The term "biomass" refers to the material produced by the growth and / or proliferation of cells. Biomass may include cells and / or intracellular contents, as well as extracellular material (but not limited to compounds secreted by cells).

[0022] The term "bioreactor" or "fermenter" refers to a closed or partially closed vessel in which cells grow and are maintained. The cells may or may not be held in a liquid suspension. In some embodiments, the cells are not held in a liquid suspension but instead, without limitation, may grow and / or be maintained in contact with, on, or within another non-liquid substrate such as a solid growth support material.

[0023] The term "carbon fixation" process, reaction, or pathway refers to an enzymatic reaction or metabolic pathway that, without limitation, converts gaseous forms of carbon such as CO2, CO, and CH4 under ambient conditions to carbon-based biochemicals that are liquid or solid, or dissolved or held in suspension in an aqueous solution under ambient conditions.

[0024] "Carbon source" refers to the type of molecule from which a microorganism obtains the carbon necessary for organic biosynthesis.

[0025] "Carboxydotrophic" refers to a microorganism capable of tolerating or oxidizing carbon monoxide. In preferred embodiments, carboxydotrophic microorganisms can utilize CO as a carbon source and / or as a reducing electron source for biosynthesis and / or respiration.

[0026] "Chemolithoautotrophy" refers to an organism that obtains energy by the oxidation of a chemical electron donor by a chemical electron acceptor and synthesizes all the organic compounds it requires for survival and growth from carbon dioxide.

[0027] In the claims and specification, all transitional clauses such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” and “holding” are understood to be open-ended; that is, they include, but are not limited to, these terms. Only the transitional clauses “consisting of” and “consisting essentially of” are considered closed or half-closed transitional clauses, respectively.

[0028] In this specification, “Consortium” means two or more different species or strains of microorganisms and / or multicellular organisms which are grown together, for example, by co-culture in the same growth medium.

[0029] The term "cultivate" refers to growing a population of cells (e.g., microbial cells) in a liquid or solid medium under conditions favorable for growth.

[0030] The term “derived from” encompasses the terms “arrived from,” “obtained from,” “can be obtained from,” “isolated from,” and “created from,” and generally indicates that a particular material has characteristics whose origin can be found in another particular material or which can be described by reference to another designated material.

[0031] The term "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.

[0032] "Extremophiles" refer to microorganisms that can grow in extreme physical or geochemical conditions (e.g., high or low temperatures, pH, or high salinity) compared to the conditions on the surface of the Earth or the ocean. Typically, these are tolerated by most life forms found on or near the Earth's surface.

[0033] The term "gasification" refers to a common high-temperature process that converts carbon-based materials into a mixture of gases, such as hydrogen, carbon monoxide, and carbon dioxide, called synthesis gas (syngas), or generator gas. This process generally involves partial combustion and / or the application of externally generated heat, along with the controlled addition of oxygen and / or vapor, so that oxygen is present, which is insufficient for the complete combustion of the carbon-based material.

[0034] "Halophilic" refers to a type of extremophilic microorganism that thrives in environments with extremely high salt concentrations.

[0035] "Heterotrophic" refers to organisms that cannot synthesize all the organic compounds they need to survive and grow from carbon dioxide, and therefore must utilize organic compounds for growth. Heterotrophic organisms cannot produce their own food; instead, they obtain food and energy by taking in and metabolizing organic matter from plants or animals, rather than fixing carbon from inorganic sources such as carbon dioxide.

[0036] "Hydrogen oxidizers" refer to microorganisms that utilize reduced H2 as an electron donor in the production of intracellular reduced equivalents and / or in respiration.

[0037] "Hyperthermophilic" refers to a type of extremophilic microorganism that lives its entire life in extremely high-temperature environments (typically above approximately 60°C (140°F)).

[0038] The term "hydrogen-oxidizing gas (knallgas)" refers to a mixture of hydrogen molecules and oxygen gas. "Hydrogen-oxidizing microorganisms" are microorganisms that, in respiration, can produce intracellular energy carriers such as adenosine-5'-triphosphate (ATP) by using hydrogen as an electron donor and oxygen as an electron acceptor. The terms "oxyhydrogen" and "oxyhydrogen microorganisms" can be used synonymously with "hydrogen oxidation" and "hydrogen-oxidizing microorganisms," respectively. Hydrogen-oxidizing microorganisms generally produce NAD.+ Molecular hydrogen is utilized by hydrogenases, which have some of the electrons provided by H2 that are used for the reduction of (and / or other intracellular reducing equivalents) and some of the electrons from H2 that are used for aerobic respiration. Hydrogen-oxidizing microorganisms generally autotrophically fix CO2 via pathways that include, but are not limited to, the Calvin cycle or the reverse citric acid cycle ["Thermophilic bacteria", Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0039] The term "lysate" refers to a liquid containing a mixture and / or solution of cellular contents resulting from cell lysis. In some embodiments, the methods described herein include the purification of a chemical or mixture of chemicals in the cell lysate. In some embodiments, the methods include the purification of amino acids and / or proteins in the cell lysate.

[0040] The term "lysis" refers to the rupture of the cell wall of a cell, allowing the plasma membrane and, if present, a significant amount of intracellular material to escape into the extracellular space. Lysis can be carried out using electrochemical, mechanical, osmotic, thermal, or viral means. In some embodiments, the methods described herein include carrying out the lysis of cells or microorganisms described herein to separate a chemical substance or a mixture of chemical substances from the contents of a bioreactor. In some embodiments, the methods include carrying out the lysis of cells or microorganisms described herein to separate amino acids or a mixture of amino acids and / or proteins from the contents of a bioreactor or cell growth medium.

[0041] "Methane-producing bacteria" refers to microorganisms that produce methane as a product of anaerobic respiration.

[0042] "Methyl-nutrient" refers to microorganisms that can use reduced one-carbon compounds such as methanol or methane as a carbon source and / or electron donor for their growth, but are not limited to these.

[0043] The terms "microorganism" and "microbe" refer to tiny, single-celled organisms.

[0044] The term "molecule" means any distinct or identifiable structural unit of substance that contains one or more atoms, including, for example, hydrocarbons, lipids, polypeptides, and polynucleotides.

[0045] "Oligopeptides" refer to peptides that contain a relatively small number of amino acid residues, for example, about 2 to 20 amino acids.

[0046] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in an enumeration, “or” or “and / or” shall be interpreted as inclusive, that is, including at least one (including more than one) of multiple elements or lists of elements, and optionally additional unenumerated items. Only terms that are explicitly indicated, such as “only one of” or “exactly one of” or, where used in the claims, “consisting of,” refer to including exactly one element of multiple elements or lists of elements. In general, where used herein, the term “or” shall be interpreted only as indicating an exclusive alternative when preceded by an exclusive term, such as “either,” “one of,” “only one of,” or “exactly one of” (i.e., “either one or neither”). When used in patent claims, "consisting essentially of" shall have its ordinary meaning as it is used in the field of patent law.

[0047] The term "organic compound" refers to any gaseous, liquid, or solid chemical compound that contains carbon atoms, with the exception of carbides, carbonates, simple oxides of carbon, cyanides, and allotropes of pure carbon (such as diamond and graphite), which are considered inorganic.

[0048] A "peptide" refers to a compound (polypeptide) consisting of two or more amino acids linked together in a chain, where the carboxyl group of each acid is linked to the next amino group by an R-OC-NH-R' type bond (for example, approximately 2 to 50 amino acids, or 21 to 50 amino acids).

[0049] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length and any three-dimensional structure, and of single-stranded or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.) nucleotides, including deoxyribonucleotides, ribonucleotides, and / or analogues or modified forms of deoxyribonucleotides or ribonucleotides, such as modified nucleotides or bases or their analogues. Because the genetic code is degenerate, multiple codons can be used to code for a particular amino acid, and the present invention encompasses polynucleotides that code for a particular amino acid sequence. Any type of modified nucleotide or nucleotide analogue can be used, such as modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, phosphorothioates, etc.), as long as the polynucleotide retains the desired functionality under the conditions of use. Labels can also incorporate, for example, radioactive or non-radioactive labels or anchors, such as biotin, for the purpose of detection or capture. The term polynucleotide also includes peptide nucleic acids (PNAs). Polynucleotides may or may not be naturally occurring. The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably herein. Polynucleotides may include 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 bonds may be substituted 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 optionally a substituted or unsubstituted alkyl (1-20C) including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. All linkages within a polynucleotide do not need to be identical.Polynucleotides may be linear, circular, or contain a combination of linear and circular portions.

[0050] As used herein, “polypeptide” refers to a composition composed of amino acids that is recognized as a protein by those skilled in the art. Conventional one- or three-letter codes for amino acid residues are used herein. The terms “polypeptide” and “protein” are used herein interchangeably and 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 modified naturally or by intervention, such as the formation of disulfide bonds, glycosylation, lipidization, acetylation, phosphorylation, or binding with labeling components. The definition also includes polypeptides containing, for example, one or more analogues of amino acids (e.g., non-natural amino acids), as well as other modifications known in the art.

[0051] The term “precursor” or “precursor of” refers to an intermediate toward the manufacture of one or more components of a finished product.

[0052] "Generator gas" refers to a gas mixture containing various ratios of H2, CO, and CO2, with a calorific value typically ranging from half to one-tenth the calorific value of natural gas per unit volume under standard conditions. Generator gas can be produced from various raw materials in various ways, including gasification, steam reforming, or self-reformation of carbon-based raw materials. In addition to H2, CO, and CO2, generator gas may contain other components, depending on the production process and raw materials, but not limited to methane, hydrogen sulfide, condensable gases, tar, and ash. The proportion of N2 in the mixture can be high or low depending on whether air is used as an oxidizer in the reactor and whether the heat for the reaction is provided by direct combustion or indirect heat exchange.

[0053] The term "produce" includes both intracellular and extracellular production of compounds, including the secretion of compounds from cells.

[0054] "Psyrophilic bacteria" typically refer to a type of extremophilic microorganism that can grow and reproduce at temperatures below approximately 10°C.

[0055] As used herein, the terms “recovered,” “isolated,” “purified,” and “separated” refer to material from which at least one naturally associated component has been removed (e.g., a protein, nucleic acid, or cell). For example, these terms may refer to material that substantially or essentially does not contain the components normally associated with it, as would be found in its original state, such as an intact biological system.

[0056] The phrases “substantially absent” or “essentially absent” with respect to any given component mean that such component is present, if present at all, only in amounts that are not functionally significant, i.e., do not have a significant adverse effect on the intended performance or function of any process or product. Typically, “substantially absent” means less than about 1% by weight of such component, e.g., less than about 0.5% by weight, less than about 0.1% by weight, and zero% by weight. The terms “substantially absent” or “essentially absent” shall be used for amounts less than 1% of the component.

[0057] "Sulfur oxidizing agents" refer to microorganisms that utilize reducing sulfur-containing compounds, such as H2S, for the production of intracellular reducing equivalents and / or as electron donors in respiration, but are not limited to these.

[0058] "Syngas" or "Synthesis gas" refers to a type of gas mixture that contains H2 and CO, similar to the generator gas, but is more specifically controlled in terms of the H2 and CO content, and the ratio and level of impurities for the synthesis of certain types of chemical products (but not limited to methanol or Fischer-Tropsch diesel). Synthesis gas generally contains H2, CO, and CO2 as its main components and can be produced through established methods such as steam reforming of methane, or through the gasification of any organic, combustible, carbon-based material (but not limited to biomass, organic matter, or peat). The hydrogen component of synthesis gas increases by the reaction of CO with vapor in the water-gas shift reaction, and simultaneously increases the CO2 in the synthesis gas mixture.

[0059] "Thermophiles" refer to a type of extremophilic microorganism that lives its entire life cycle at relatively high temperatures, typically between 45°C and 122°C.

[0060] "Wild type" refers to microorganisms that exist in nature.

[0061] "Yield" refers to the amount of product produced from a given material relative to the total amount of material that would be produced if all of the supplied material were converted into the product. For example, the yield of a product can be expressed as the percentage of the product produced relative to the theoretical yield that would occur if 100% of the supplied material were converted into the product.

[0062] High-protein food products In some embodiments, high-protein food compositions and methods for producing them are provided. “Protein products” derived from one or more microorganisms described herein (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) can be processed or incorporated into high-protein edible food compositions for consumption by humans and / or animals. Food compositions (food products) may be, for example, food commodities, and / or food ingredients, and / or nutritional products, and / or animal feed, and / or pet food products. In some embodiments, the food composition may contain at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% (by weight, e.g., by weight %) on a dry weight basis.

[0063] In certain embodiments, the protein products described herein are used in the production of vegetarian or vegan food products. In certain embodiments, they are used in the production of organic food products and / or pesticide-free and / or herbicide-free and / or fungicide-free and / or antibiotic-free and / or non-genetically modified (non-GMO) food products. In certain embodiments, they are used in locally sourced food products. In certain embodiments, they are used in probiotic food products or prebiotic food products (e.g., prebiotic nutritional products).

[0064] A process is provided for producing a high-protein food product comprising one or more protein products from one or more microorganisms described herein. In certain embodiments, the food product does not contain animal protein or fat. In certain embodiments, the protein product(s) are incorporated into food products such as dairy products, dairy substitutes, meat products (such as livestock, game meat, poultry, fish, or seafood products), meat substitutes and / or imitation meat products (imitation livestock, game meat, poultry, fish, or seafood products), bakery products, confectionery, health protein bars, protein powders, sports and / or energy drinks, and / or protein shakes and / or smoothies. In certain embodiments, the protein product(s) are textured for incorporation into meat products and / or imitation meat products. In certain embodiments, the high-protein component(s) can be used as meat fillers in beef patties.

[0065] In certain embodiments, the high-protein food products described herein do not contain animal fat. In certain embodiments, they have a relatively high ratio of polyunsaturated fats to saturated fats. In certain embodiments, they have a high-quality protein content nearly comparable to milk protein. In certain non-limiting embodiments, their amino acid content is substantially similar, very close to, or identical to that recommended as ideal by the United Nations Food and Agriculture Organization. In certain embodiments, food products produced using the protein products of the present invention represent health foods and / or low-calorie foods. In certain embodiments, the protein products have a mild flavor and / or a pale cream color and / or easy dispersibility and / or relatively high water absorption and / or relatively high fat adsorption. In certain embodiments, the protein products may be formed into fibers and / or thermally extruded and / or coagulated into gels. In certain such non-limiting embodiments, gel coagulation occurs at a pH that drops to a range of about 3 to about 6 when heated. In certain embodiments, one or more properties of the protein product, but not limited to these, make it sufficiently suitable for incorporation into food products such as dairy products, dairy substitutes, meat products, meat substitutes and / or imitation meat products, bakery products, confectionery, health protein bars, protein powders, sports and / or energy drinks, and / or protein shakes and / or smoothies. In certain embodiments, the protein product is textured for incorporation into meat products and / or imitation meat products. In certain embodiments, the protein product can be used as a meat filler, for example, as a meat filler in beef patties. In certain non-limiting embodiments, about 30 parts of the protein product can be combined with 70 parts of meat, for example, ground beef, and in other embodiments, about 10 parts of the protein product can be combined with 90 parts of meat (for example, ground beef).In certain non-limiting embodiments, the protein product is combined with beef and / or other meat products in a ratio that meets the requirements set forth by the USDA and / or in accordance with the rules governing the National School Lunch Program (Type A School Lunch). In certain non-limiting embodiments, the protein product is included in a formulation having a combined protein equivalent ratio (PER) of approximately 2.6. In certain non-limiting embodiments, the water-absorbing and / or fat-binding properties of the protein product help reduce shrinkage (fat and water loss) during cooking and / or enhance the moisture and texture of the cooked patty or other meat or food product.

[0066] In certain embodiments, the protein products produced as described herein are included in recipes and / or formulations together with one or more of the following ingredients: water; tomatoes; tomato juice; tomato sauce; beans; spices, but not limited to chili spice; seasonings; animal proteins; beef; poultry (e.g., chicken, turkey, duck, goose); pork; fish; seafood; soybeans; wheat; wheat flour; yeast; yeast extract; spirulina; margarine; butter; dairy products; cheese; sugar; brown sugar; honey; eggs; salt; vanilla; chocolate; baking soda; baking powder; condensed milk; and / or caramel. In certain embodiments, the combined ingredients undergo one or more of the following processes: hydration; blending; mixing; beating; sifting; sprinkling; heating; cooking; frying; deep-frying; baking; simmering; browning; boiling. In certain embodiments, the ingredients are baked at approximately 350°F (177.7°C) and / or baked or cooked at approximately 375°F (190.5°C) to 450°F (232.2°C). In certain embodiments, the protein product is used as a meat filler or meat substitute in one or more of the following: patties; chili con carne; pizza toppings; ground beef; chicken nuggets; and fish sticks. In certain embodiments as meat fillers and / or substitutes, the protein product replaces about 50% or more than 50% of the meat component in a food product (e.g., any of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%), and in other embodiments, replaces less than 50% (e.g., any of less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than 10%).

[0067] In certain embodiments, protein product components impart improved nutrition, water absorption, fat-binding properties, texture, and / or eating quality to food products such as cereal-based products. In certain embodiments, protein product components are used for nutritional fortification or are incorporated into cereal-based products. In certain embodiments, cereal-based products are breakfast cereals, cookies, cakes, pies, brownies, muffins, or bread. In certain embodiments, protein products are used as a substitute for milk protein (e.g., sodium caseinate) and / or as a vitamin and / or mineral supplement in milk or dairy products. In certain such embodiments, protein product components are used in, but are not limited to, non-fat dried milk, powdered milk, or dairy-type beverages, e.g., instant breakfast mixes, e.g., soy milk, rice milk, and almond milk, e.g., soy milk, rice milk, and almond milk, e.g., soy milk, rice milk, and almond milk, e.g., soy milk, rice milk, and almond milk, e.g., soy milk, rice milk, and almond milk, e.g., soy milk, soy milk, and almond milk, and / or soy milk. In certain embodiments, protein product components are used in nutritionally fortified (e.g., fortified with protein, vitamins, and / or minerals) candies, desserts, or snacks.

[0068] In some embodiments, a protein product derived from one or more microorganisms described herein (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) is optionally processed to produce a food product or its components in a process that includes heating under shear stirring and then extruding to produce a food with a desired texture (e.g., chewy, crunchy, crisp, resistant to dispersion in water, etc.). In exemplary, non-limiting embodiments, an aqueous paste of a protein product (e.g., containing about 20% (w / w) to about 50% (w / w), about 20% (w / w) to about 40% (w / w), about 30% (w / w) to about 50% (w / w), about 20% (w / w) to about 35% (w / w), or about 35% (w / w) to about 50% (w / w) of water), optionally a plant-based material such as plant protein (e.g., soybean meal, sesame meal, cottonseed meal, corn meal, wheat meal, and / or pi) may be used. Combined with a nut meal, etc., the product is heated to a temperature of approximately 150°F (65.6°C) to approximately 400°F (204.4°C), or approximately 225°F (107.2°C) to approximately 275°F (135.0°C) for approximately 10 seconds to approximately 300 seconds, and a shear force is applied (optionally simultaneously with heating) at a shear rate of approximately 10 rpm to approximately 60 rpm and a torque of approximately 200 to approximately 2000 milligrams, and the heated and sheared protein product is extruded through a die to provide a molded extruded product. Optionally, the extruded product is exposed to an oxygen-containing gas stream. In one embodiment, the oxygen-containing gas stream is, for example, an air stream (e.g., a dry air stream) at a temperature of approximately 80°F (26.7°C) to approximately 212°F (100°C) for approximately 0.5 minutes to approximately 10 minutes.

[0069] In some embodiments, a protein product derived from one or more microorganisms described herein (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) is processed to produce a food product or a component thereof in a process comprising: combining the protein product with one or more additional protein sources (but not limited to, e.g., peas, rice, glutinous rice, wheat, gluten, soybeans, hemp, canola, insects, algae, and / or buckwheat); heating the mixture (e.g., about 150°F (65.6°C) to about 400°F (204.4°C)); and subjecting the mixture to shear forces in an extruder to create a textured product having a desired texture and / or functional characteristics (e.g., chewy, crunchy, crisp, resistant to dispersion in water, etc.).

[0070] In some embodiments, free amino acids are included as part of a protein product or as a supplement to a protein product to impart a desired flavor. In one non-limiting embodiment, glutamic acid is included to impart umami to the food product.

[0071] In some embodiments, for example, in meat substitutes or artificial meat products, hydrogels, lipogels, and / or emulsions are included in the composition, for example, as an agent-releasing system (for releasing colorants, flavorings, fatty acids, leavening agents, gelling agents (for example, bicarbonates (e.g., potassium bicarbonate), calcium hydroxide, and / or alginates (e.g., sodium or potassium alginate)) where the agent(s) may be released during the cooking of the food product to simulate animal meat.

[0072] In some embodiments, the food product comprises, but is not limited to, one or more plant protein sources such as peas, rice, glutinous rice, wheat, gluten, soybeans, hemp, canola, insects, algae, and / or buckwheat, in combination with a protein product produced by the microorganisms described herein (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof), the protein product imparting flavor, for example, a meat-like flavor (such as the flavor of livestock, game meat, poultry, or seafood meat).

[0073] In some embodiments, the food product, for example, a meat substitute or artificial meat product, may be a heme compound, such as a heme-containing polypeptide. In one embodiment, the food product contains heme (e.g., a heme-containing polypeptide) from a microorganism from which a protein product is derived. In certain such embodiments, the microorganism is a Cupriavidus microorganism, such as Cupriavidus necatar.

[0074] In some embodiments, meat substitutes or artificial or imitation meat products (e.g., livestock (e.g., beef, pork), game meat, poultry, fish, or seafood-like products) include protein products produced by the microorganisms described herein (e.g., one or more of single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof). In some embodiments, the meat-like product is a vegan product that does not contain any animal-derived components. In some embodiments, fortified meat products containing animal protein are provided (e.g., beef, poultry, pork, fish, seafood, or egg products, where a portion of the product is a protein product component produced by the microorganisms described herein) (e.g., one or more of single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof). For example, protein products may be included as fillers in fortified meat products or meat-like products, for example, by replacing at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of any meat component or artificial or imitation meat component (e.g., plant-based artificial or imitation meat component) to produce fortified meat products or meat-like / imitation meat products, respectively. In some embodiments, the microorganisms are CO2-grown or air-grown microorganisms, for example, oxyhydrogen microorganisms. Non-exclusive examples of meat substitutes include 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, and 10,039,306. These are described in Nos. 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 herein by reference in their entirety.

[0075] In some embodiments, at least some, all, or substantially all of the protein products in the food products described herein (including, but not limited to, meat substitutes or artificial meat products) are protein products derived from the Cupriavidus microorganism, e.g., Cupriavidus necatar, e.g., DSM531 or DSM541 (e.g., one or more of single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof).

[0076] In some embodiments, at least some, all, or substantially all of the protein products in the food products described herein, such as meat substitutes or artificial meat products, are derived from lactic acid bacteria, for example, but not limited to, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus bacteria (e.g., one or more of single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof). In some embodiments, the lactic acid bacteria are generally recognized as safe (GRAS) bacteria.

[0077] In some embodiments, at least some or all of the protein products in the food products described herein (including, but not limited to, meat substitutes or artificial meat products) include protein products derived from Fusarium, Rhizopus, or Aspergillus fungal microorganisms, including, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof). In some embodiments, the fungal microorganisms are GRAS microorganisms.

[0078] Meat analog products Artificial meat products are provided that are similar to and / or have the flavor of animal meat (e.g., livestock, game meat, poultry, fish, or seafood). The artificial meat products comprise protein products derived from one or more microorganisms described herein (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) that simulate the texture and / or physical characteristics of animal meat, such as flavor, aroma, texture, and appearance.

[0079] In some embodiments, the artificial meat product comprises at least about 10% by weight, at least about 15% by weight, at least about 20% by weight, or at least about 25% by weight of the microbial protein products described herein, optionally bound together by one or more binders, to produce a food product having one or more similar textures and / or functional characteristics compared to animal meat. In some embodiments, the artificial meat product is similar to animal meat, e.g., ground meat (e.g., ground beef, ground pork, ground turkey). In some embodiments, the artificial meat product consists mainly or entirely of components derived from non-animal sources. In alternative embodiments, the artificial meat product consists of components that are partially derived from animal sources but supplemented with components derived from non-animal sources. In some embodiments, the artificial meat product further comprises one or more agent-releasing systems and / or other components. In various embodiments, the artificial meat products herein may be sliced, cut, ground, shredded, grated, or otherwise processed, or left unprocessed. Examples of sliced ​​forms, but not limited to these, include dried meat, cured meat, and sliced ​​lunch meat or deli meat. In some embodiments, the artificial meat food products provided herein are formed by, for example, shredding and then joining together, cutting into chunks, grinding and forming, or dicing, in order to produce a product that resembles animal jerky in appearance and / or texture.

[0080] In some embodiments, the artificial meat product is vegan. In some embodiments, the artificial meat product does not contain GMO ingredients. In some embodiments, the artificial meat product does not contain ingredients derived from nuts. In some embodiments, the artificial meat product contains less than about 0.6% by weight or less than about 0.5% by weight of sodium. In some embodiments, the meat-like food product is gluten-free or substantially gluten-free. In some embodiments, the meat-like food product is soy-free or substantially soy-free.

[0081] In some embodiments, the artificial meat food products provided herein contain about 5% to about 30% by weight of lipids, for example, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 30%, about 5% to about 15%, about 10% to about 20%, about 20% to about 30%, about 5% to about 15%, about 10% to about 20%, about 20% to about 30%, about 5% to about 15%, about 15% to about 30%, about 5% to about 25%, or about 10% to about 30% of lipids. In some embodiments, the artificial meat product contains about 0.5% to about 10% of total carbohydrates, for example, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 2% to about 8%, or about 3% to about 6% of total carbohydrates. In some embodiments, the artificial meat product contains about 0.5% to about 5% dietary fiber, for example, about 0.5% to about 1%, about 1% to about 5%, about 5% to about 10%, about 2% to about 8%, or about 3% to about 6% dietary fiber.

[0082] The artificial meat products provided herein contain a moisture content (MC) of at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight. In some embodiments, the artificial meat products contain an MC similar to that of animal meat (e.g., livestock, game meat, poultry, fish, or seafood).

[0083] In some embodiments, the artificial meat product comprises one or more colorants. In some embodiments, the artificial meat product comprises one or more color enhancers. In some embodiments, the meat-like food product comprises 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 grape seed extract, beet extract and tomato extract, beet extract and lycopene, beet extract and beta-carotene, beet extract and anthocyanin, beet extract and anthocyanin and annatto, beet extract and annatto and lycopene, beet extract and ascorbic acid, anthocyanin and annatto, beet extract and annatto and ascorbic acid, beet extract and annatto and beta-carotene, beet extract and turmeric and ascorbic acid, and anthocyanin, 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, colorants, color stabilizers, and / or color accelerators are present in non-uniform 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, or 99:1). In some embodiments, the artificial meat product includes browning agents, e.g., but not limited to, pentoses (e.g., ribose, arabinose, xylose), hexoses (e.g., glucose, fructose, mannose, galactose), dextrin, and commercially available browning agents (e.g., Red Arrow dextrose, wood-derived agents).

[0084] In some embodiments, the artificial meat products herein include, but are not limited to, one or more plant protein sources such as peas, rice, glutinous rice, wheat, gluten, soybeans, hemp, canola, insects, algae, and / or buckwheat, combined with protein products produced by microorganisms described herein, where the protein products impart a meat-like flavor to the composition.

[0085] In some embodiments, the artificial meat products of this specification include a heme compound, such as a heme-containing polypeptide. For example, the heme compound (e.g., a heme-containing polypeptide) may be derived from a microorganism from which the protein product is derived. In certain such embodiments, the heme compound is derived from a Cupriavidus microorganism, such as Cupriavidus necatar. In certain such embodiments, the heme compound is hemoglobin or flavohemoglobin.

[0086] protein products Protein products (e.g., one or more single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) are derived from and / or include biomass and / or protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, and / or oligopeptides from one or more microorganisms as described herein.

[0087] According to one embodiment, the protein product contains about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% of amino acids, such as free amino acids. According to one embodiment, the protein product comprises a peptide containing oligopeptides in proportions of approximately 50% to approximately 55%, approximately 55% to approximately 60%, approximately 60% to approximately 65%, approximately 65% ​​to approximately 70%, approximately 70% to approximately 75%, approximately 75% to approximately 80%, approximately 80% to approximately 85%, approximately 85% to approximately 90%, approximately 90% to approximately 95%, approximately 95% to approximately 98%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 98%. According to one embodiment, the protein product comprises a peptide comprising a polypeptide containing about 20 to about 50 amino acids (e.g., 21 to 50 amino acids) in proportion to about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%.According to one embodiment, the protein product comprises a peptide comprising a polypeptide containing about 50 to about 200 amino acids (e.g., 51 to 200 amino acids) in amounts of about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%. According to one embodiment, the protein product comprises a peptide comprising a polypeptide containing about 200 to about 500 amino acids (e.g., 201 to 500 amino acids) in amounts of about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%. According to one embodiment, the protein product comprises a peptide comprising a polypeptide containing less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids, in proportion to about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 90% to about 95%, about 95% to about 98%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98%.According to one embodiment, the protein product comprises a combination of free amino acids, oligopeptides, and polypeptides having 21 to 50 amino acids, with the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to the free amino acid ratio being the ratio of oligopeptides to oligopeptides being the ratio being the free amino acid ratio being the ratio being the free amino acid ratio being the ratio being the free amino acid ratio being the oligopeptide The ratio of polypeptides containing amino acids is approximately 1:1:1, or approximately 0-3:0-3:0-3, or approximately 3-6:0-3:0-3, or approximately 0-3:3-6:0-3, or approximately 0-3:0-3:0-3, or approximately 0-3:0-3:0-3:0-6, or approximately 3-6:0-6:0-3, or approximately 0-3:0-3:0-6:0-3 6, or approximately 3-6: approximately 0-3: approximately 3-6, or approximately 6-9: approximately 0-3: approximately 0-3, or approximately 0-3: approximately 6-9: approximately 0-3, or approximately 0-3: approximately 0-3: approximately 6-9, or approximately 6-9: approximately 6-9: approximately 0-3, or approximately 0-3: approximately 6-9: approximately 6-9, or approximately 6-9: approximately 0-3: approximately 6-9.

[0088] In some embodiments, the protein product includes free amino acids. In certain embodiments, the amino acids may be produced by the microorganisms described herein, and in some embodiments, may be secreted by the microorganisms described herein. Non-limiting examples of microbial amino acid production can be found in PCT application no. WO2014 / 145194, which is incorporated herein in whole by reference.

[0089] In some embodiments, the protein product exhibits water and / or oil absorption at a level suitable for incorporation into food compositions described herein, such as artificial meat or meat substitute compositions, but not limited to these. For example, the water retention capacity of the protein product may be about 1 to about 10, for example, about 2 to about 4 times by weight.

[0090] In one embodiment, the protein product includes heme (e.g., heme-containing polypeptide) produced by a microorganism from which the protein product originates, such as a Cupriavidus microorganism, for example, Cupriavidus necatar.

[0091] In some embodiments, but not limited to these, at least some, all, or substantially all of the proteins in the protein products described herein, such as single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, and / or oligopeptides, are derived from Cupriavidus microorganisms, for example, Cupriavidus necatar, for example, DSM531 or DSM541, but not limited to these.

[0092] In some embodiments, but not limited to these, at least some, all, or substantially all of the proteins in the protein products described herein, such as single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, and / or oligopeptides, are derived from lactic acid bacteria, such as, but not limited to, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacteria are GRAS bacteria.

[0093] In some embodiments, but not limited to these, at least some, all, or substantially all of the protein products in the protein products described herein, such as single-cell proteins, cell lysates, protein concentrates, protein isolates, protein extracts, protein hydrolysates, free amino acids, peptides, and / or oligopeptides, are derived from Fusarium, Rhizopus, or Aspergillus fungal microorganisms, including, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae. In some embodiments, the fungal microorganisms are GRAS microorganisms.

[0094] Protein concentrate In certain embodiments, a method is used to extract non-protein fractions (e.g., lipids, nucleic acids, polysaccharides) without solubilizing the major protein fraction. The recovered insoluble protein fraction is a protein concentrate. In certain embodiments, the protein concentrate is produced from protein-containing biomass produced by one or more microorganisms, as described herein.

[0095] In certain embodiments, the protein concentrate is produced via a solvent extraction process. In certain such embodiments, the solvent extraction process includes, for example, alcohol extraction or washing, such as aqueous alcohol washing. In certain embodiments, acid treatment is used to produce the protein concentrate. In certain embodiments, the protein concentrate is produced via a thermal denaturation process.

[0096] In certain embodiments, one or more of the steps of solvent extraction, acid treatment, and / or thermal denaturation are carried out and may be used sequentially or in parallel to produce a protein concentrate. In certain embodiments, the thermal denaturation step is carried out in the production of the protein concentrate following the solvent extraction step. In certain embodiments, the solvent extraction step is carried out in the production of the protein concentrate following the thermal denaturation step. In certain embodiments, the thermal denaturation step and the acid treatment are combined in the production of the protein concentrate. In certain such embodiments, the insoluble material resulting from the heat + acid treatment is subjected to the solvent extraction step. In certain embodiments, the protein concentrate produced via one or more solvent extraction, acid treatment, and / or thermal denaturation steps is washed with water.

[0097] In certain embodiments, the protein concentrate described herein includes at least some or most of the oil and / or water-soluble non-protein components present in the starting biomass removed by the protein concentrate process.

[0098] In certain embodiments, the protein concentrates described herein contain at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight of protein, based on the water-free state. Crude protein content can be defined as the total nitrogen percentage (%N) of the material multiplied by a Jones coefficient equal to 6.25, i.e., crude protein = 6.25 * %N. In certain embodiments, the protein concentrates described herein contain a crude protein content of at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight, based on the water-free state. The determination of the total amino acid content of proteinaceous materials is well established in the science of biochemical analysis (e.g., using AOAC method 994.12). In certain embodiments, the protein concentrates described herein contain a total amino acid content of at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight of the concentrate, based on the water-free state. In certain embodiments, the protein concentrate produced according to the present invention contains a higher protein content and / or a higher crude protein content and / or a higher total amino acid content than the soy protein concentrate.

[0099] In certain embodiments, the protein concentrates described herein contain a carbohydrate content of less than about 20% by weight, less than about 10% by weight, less than about 5% by weight, or less than about 1% by weight. In certain embodiments, the proteins described herein contain an ash content of less than about 10% by weight, less than about 8% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight. In certain embodiments, the protein concentrates described herein contain a lipid content of less than about 10% by weight, less than about 8% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight.

[0100] In certain embodiments, a solvent extraction process is applied to the production of protein concentrates. The solvent may include one or more alcohols or aqueous solutions containing one or more alcohols. The production of protein concentrates using alcoholic solvents is based on the ability of solutions containing lower aliphatic alcohols (e.g., methanol, ethanol, isopropyl alcohol) to extract lipid and soluble sugar fractions without solubilizing the proteins and / or by denaturing them. In certain embodiments, the concentration of alcohol used in the solvent in the solvent extraction process is at least about 10% by weight, at least about 20% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, or at least about 99% by weight.

[0101] Starting from wet or dry microbial biomass produced as described herein, in certain embodiments, the protein concentration process comprises one or more of the following steps: liquid-solid extraction, removal and recovery of the solvent from the liquid extract, removal and recovery of the solvent from the solid (e.g., protein concentrate), and drying and grinding of the solid (e.g., protein concentrate).

[0102] In certain embodiments, solid-liquid extraction is performed in batch or sequentially. In certain embodiments, solid-liquid extraction is performed using one or more of the following: horizontal belt extractors; basket extractors; stationary extractors; and / or rotary cell extractors.

[0103] Heat treatment can reduce the solubility of sugar by binding it to proteins (e.g., through the Maillard reaction) or by caramelization. Such condensation reactions can reduce solvent extraction of sugar. They can also result in a darker color for concentrates, which may be undesirable in certain applications. In certain embodiments, heat treatment is not performed until after solvent extraction. In certain embodiments, the protein concentrate process utilized avoids the occurrence of the Maillard reaction.

[0104] In certain embodiments, a nonpolar solvent is used in a solvent extraction step. In certain embodiments, a nonpolar solvent is used in combination with an alcohol solvent. In certain embodiments, a nonpolar solvent is used in combination with an aqueous alcohol solution. In certain embodiments, a nonpolar solvent is used to extract neutral lipids from an extract produced using alcohol and / or an aqueous alcohol solution. In certain non-limiting embodiments, a nonpolar solvent having a boiling point range (i.e., distillation range) of 65°C to 70°C is used. In certain non-limiting embodiments, a nonpolar solvent consisting mainly of 6-carbon alkanes is used. In certain embodiments, hexane is used as a nonpolar solvent. In certain such embodiments, the hexane used as a nonpolar solvent conforms to the stringent quality specifications required for the extraction of edible oils from soybeans and other plant-based sources (including, but not limited to, boiling (distillation) range, maximum non-volatile residue, flash point, maximum sulfur, maximum cyclic hydrocarbon, color, and specific gravity).

[0105] In certain embodiments, "supercritical extraction," which uses liquid carbon dioxide under high pressure, is utilized for solvent extraction.

[0106] It is known that diffusion is often the rate-limiting process in the solvent extraction of biological materials. Therefore, typically, considerable effort and energy are directed towards reducing the size of the biomass being extracted. For example, soybeans and oilseeds often roll into thin flakes, and are therefore reduced and thinned to facilitate diffusion. In certain embodiments, for microscopic unicellular microorganisms described herein, any size reduction before solvent extraction becomes unnecessary. In certain embodiments, the solvent extraction is more efficient, i.e., requires less solvent and / or recovers a higher percentage of extractable solute than equivalent solvent extractions performed on higher plant or animal biomass.

[0107] In certain embodiments, cell aggregates, i.e., microbial biomass produced as described herein, are retained in a liquid suspension when solvent-extracted or, when dried, supplied to the solvent extraction process as loose forces having an open porous structure.

[0108] In certain embodiments, the extraction rate is increased by performing one or more stirs and / or increasing the temperature. Higher temperatures may result in higher solubility and / or a higher diffusion coefficient of the extractable material (e.g., lipids).

[0109] Water-free (absolutely) low-liphatic alcohols such as ethanol or isopropanol are fairly suitable solvents for lipids at high temperatures, but the solubility of oils in these solvents decreases significantly as the temperature drops. In certain embodiments, lipid extraction is carried out at high temperatures with one or more alcohols, including but not limited to ethanol, isopropanol, and / or methanol. In certain such embodiments, the lipid extract is cooled and lipid saturation occurs. In certain such embodiments, excess lipids are separated as a separate phase, which can be recovered by a solid-liquid separation process, including but not limited to centrifugation. In certain such embodiments, the solvent, i.e., alcohol(s), is reheated and sent back for solvent extraction.

[0110] When using a concentration gradient to move extractable substances from a solid, maintaining a high gradient can facilitate the extraction process. In certain embodiments, this effect is utilized by employing the principle of countercurrent multistage extraction. In certain embodiments, the solvent extraction process is divided into several contact stages. In certain embodiments, each stage includes mixing the solid, e.g., microbial biomass and / or protein concentrate, as well as the solvent phase, and separating the two flows after extraction. In certain embodiments, as the process moves from one stage to the next, the solid, e.g., microbial biomass and / or protein concentrate, and the solvent flow in opposite directions. Therefore, the microbial biomass and / or protein concentrate with the lowest extractable content (e.g., lipids) comes into contact with the most dilute solvent, resulting in a higher extractable yield (e.g., lipid yield) and higher propulsion throughout the extractor.

[0111] In certain embodiments, solvent extraction is carried out using batch, semi-continuous, and / or continuous solvent extractors.

[0112] In a batch process, a specific amount of microbial biomass and / or biological material comes into contact with a specific amount of fresh solvent. In certain embodiments, the extract is drained, distilled, and the solvent is recirculated through the extractor until the residual extractable content (e.g., lipid content) in the batch of microbial biomass and / or biological material is reduced to a target level.

[0113] In certain embodiments, a semi-continuous solvent extraction system consisting of several batch extractors connected in series is used. In certain such embodiments, the solvent and / or extract flows from one extractor to the next in a series. In certain non-limiting embodiments, a French stationary basket extractor is used.

[0114] In certain embodiments, a continuous solvent extraction process is utilized in which microbial biomass and / or biological material and / or protein concentrates and solvents are continuously supplied to an extractor. In certain embodiments, one or more of the following are used for solvent extraction: belt extractors, e.g., De Smet extractors, but not limited to these; mobile basket extractors, e.g., Lurgi mobile basket extractors or TOM (Turning Over of Material) HLS extractors, but not limited to these; and / or carousel extractors.

[0115] In certain embodiments, the protein concentrate produced as described in the present invention has a residual lipid content of about 0.6% by weight or less, about 0.25% to about 0.6% by weight, or about 0.25% by weight or less. In certain embodiments of the protein concentrate production described herein utilizing solvent extraction, the solvent loss per extraction is about 0.3% or less per extraction, or about 0.07% to about 0.3% per extraction, or about 0.07% or less per extraction.

[0116] In certain embodiments, at least two streams, such as an extract (e.g., lipid extract) stream and a solid (e.g., protein concentrate) stream, leave the solvent extraction step. In certain such embodiments, the solid stream contains solvent residue. In certain embodiments, one or more processes are used to remove and recover the solvent from one or the other, or both, streams.

[0117] In certain embodiments, the alcohol is removed from the liquid extract by evaporation and rectified by distillation. The alcohol is then brought to a suitable concentration for further extraction. In certain embodiments, the recovered solvent is recycled through an extractor. In certain embodiments, the distillation residue contains an aqueous solution containing lipids and / or nucleic acids, sugars, and / or other soluble substances. In certain embodiments, the aqueous residue is concentrated to approximately 50% total soluble solids. In certain embodiments, the lipids and / or aqueous residue are used as a caloric component and / or as a binder in animal feed. In certain embodiments, the lipids and / or aqueous residue are returned to a bioreactor. In certain embodiments, the lipids and / or aqueous residues are returned to a bioreactor where they can be used for mixed trophic growth. In certain such cases, mixed trophic growth includes, but is not limited to, growth on H2 and organic substrates, such as lipids and / or nucleic acids. In certain embodiments, the extract contains 30% or less lipids. In certain embodiments, for every ton of lipid recovered, approximately 2.5 tons of solvent are recovered by distillation. In certain embodiments, one or more methods of solvent removal are used, but are not limited to these, including flash evaporation, vacuum distillation, and / or vapor stripping.

[0118] In certain embodiments, the solvent is removed from the solid resulting from one or more solvent extraction steps. In certain embodiments, flash desolvation is used to remove solvent residue. In certain embodiments, superheated vapor of an alcohol-water mixture is applied to the protein concentrate produced as described herein. In certain embodiments, steam distillation is used to remove solvent residue or trace amounts of solvent from the solid resulting from the solvent extraction. In certain such embodiments, the solid recovered from the solvent extraction is used to produce a protein concentrate. In certain embodiments, desolvation of the solid recovered from the solvent extraction is carried out via flash desolvation (FD). In certain such embodiments, the solid with solvent residue coming out of the extractor is fluidized in a stream of superheated solvent vapor, where the superheating of the vapor provides energy to evaporate the solvent from the solid. In certain such embodiments, the turbulent nature of the solid-vapor flow facilitates rapid heat and mass transfer. In certain embodiments, a short stripping step is used for complete solvent removal. In certain embodiments, rapid cooling is performed after the removal of residual solvent. In certain embodiments, any excess water remaining in the protein concentrate after desolvation is removed by methods such as, but not limited to, hot air drying, drying under a thermally inert gas, vacuum drying, or freeze-drying.

[0119] In certain embodiments, an acid treatment or acid washing process is used to precipitate proteins from a solution, and the precipitated proteins are used to produce protein concentrates. In certain embodiments, the pH of the broth or biomass or biomass soluble product emerging from the bioreactor is approximately pH=7, or in the range of approximately pH6 to pH8. Proteins generally exhibit minimal water solubility in the pH range corresponding to their isoelectric point range. In certain embodiments, the isoelectric point range of proteins produced as described herein (e.g., proteins produced using a CO2 carbon source) occurs in the range of pH less than pH=6, or pH3 to pH6, pH4 to pH5, pH4.2 to pH4.5, or pH approximately 4, or around pH4.2 or around pH4.5. In certain embodiments, one or more acids, but not limited to phosphoric acid, sulfuric acid, hydrochloric acid, acetic acid, and / or carbonic acid / CO2 (aqueous solution), are used to lower the pH of the culture broth and / or biomass and / or biomass soluble product to the isoelectric point range. In certain embodiments, proteins are precipitated by lowering the pH to the isoelectric point range, and nucleic acids, sugars, and / or other soluble nonproteins are extracted using acid-added water as a solvent, so as to maintain the pH in the isoelectric point range. In certain embodiments, a rotary vacuum filter or decantation centrifuge is used for solid-liquid separation from the solid containing the precipitated proteins.

[0120] In certain embodiments, the dissolved or defatted microbial biomass produced as described herein is mixed with acidified water in a stirring vessel. In certain such embodiments, the slurry is then fed to a decanter centrifuge to separate the extracted solid from the extract. In certain such embodiments, the solid is continuously discharged from the decanter centrifuge. In certain such embodiments, the discharged solid has a dry matter content of about 10% by weight, about 20% by weight, about 30% by weight, or about 10% to about 30% by weight. In certain such embodiments, the solid thus recovered is dried to produce an isoelectric protein concentrate. In certain embodiments, the isoelectric solid cake is resuspended in water to neutralize the acidity, and in a second step of centrifugation, a neutral protein concentrate cake is produced. In certain such embodiments, the protein concentrate has a protein content of at least about 60% by weight, at least about 70% by weight, at least about 75% by weight, or at least about 80% by weight on a dry matter basis.

[0121] In certain embodiments, the protein solubility of the neutralization product, as indicated by the nitrogen solubility index (NSI), is at least about 40%, at least 50%, at least 60% NSI, or an NSI value greater than about 60%.

[0122] In certain embodiments, liquid extracts containing soluble components such as nucleic acids, sugars, and minerals, and protein fractions soluble at pH less than 6, pH 3 to pH 6, pH less than 5, pH about 4.5, or pH 4 to pH 5, are returned to the original bioreactor and / or another bioreactor for the production of mixed-trophic or heterotrophic microbial biomass. In certain such embodiments, additional proteinaceous biomass is generated.

[0123] In certain embodiments, a thermal denaturation and / or water extraction process is used to produce a protein concentrate. In certain embodiments, the protein produced as described herein is made insoluble by thermal denaturation using moist heat. In certain embodiments, the microbial biomass produced as described herein is heated in boiling water or in a pressure cooker or autoclave. In certain embodiments, the microbial biomass produced as described herein is subjected to continuous high-temperature, short-time moist heat treatment, for example, using an extruder-cooker. In certain embodiments, the heat treatment of the microbial biomass exposes the biomass to temperatures of at least about 90°C, at least about 100°C, at least about 110°C, at least about 120°C, at least about 130°C, or at least about 140°C, or temperatures of about 100°C to about 121°C, or up to 150°C. In certain embodiments, the duration of the heat treatment is at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 1 hour, at least about 3 hours, at least about 5 hours, or about 24 hours, or the duration is less than about 48 hours or less than about 72 hours. In certain embodiments, the heat-treated microbial biomass produced as described herein is extracted with hot water that dissolves nucleic acids, sugars, and / or other non-protein-soluble substances. In certain embodiments, after heat treatment and protein denaturation, solid-denatured proteins are separated from non-protein-soluble substances by utilizing a solid-liquid separation step well known in science for producing protein concentrates from soybeans and other plant-based protein sources. Examples of apparatus and processes that may be used in the present invention to separate protein-rich solids from protein-depleted liquids include, but are not limited to, one or more of rotary vacuum filters, decantation centrifuges, continuous centrifuges, and belt presses.

[0124] In certain embodiments, a protein-rich solid (i.e., cake) produced by one or more of the aforementioned processes, specifically solvent extraction, thermal denaturation, and acid / isoelectroprecipitation, is passed through an extruder. In certain such embodiments, the extruded material is cooled and then ground.

[0125] In certain embodiments, the protein-rich solid (i.e., cake) and / or extruded material, optionally produced by subsequent extrusion, by one or more of the aforementioned processes, specifically solvent extraction, thermal denaturation, and acid / isoelectric point precipitation, is dried using a drying process well established in the production of protein concentrates from soybeans and other plant-based sources. In certain embodiments, the protein-rich cake is wet-ground to obtain a fine slurry. In certain such embodiments, the slurry is spray-dried. In certain embodiments, the protein-rich cake or extruded material is freeze-dried. In certain embodiments, the protein-rich cake or extruded material is dried in a forced-circulation dryer. In certain embodiments, the protein-rich cake or extruded material is dried to a moisture content of about 10% or less.

[0126] In certain embodiments, the protein-rich cake or extruded material is ground into a fine powder. In certain such embodiments, at least about 97% of the ground product is passed through a standard 100-mesh screen. In other embodiments of the invention, the protein-rich cake or extruded material is converted into a grit form with coarser granulation. In certain embodiments, the protein-rich cake or extruded material is converted into powder or grit using one or more of the following grinders: a hammer mill, a pin mill, an impact turbo mill, and / or a similar grinder. In certain embodiments, about 3% or less of the ground product is retained by a 100-mesh screen. In certain embodiments, an air separation system is used to separate the fine product from the coarse fraction. In certain embodiments, the coarse fraction is recirculated back through the mill or grinder.

[0127] In certain embodiments, the final form of the protein concentrate resulting from the process described herein may be granular, flour-like, spray-dried, or textured.

[0128] In certain embodiments, the protein concentrate produced as described herein contains less than about 1% by weight of lipids. In certain embodiments, the protein concentrate produced as described herein contains less than about 20% by weight, less than about 15% by weight, less than about 10% by weight, less than about 5% by weight, or less than 1% by weight of lipids. In certain embodiments, the lipid content of the protein concentrate produced as described herein varies from about 1% by weight to about 10% by weight, or in certain embodiments, from about 4.5% to about 9% or from about 5% to about 6%.

[0129] In certain embodiments, a plant-based oil or fat is combined with a protein concentrate produced as described herein, and the lipid content of the combined protein concentrate and vegetable oil or fat formulation differs by about 4.5% to about 9% by weight, about 5% to about 6% by weight, about 9% to about 15% by weight, about 15% by weight, and about 20% by weight. In certain embodiments, the formulation of protein concentrate and vegetable oil or fat has a total lipid content of about 15% by weight.

[0130] In certain embodiments, but not limited to these, lecithin such as soy lecithin or egg lecithin is combined with the protein concentrate produced as described herein. In certain such embodiments, the addition of lecithin enhances the dispersibility and emulsifying properties of the protein concentrate. In certain such embodiments, the lecithin content of the formulation containing the protein concentrate and lecithin varies by up to about 15% by weight.

[0131] In certain embodiments, the oil and / or phospholipid content of the microbial biomass produced as described herein may have an egg and / or shortening-type effect and act as an emulsifier.

[0132] It is known that a high lipid content in protein concentrates can reduce their storage stability. In certain embodiments, low-lipid protein concentrates are produced with increased storage stability.

[0133] In certain embodiments, the protein concentrate produced as described herein may have an NSI of up to about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In certain embodiments, the protein concentrate produced as described herein may have an NSI of about 10% to about 20%, about 10% to about 30%, about 20% to about 30%, or about 10% to about 15%.

[0134] In certain embodiments, the dispersibility and functionality of the protein concentrate are enhanced by steam injection or jet cooking, and / or high-shear homogenization.

[0135] Protein hydrolysates In some embodiments, at least some, all, or substantially all of the protein products are produced by hydrolyzing a protein of at least one microorganism described herein (e.g., single-cell protein, cell lysate, protein concentrate, protein isolate, and / or protein extract). For example, hydrolysis of a cell protein may produce peptides, oligopeptides, and / or free amino acids.

[0136] The hydrolysis of microbial proteins can be carried out by acidic, basic, and / or enzymatic processes. Methods for hydrolyzing proteins are well known in the art. Non-limiting examples of methods and hydrolysis compositions for microbial proteins can be found in U.S. Provisional Applications 62 / 901,169 and 62 / 943,754, and PCT U.S. Application 20 / 50902, which are incorporated herein by reference in their entirety.

[0137] In some embodiments, the hydrolysis method may involve increasing or decreasing the pH of a protein suspension, such as a suspension of microbial biomass, thereby producing an alkaline or acidic suspension, respectively. The starting biomass suspension may contain a suitable amount of biomass in a liquid, such as 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%, for example, about 0.2% to about 8%, about 0.5% to about 6%, about 1% to about 6%, about 2% to about 6%, about 3% to about 5%, about 4% to about 8%, about 6% to about 8%, about 5% to about 7%, or about 5% to about 8%.

[0138] In some embodiments, microbial cells in the biomass are dissolved at the start of the process, for example, before increasing or decreasing the pH, which facilitates the recovery of proteins from the biomass into a suspension composition.

[0139] In certain embodiments, an alkaline or acidic suspension may be subjected to heat for an appropriate amount of time to produce a protein hydrolysis composition. The suspension can be concentrated, dried (e.g., lyophilized), or used directly as a liquid suspension. In certain embodiments, an alkaline or acidic suspension is subjected to heat and pressure, for example, by autoclaving the alkaline or acidic suspension, to produce a protein hydrolysis composition. In some embodiments, the suspension is neutralized with a buffer to lower or raise the pH after heating or heat / pressure treatment. In certain embodiments, the pH is lowered sufficiently (for alkaline suspensions) or raised (for acidic suspensions) to allow for subsequent enzymatic treatment of the suspension with a hydrolytic enzyme, such as a protease (e.g., alkaline protease, acidic protease, or metalloprotease). After enzymatic hydrolysis, a protein hydrolysis composition is produced. In other embodiments, the biomass suspension is hydrolyzed with a proteolytic enzyme, such as a protease (e.g., alkaline protease, acidic protease, or metalloprotease), without prior alkali or acid treatment.

[0140] In certain embodiments, the hydrolyzed protein in the protein hydrolysate is mainly located in the soluble fraction of the suspension. The resulting suspension can be clarified, for example, by centrifugation to obtain a supernatant fraction containing the hydrolyzed protein. In some embodiments, the suspension (hydrolysate) is clarified following the hydrolysis treatment (e.g., alkaline or acid hydrolysis, optionally enzymatic treatment (e.g., protease) or enzymatic hydrolysis alone) 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 or filtration. In some embodiments, after the suspension has been clarified, for example, after centrifugation, the supernatant can be separated from the pellet.

[0141] In some embodiments, a clarified liquid composition containing hydrolyzed protein (e.g., a soluble fraction such as the supernatant of a separated suspension) is dried, for example, lyophilized, to produce a dry composition or a substantially dry composition. In some embodiments, the lyophilized composition has a water content of about 10% or less, e.g., about 8% or less, about 6% or less, about 5% or less, or about 3% or less. In some embodiments, the lyophilized protein hydrolyzed composition has a water content of about 1% to about 10%, e.g., about 1% to about 8%, about 1% to about 6%, about 2% to about 5%, about 2% to about 6%, about 3% to about 5%, about 4% to about 8%, about 6% to about 8%, about 5% to about 7%, or about 5% to about 8%.

[0142] 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, for example, about 75% or less, about 50% or less, about 40% or less, or about 30% or less. In some embodiments, each of the aforementioned water content ranges may be at least about 20%, at least about 25%, at least about 30%, at least about 40%, or at least about 50% (up to the range where such aforementioned ranges exceed such lower limits). In some embodiments, the dehydrated product is dried using, for example, heat and / or evaporation, by one or more of the following methods: 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 in a rotary evaporator so that, for example, about 50% to about 65% or more of the water is removed. In some embodiments, further dehydration is achieved by freeze-drying, for example, so that the freeze-dried protein hydrolysis composition has a moisture content of about 1% to about 10%, for example, about 1% to about 8%, about 1% to about 6%, about 2% to about 5%, about 2% to about 6%, about 3% to about 5%, about 4% to about 8%, about 6% to about 8%, about 5% to about 7%, or about 5% to about 8%.

[0143] In some embodiments, at least some or all of the proteins from which the protein hydrolysates are produced (e.g., single-cell proteins, cell lysates, protein concentrates, protein isolates, and / or protein extracts) are derived from Cupriavidus microorganisms, for example, Cupriavidus necater, but not limited to these, e.g., DSM531 or DSM541. In some embodiments, the protein hydrolysis composition (e.g., comprising peptides, oligopeptides, and / or free amino acids) is derived from proteins of Cupriavidus microorganisms, for example, Cupriavidus necater, but not limited to these, e.g., DSM531 or DSM541.

[0144] In some embodiments, at least some, all, or substantially all of the protein from which the protein hydrolysate is produced (e.g., single-cell protein, cell lysate, protein concentrate, protein isolate, and / or protein extract) is derived from lactic acid bacteria, for example, but not limited to, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the protein hydrolysis composition (e.g., containing peptides, oligopeptides, and / or free amino acids) is derived from lactic acid bacteria, for example, but not limited to, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacteria are GRAS bacteria.

[0145] In some embodiments, at least some or all of the proteins from which protein hydrolysates are produced (e.g., single-cell proteins, cell lysates, protein concentrates, protein isolates, and / or protein extracts) are derived from Fusarium, Rhizopus, or Aspergillus fungal microorganisms, for example, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae. In some embodiments, the protein hydrolysis composition (e.g., comprising peptides, oligopeptides, and / or free amino acids) is derived from proteins from Fusarium, Rhizopus, or Aspergillus fungal microorganisms, for example, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae.

[0146] In some embodiments, the protein hydrolysates herein typically contain, or consist of, peptides within a size range that is non-allergenic, for example, non-allergenic to humans. In some embodiments, the protein hydrolysates incorporated into a food composition as described herein contain peptides and free amino acids, the peptides being within a size range that is typically non-allergenic. In some embodiments, the non-allergenic peptides are within a size range with an average molecular weight distribution of about 800 to about 1500 Da. For example, the peptides obtained by the protein hydrolysis described herein may have an average molecular weight smaller than any of the following: about 1500, 1400, 1300, 1200, 1100, 1000, 900, or 800 Da.

[0147] In some embodiments, salts are removed from the protein hydrolysate before incorporating the hydrolysate into the food composition as described herein (e.g., when an acid or alkaline salt is used for hydrolysis). For example, the protein hydrolysate can be purified by filtration (e.g., ultrafiltration) or dialysis to remove salts and / or other impurities.

[0148] microorganisms The proteinaceous materials used in this method and incorporated into the compositions described herein (protein products described herein) are derived from one or more microorganisms. The microorganisms from which single-cell proteins, cell lysates, protein concentrates, protein isolates, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof are derived may be photosynthetic autotrophs, heterotrophs, methanetrophs, methyltrophs, carboxytrophs, or chemoautotrophs. In some embodiments, the microorganisms include oxyhydrogen microorganisms. The microorganisms may be wild-type, genetically modified (e.g., recombinant), or a combination thereof.

[0149] Microbial biomass can be collected from a culture of one or more suitable microorganisms, for example, in a fermenter or bioreactor. The biomass can be collected using any suitable method, such as a centrifuge, to separate the cell aggregate from the culture medium. In some embodiments, the collected biomass can be used to produce protein hydrolysis compositions. In some embodiments, the collected biomass is spray-dried or freeze-dried to produce dried biomass, which can then be used as an ingredient for producing the food compositions described herein or for producing protein hydrolysis compositions. In some embodiments, protein products (e.g., single-cell proteins, cell lysates, protein extracts, protein-containing extracts, protein concentrates, protein isolates, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof) are produced from the collected biomass.

[0150] In some embodiments, the microorganism or its protein product comprises a strain within the genus Cupriavidus, Ralstonia, or Hydrogenobacteria. In some embodiments, the microorganism comprises the species Cupriavidus necater or Cupriavidus metallidurans. In some embodiments, the microorganism comprises a strain of the species Cupriavidus necater DSM531 or DSM541. In some embodiments, the microorganism comprises the species Cupriavidus metallidurans. In some embodiments, the microorganism comprises a strain of the species Cupriavidus metallidurans DSM2839.

[0151] In some embodiments, the microorganism or its protein product comprises a strain within the genus Xanthobacter. In some embodiments, the microorganism comprises Xanthobacter autotrophicus. In some embodiments, the microorganism comprises a strain of Xanthobacter autotrophicus species DSM 432.

[0152] In some embodiments, the microorganism or its protein product comprises Rhodococcus or Gordonia microorganisms. In some embodiments, the microorganism comprises Rhodococcus opacus. In some embodiments, the microorganism comprises Rhodococcus opacus (DSM43205) or Rhodococcus species (DSM3346). In some embodiments, the microorganism comprises Rhodococcus opacus; Hydrogenovibrio marinus; Rhodopseudomonas capsulata; Hydrogenobacter thermophilus; or Rhodobacter sphaeroides. In some embodiments, the microorganism comprises strains within the Burkholderiaceae family.

[0153] In some embodiments, the microorganism or its protein product may include, but is not limited to, lactic acid bacteria such as Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus. In some embodiments, the lactic acid bacteria are GRAS bacteria.

[0154] In some embodiments, the microorganisms or their protein products are Fusarium, Rhizopus, or Aspergillus fungal microorganisms, for example, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae. In some embodiments, the fungal microorganisms are GRAS microorganisms.

[0155] In some embodiments, a microbial consortium (i.e., two or more microorganisms grown together) is used as a source of protein products in the methods and compositions described herein. The consortium may comprise one or more microbial species or strains described herein, or one or more microorganisms having one or more microbial traits described herein. In some embodiments, the consortium may comprise two or more microbial species or strains described herein, or two or more microorganisms having one or more microbial traits described herein.

[0156] In some embodiments, the microorganisms described herein can accumulate protein up to about 50% by weight or more of the total cell mass. In some embodiments, the microorganisms described herein can accumulate protein up to about 60% by weight or more of the total cell mass. In some embodiments, the microorganisms can accumulate protein up to about 70% by weight or more of the total cell mass. In some embodiments, the microorganisms can accumulate protein up to about 80% by weight or more of the total cell mass. In some non-limiting embodiments, the microorganisms exhibiting these characteristics are Cupriavidus microorganisms, for example, Cupriavidus necatar, for example, Cupriavidus necatar DSM531 or DSM541.

[0157] In certain embodiments, the biomass produced (e.g., microbial cells) has a higher protein content and / or lower fat content than soybeans. In certain embodiments, the biomass produced (e.g., microbial cells) has a protein content higher than any of about 40% by weight, about 50% by weight, about 60% by weight, about 70% by weight, or about 80% by weight, and a fat content lower than any of about 20% by weight, about 15% by weight, about 10% by weight, or about 5% by weight. For example, biomass may contain approximately 40% or at least approximately 40% by weight of protein and approximately 20% or less than approximately 20% by weight of fat, or approximately 40% or at least approximately 40% by weight of protein and approximately 15% by weight of fat, or approximately 40% or at least approximately 40% by weight of protein and approximately 10% or less than approximately 10% by weight of fat, or approximately 40% or at least approximately 40% by weight of protein and approximately 5% or less than approximately 5% by weight of fat, or approximately 50% or at least approximately 50% by weight of protein and approximately 20% or less than approximately 20% by weight of fat, or approximately 50% or at least approximately 50% by weight of protein and approximately 15% by weight of fat, or approximately 50% or at least approximately 50% by weight of protein. Amount and fat content of approximately 10% by weight or less than approximately 10% by weight, or protein content of more than approximately 50% by weight or at least approximately 50% by weight and fat content of approximately 5% by weight or less than approximately 5% by weight, or protein content of more than approximately 60% by weight or at least approximately 60% by weight and fat content of approximately 20% by weight or less than approximately 20% by weight, or protein content of more than approximately 60% by weight or at least approximately 60% by weight and fat content of approximately 15% by weight or less than approximately 15% by weight, protein content of more than approximately 60% by weight or at least approximately 60% by weight and fat content of approximately 10% by weight or less than approximately 10% by weight, or protein content of more than approximately 60% by weight or at least approximately 60% by weight and fat content of approximately 5% by weight or less than approximately 5% by weight, or protein content of approximately 70% by weight or at least approximately 70% by weight and fat content of approximately 20% by weight or less than approximately 20% by weight,Alternatively, it may have a protein content of more than approximately 70% by weight or at least approximately 70% by weight and a fat content of less than approximately 15% by weight, or a protein content of more than approximately 70% by weight or at least approximately 70% by weight and a fat content of about 10% by weight or less than approximately 10% by weight, or a protein content of more than approximately 70% by weight or at least approximately 70% by weight and a fat content of about 5% by weight or less than approximately 5% by weight, or a protein content of more than approximately 80% by weight or at least approximately 80% by weight and a fat content of about 15% by weight or less than approximately 15% by weight, or a protein content of more than approximately 80% by weight or at least approximately 80% by weight and a fat content of about 10% by weight or less than approximately 10% by weight, or a protein content of more than approximately 80% by weight or at least approximately 80% by weight and a fat content of about 5% by weight or less than approximately 5% by weight. In some non-limiting embodiments, microorganisms exhibiting these characteristics are Cupriavidus microorganisms, such as Cupriavidus necata, for example, Cupriavidus necata DSM531 or DSM541.

[0158] In some embodiments, the microorganisms described herein can grow spontaneously on H2 / CO2 and / or synthesis gas and / or generator gas. In some embodiments, the microorganisms can spontaneously accumulate polyhydroxyalkanoates (PHAs) (e.g., polyhydroxybutyrates (PHBs)) up to about 50% by weight or more of their cellular biomass. In some embodiments, the microorganisms have a natural ability to induce high-flux carbon via acetyl-CoA metabolic intermediates, which can lead to fatty acid biosynthesis, along with several other synthetic pathways, e.g., PHA, e.g., PHB synthesis and / or amino acid biosynthesis. In some embodiments, microorganisms exhibiting these traits are Cupriavidus microorganisms, e.g., Cupriavidus necater, e.g., Cupriavidus necater DSM531 or DSM541. In some embodiments, the microorganisms do not produce and / or accumulate PHAs (e.g., PHBs).

[0159] In some embodiments, the microorganism or its protein product comprises a strain within Corynebacterium autotrophicum. In some non-limiting embodiments, the microorganism comprises Corynebacterium autotrophicum and / or Corynebacterium glutamicum. In some embodiments, the microorganism comprises Hydrogenovibrio marinus. In some embodiments, the microorganism comprises Rhodopseudomonas capsulata, Rhodopseudomonas palustris, or Rhodobacter sphaeroides.

[0160] In some embodiments, the microorganism or its protein product includes one or more of the following genera: Cupriavidus, Rhodococcus, Hydrogenovibrio, Rhodopseudomonas, Hydrogenobacter, Goldonia, Arthrobacter, Streptomyces, Rhodobacter, and / or Xanthobacter.

[0161] In some embodiments, the microorganism or its protein product includes microorganisms of the Actinomycete class. In some embodiments, the microorganism includes microorganisms of the Corynebacterium suborder (Corynebacterium, Gordoniaceae, Mycobacteriaceae, and Nocardiaceae). In some embodiments, the microorganism includes microorganisms of the Nocardiaceae family. In some embodiments, the microorganism includes microorganisms derived from one or more of the following classifications: Corynebacterium, Gordonia, Rhodococcus, Mycobacterium, and Tucumrella. In some embodiments, the microorganisms are Rhodococcus species such as Rhodococcus opacus, Rhodococcus aurantiacus, Rhodococcus baikonurensis, Rhodococcus boritolerans, Rhodococcus equi, Rhodococcus coprophilus, Rhodococcus corynebacterioides, Nocardia corynebacterioides (synonym: Nocardia corynebacterioides), and Rhodococcus erythropolis. Rhodococcus 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 rhodochrous; Rhodococcus rhodnii; (Synonym: Nocardia rhodnii); Rhodococcus ruber (Synonym: Streptothrix rubra); Rhodococcus genus RHA1; Rhodococcus triatmae (Rhodococcus Examples include *Rhodococcus triatomae*, *Rhodococcus tukisamuensis*, *Rhodococcus wratislaviensis* (synonyms: *Tsukamurella wratislaviensis*, *Rhodococcus yunnanensis*, or *Rhodococcus zopfii*). In some embodiments, the microorganisms include *Rhodococcus opacus* DSM43205 or DSM43206. In some embodiments, the microorganisms include *Rhodococcus* strain DSM3346.

[0162] In some embodiments, the microorganism or its protein product comprises a microorganism (e.g., any of the microbial genera or species described herein) that can grow spontaneously on H2 / CO2 and / or synthesis gas and / or generator gas, and can spontaneously accumulate lipids in at least about 10% by weight, about 20% by weight, about 30% by weight, about 40% by weight, about 50% by weight, about 60% by weight, about 70% by weight, about 80% by weight, or more of the cellular biomass. In some embodiments, the microorganism comprises a microorganism (e.g., any of the microbial genera or species described herein) that has the innate ability to deliver high-flux carbon to the fatty acid biosynthesis pathway. In some embodiments, the microorganisms exhibiting these characteristics are Rhodococcus microorganisms, such as Rhodococcus opacus (e.g., Rhodococcus opacus DSM43205 or DSM43206 or DSM44193), or Cupriavidus microorganisms, such as Cupriavidus necata (e.g., Cupriavidus necata DSM531 or DSM541).

[0163] In some embodiments, the microorganism or its protein product includes an oxyhydrogen strain or a hydrogen oxidation strain. In some embodiments, the microorganisms include one or more of the following hydrogen-oxidizing microorganisms: Aquifex pyrophilus, Aquifex aeolicus, other Aquifex species; Cupriavidus necator, Cupriavidus metallidurans, or other Cupriavidus species; Corynebacterium autotrophicum, or other Corynebacterium species; Gordonia desulfuricans, Gordonia polyisoprenivorans, Gordonia rubripertincta, Gordonia hydrophobica, Gordonia westphalica westfalica), or other species of Gordonia; Nocardia autotrophica, Nocardia opaca, or other species of Nocardia; purple non-sulfur phototrophic bacteria, for example, but not limited to these, Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophilus Rhodopseudomonas (acidophila), or other Rhodopseudomonas species; 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 pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophile, or other Pseudomonas species; Hydrogenomonas pantotropha, Hydrogenomonas 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 Hydrogenothermus marinus, or other Hydrogenothermus species;Helicobacter pylori or other Helicobacter species; Xanthobacter autotrophicus, Xanthobacter flavus or other Xantobacter species; Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava or other Hydrogenophaga species; Bradyrhizobium japonicum or other Bradyrhizobium species; Ralstonia eutropha or other Ralstonia species; Alcaligenes eutrophus, Alcaligenes fasciis 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, or other Arthrobacter species; Azospirillum lipoferum or Azospirillum species; Variovorax paradoxus or other Variovorax species; Acidovorax facilis (facilis), or other species of the genus Acidoboracus;Bacillus schlegelii, Bacillus tusciae, and other Bacillus genera; Calderobacterium hydrogenophilum or other Calderobacterium genera; Derxia gummosa or other Derxia genera; Flavobacterium autothermophilum or other Flavobacterium genera; Microcyclus aquaticus or other Microcyclus genera; Mycobacterium gordoniae or other Mycobacterium genera; Paracoccus denitrificans or other Paracoccus genera; Persephonella marina, Persephonella guaymasensis, or other Persephonella genera; Renobacter vacuolatum or other Renobacter species; Seliberia carboxydohydrogena or other Seliberia species; Streptomycetes coelicoflavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetes thermocarboxydus, or other Streptomycetes species; Thermoclinis ruber or other Thermoclinis species; Woutersia species; cyanobacteria, for example, but not limited to, Anabaena oscillarioides, Anabaena spiroide, Anabaena cylindrica, or other Anabaena species, and Arthrospira platensis, Arthrospira maxima, or other Arthrospira species;Green algae, for example, but not limited to, Scenedesmus obliquus or other species of Scenedesmus; Chlamydomonas reinhardii or other species of Chlamydomonas; species of Ankistrodesmus; and Rhaphidium polymorphium or other species of Rhaphidium. In some embodiments, a consortium of microorganisms including any of the above-mentioned acid-hydrogen microorganisms is used to produce the protein products described herein.

[0164] In some embodiments, the microorganism or its protein product comprises one or more of the following genera: Cupriavidus; Xanthobacter; Diesia; Goldonia; Mycobacterium; Nocardia; Pseudonocardia; Arthrobacter; Alcaniborax; Rhodococcus; Streptomyces; Rhodopseudomonas; Rhodobacter; and Acinetobacter; or a consortium of microorganisms comprising one or more of these genera.

[0165] In some embodiments, the microorganism or its protein product includes one or more of the following: Arthrobacter methylotrophus DSM14008; Rhodococcus opacus DSM44304; Rhodococcus opacus DSM44311; Xanthobacter autotrophicus DSM431; Rhodococcus opacus DSM44236; Rhodococcus ruber DSM43338; ​​Rhodococcus opacus DSM44315; Cupriavidus metallidurans metallidurans) DSM2839; Cupriavidus necator DSM531; Cupriavidus necator DSM541; Rhodococcus aetherivorans DSM44752; Gordonia desulfuricans DSM44462; Gordonia polyisoprenivorans DSM44266; Gordonia polyisoprenivorans DSM44439; Gordonia rubripertincta DSM46039; Rhodococcus percolatus DSM 44240; Rhodococcus opacus DSM43206; Gordonia hydrophobica DSM44015; Rhodococcus zopfii DSM44189;Gordonia westfalica DSM44215, Xanthobacter autotrophicus DSM1618; Xanthobacter autotrophicus DSM2267; Xanthobacter autotrophicus DSM3874; Streptomycetes coelicoflavus DSM41471; Streptomycetes griseus DSM40236; Streptomycetes species, DSM40434; Streptomycetes xanthochromogenes DSM40111; Streptomycetes thermocarboxydus (thermcarboxydus) DSM44293; Rhodobacter sphaeroides DSM158. In some embodiments, the microorganism or its protein product comprises 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.

[0166] It is characterized by several different microorganisms (i.e., carboxylotrophic microorganisms) that can grow on carbon monoxide as an electron donor and / or carbon source. In some cases, carboxylotrophic microorganisms can also use H2 as an electron donor and / or grow mixotrophically. In some cases, carboxylotrophic microorganisms are facultative chemosynthetic autotrophs [Biology of the Prokaryotes, edited by J Lengeler, G. Drews, H. Schlegel, John Wiley & Sons, Jul 10, 2009, the whole of which is incorporated herein by reference]. In some embodiments, the microorganism or its protein product includes one or more of the following carboxylotrophic microorganisms: Acinetobacter; Alcaligenes carboxydus or other Alcaligenes; Arthrobacter; Azomonas; Azotobacter; Bacillus schlegeli or other Bacillus; Hydrogenophaga pseudoflava or other Hydrogenophaga; Pseudomonas carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas comprancelis, Pseudomonas gazotropha, Pseudomonas thermocarboxydovorans, or other Pseudomonas; Rhizobium japonica Rhizobium japonicum or other Rhizobium species; and Streptomyces G26, Streptomyces thermoautotrophicus, and other Streptomyces species. In some embodiments, the microorganism or its protein product comprises a consortium of microorganisms including carboxylotrophic microorganisms such as one or more of the above-mentioned carboxylotrophic microorganisms. In certain embodiments, carboxylotrophic microorganisms capable of chemosynthetic autotrophy are used.In certain embodiments, carboxylotrophic microorganisms capable of utilizing H2 as an electron donor in respiration and / or biosynthesis are used.

[0167] In some embodiments, the microorganism or its protein product includes mandatory and / or facultative chemosynthetic autotrophic microorganisms such as one or more of the following: Acetanaerobium; Acetobacterium; Macetogenium; Achromobacter; Acidianus; Acinetobacter; Actinomajura; Aeromonas; Alcaligenes; Alcaliqenes; Aquaspirillum; Alcobacter; Aureobacterium; Bacillus; Begiatoa; Butyribacterium genus *Ribacterium*; *Carboxidothermus*; *Clostridium*; *Comamonas*; *Cupriavidus*; *Dehalobacter*; *Dehalococcoide*; *Dehalospirillum*; *Desulfobacterium*; *Desulfomonile*; *Desulfotomaculum*; *Desulfovibrio*; *Desulfosarcina*; *Ectothiorhodospira*; *Enterobacter*; *Eubacterium*; *Feroplasma*; *Halothibacillus*; *Hydrogenomonas*; *Leptospirum*; *Metalosphaerus* Ra genus; Metabacterium genus; Metanobrevibacter genus; Metanococcus genus; Metanococcoides genus; Metanogenium genus; Metanolobus genus; Metanomicrobium genus; Metanoplanus genus; Metanosarsina genus; Metanospirrhirum genus; Metanothermus genus; Metanotrix genus; Micrococcus genus; Nitrobacter genus; Nitrobacter genus; Nitrococcus genus; Nitrosococcus genus; Nitrospina genus; Nitrospira genus; Nitrosolobus genus; Nitrosomonas genus; Nitrosospira genus; Nitrosovibrio genus; Nitrospina genus; Oreomonas genus; Paracoccus; Peptostreptococcus; Planctomyces; Pseudomonas; Ralstonia; Rhodobacter; Rhodococcus; Rhodocyclus; Rhodomicrobium; Rhodopseudomonas; Rhodospiryllum; Shuwanella; Siderococcus; Streptomyces; Sulfobacillus; Sulfolobus; Thermotrix; Thiobacillus; Thiomiclospira; Thioproca; Thiosfera; Thiotrix; Thiobalaam; Sulfur oxidizers; Hydrogen oxidizers; Ionic oxidizers; Acetate-producing genera; and methane-producing genera;A consortium of microorganisms including chemoautotrophs; chemosynthetic autotrophs inhabiting at least one of hydrothermal vents, geothermal vents, hydrothermal springs, cold seeps, subsurface aquifers, salt lakes, salt layers, and soils; and extremophilic microorganisms selected from one or more of thermophiles, hyperthermophiles, acidophiles, halophiles, and psychrophiles. In some embodiments, the microorganism or its protein products include a consortium of microorganisms including chemosynthetic autotrophic microorganisms such as one or more of the chemosynthetic autotrophic microorganisms described above.

[0168] In some embodiments, microorganisms or their protein products include extremophiles that can withstand extreme environmental parameters such as temperature, radiation, pressure, gravity, vacuum, dryness, salinity, pH, oxygen tension, and / or chemicals. Such microorganisms include hyperthermophiles, e.g., Pyrolobus fumarii; thermophiles, e.g., Synechococcus lividis; mesophiles and psychrophiles, e.g., Cyclobacter and / or hyperthermophilic sulfur-metabolizing bacteria, e.g., Thermoproteus, Pyrodictium, Sulfolobus, and Asidianus; radiation-tolerant organisms, e.g., Deinococcus radiodurans; pressure-tolerant microorganisms, e.g., piezophiles or barophiles; desiccant-tolerant anhydrous organisms, e.g., xerophiles, e.g., Artemia salina. Examples include salina; microorganisms and fungi; salt-tolerant microorganisms, e.g., halophilic bacteria, e.g., halophilic archaea and Dunaliella salina; pH-tolerant microorganisms, e.g., alkaliphilic microorganisms, e.g., Natronobacterium, Bacillus falmas OF4, Spirulina, and acidophilic bacteria, e.g., Cyanidium caldarium and Ferroplasma; gas-tolerant microorganisms, e.g., tolerant to pure CO2, e.g., Cyanidium caldarium; and metal-tolerant microorganisms (metal-tolerant), e.g., Ferroplasma acidarmanus and Ralstonia.

[0169] In certain embodiments, the microorganism or its protein products include cell lines selected from eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, violet sulfur bacteria, violet non-sulfur bacteria, extremophiles, yeasts, fungi, and proteobacteria, their manipulated organisms, and synthetic organisms. In certain embodiments, spirulina is used.

[0170] In certain embodiments, the microorganism or its protein products include, but are not limited to, green non-sulfur bacteria such as the following genera: Chloroflex, Chloronema, Oscirochloris, Heliotrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.

[0171] In certain embodiments, the microorganism or its protein products include, but are not limited to, green sulfur bacteria such as: Chlorobium, Clathrochloris, and Prosthecochloris.

[0172] In certain embodiments, the microorganism or its protein products include, but are not limited to, purple sulfur bacteria such as: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodobrum, Thermochromatium, Thiocappa, Thiorhodococcus, and Thiocystis.

[0173] In certain embodiments, the microorganism or its protein products include, but are not limited to, purple nonsulfur bacteria such as the following genera: Phaeospirium, Rhodovaca, Rhodobacter, Rhodomicrobium, Rhodopira, Rhodopseudomonas, Rhodothalassium, Rhodospirylum, Rodovibrio, and Roseospira.

[0174] In some embodiments, the microorganism or its protein product is methane-nutrient and / or methyl-nutrient. In some embodiments, the microorganism belongs to the genus Methylococcus. In some embodiments, the microorganism is Methylococcus capsulatus. In some embodiments, the microorganism is methyl-nutrient. In some embodiments, the microorganism belongs to the genus Methylobacterium. In some embodiments, the microorganism includes one or more of the following species: Methylobacterium zatmanii; Methylobacterium extorquens; Methylobacterium chloromethanicum.

[0175] In some embodiments, the microorganism or its protein product is a hydrogen-oxidizing chemoautotroph and / or carboxylotrophic and / or methyllotrophic and / or methanelotrophic.

[0176] In certain embodiments, the microorganism or its protein products include microorganisms that can grow heterotrophically using polycarbonate organic molecules as a carbon source, such as, but not limited to, sugars, such as, but not limited to, glucose and / or fructose and / or sucrose. In some embodiments, the microorganism can grow on untreated 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 is capable of mixed trophic growth and can grow mixed trophically on, for example, an organic carbon source and an inorganic energy source (e.g., an inorganic electron donor).

[0177] In certain embodiments, the microorganism or its protein product may be one or more of the following: eukaryotic plants, algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, violet sulfur bacteria, violet non-sulfur bacteria, extremophiles, archaea, yeast, fungi, proteobacteria, their manipulated organisms, and synthetic organisms.

[0178] In some embodiments, the microorganisms include or consist of Gram-positive bacteria. In other embodiments, the microorganisms include or consist of Gram-negative bacteria.

[0179] In certain embodiments, the microorganism or its protein products include naturally occurring and / or non-genetically modified (non-GMO) microorganisms and / or are non-pathogenic and / or grow under specific environmental conditions provided by a bioprocess that is not present in the surrounding environment.

[0180] In certain embodiments, a microorganism or consortium of microorganisms is isolated from an environmental sample and enriched with the desired microorganism using methods known in the field of microbiology, such as growth in the presence of a targeted electron donor, including but not limited to: H2, CO, synthesis gas and / or methane, and / or electron acceptors such as O2, nitrates, ferric, and / or CO2, and / or environmental conditions (e.g., temperature, pH, pressure, dissolved oxygen (DO), salinity, presence of various impurities and contaminants).

[0181] In certain embodiments, the microorganism or consortium of microorganisms includes probiotic microorganisms. In certain embodiments, the microorganism or consortium of microorganisms includes "generally recognized as safe" (GRAS) microorganisms, such as GRAS microorganisms of bacteria and / or fungi. In certain embodiments, a microorganism or consortium of microorganisms includes, but is not limited to, yeasts, 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 fungi, but is not limited to, one or more of the following: Aspergillus oryzae; Aspergillus sojae; Fusarium venenatum A3 / 5 (Fusarium venenatum A3 / 5); Neurospora intermedia var.Bacteria such as: Bacillus oncomensis; Rhizopus oligosporus; Rhizopus oryzae; Aspergillus luchuensis; and / or one or more of the following: Bacillus amyloliquefaciens; Bacillus subtilis; Bifidobacterium animalis (lactis); Bifidobacterium bifidum; Bifidobacterium breve; Bifidobacterium longum; Lactobacillus acidophilus; Lactobacillus brevis Lactobacillus brevis; Lactobacillus casei; Lactobacillus delbrueckii subsp. bulgaricus.Bulgaricus); Lactobacillus fermentum; Lactobacillus helveticus; Lactobacillus kefiranofaciens; Lactobacillus lactis; Lactobacillus plantarum; Lactobacillus rhamnosus; Lactobacillus reuteri; Lactobacillus sakei; Lactobacillus sanfranciscensis; Lactococcus lactis (Streptococcus lactis) Examples include: lactis (Streptococcus lactis subsp. Diacetylactis), Leuconostoc, Leuconostoc carnosum, Leuconostoc cremoris, Leuconostoc mesenteroides, Pediococcus, Propionibacterium freudenreichii, Arthrospira (Spirulina) platensis, Streptococcus faecalis, and Streptococcus thermophilus.

[0182] Protein-containing biomass from which protein products are derived can be produced by a consortium of different microbial species. The consortium may optionally include multicellular organisms. In some embodiments, the consortium includes one or more of the following: oxyhydrogen microorganisms, carboxylotrophic; methanetrophic; methyltrophic; chemoautotrophic; photosynthetic autotrophic; and heterotrophic organisms.

[0183] In some embodiments, the protein product also contains one or more vitamins produced by the microorganism from which the protein product originates. In some non-limiting embodiments, the microorganism exhibiting these characteristics is a Cupriavidus microorganism, e.g., Cupriavidus necatar (e.g., Cupriavidus necatar DSM531 or DSM541). In some non-limiting embodiments, the vitamins are B vitamins, such as vitamins B1, B2, and / or B12, but are not limited to these. In non-limiting examples, vitamins B (e.g., B1, B2, and / or B12) may be produced by a Cupriavidus microorganism, e.g., Cupriavidus necatar (e.g., Cupriavidus necatar DSM531 or Cupriavidus necatar DSM541).

[0184] microbial culture Any suitable method can be used to culture microorganisms. Microorganisms can be grown under any suitable conditions in an environment suitable for biomass growth and production. In some embodiments, microorganisms can be grown under autotrophic culture conditions, heterotrophic culture conditions, or a combination of autotrophic and heterotrophic culture conditions. Heterotrophic cultures may contain one or more suitable carbon and energy sources such as sugars (e.g., glucose, fructose, sucrose, etc.). Autotrophic cultures may contain C1 chemicals such as carbon monoxide, carbon dioxide, methane, methanol, formate, and / or formic acid, and / or mixtures containing C1 chemicals such as various synthesis gas compositions or various generator gas compositions produced from low-value carbon and energy sources, for example, through gasification, partial oxidation, pyrolysis, or steam reforming of low-value carbon sources. Examples of low-value carbon and energy sources include, but are not limited to, lignocellulose energy crops, crop residues, bagasse, sawdust, forestry residues, or food. These can be used as oxygen and energy sources by oxyhydrogen microorganisms, hydrogen-oxidizing microorganisms, or carbon monoxide-oxidizing microorganisms. Suitable methods and apparatus for culturing microorganisms and producing biomass for use in the methods of the present invention are described, for example, in U.S. PCT applications 2010 / 001402, 2011 / 034218, 2013 / 032362, 2014 / 029916, 2017 / 023110, 2018 / 016779, and 9,157,058, each of which is incorporated herein by reference in whole. In some embodiments, the organisms can be grown by photosynthesis in a bioreactor, hydroponics system, greenhouse, or cultivated land, or they can be collected from waste or natural sources.

[0185] 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 quantities of the desired molecules and / or biomass can be produced using large-scale production in bioreactor vessels.

[0186] In certain embodiments, bioreactor vessels are 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, airlift reactors; biological scrubber columns; bubble columns; agitated tank reactors; continuous agitated tank reactors; counterflow, upward flow, and expanding bed reactors; digesters, in particular digester systems, e.g., those known in the field of bioremediation; filters, e.g., trickle filters, rotary biological contact filters, rotating disks, soil filters, but are not limited to these; fluidized bed reactors; gaslift fermenters; immobilized cell reactors; loop reactors; membrane biofilm reactors; packed bed reactors; packed bed reactors; plugged flow reactors; static mixers; trickle bed reactors; and / or vertical shaft bioreactors.

[0187] For example, microbial cultures intended for the commercial production of biomass and / or organic compounds such as protein products described herein, specifically single-cell proteins, cell lysates, protein extracts, protein-containing extracts, protein concentrates, protein isolates, protein hydrolysates, free amino acids, peptides, oligopeptides, or combinations thereof, and / or other nutrients, but not limited to vitamins (e.g., vitamin B, e.g., B1, B2, and / or B12), can be carried out in bioreactors on a large scale (e.g., bioreactor capacities of 500 L, 1,000 L, 5,000 L, 10,000 L, 50,000 L, 100,000 L, 1,000,000 L or more).

[0188] In certain embodiments, chemoautotrophic and / or heterotrophic and / or carboxydotrophic and / or methanetrophic and / or methyltrophic microorganisms are grown in a liquid medium in a bioreactor using the method described herein.

[0189] In some embodiments, bioreactors containing microorganisms are constructed of opaque materials that maintain the culture in near or complete darkness. Bioreactors constructed of opaque materials such as steel and / or other metal alloys and / or reinforced concrete and / or fiberglass and / or various high-strength plastic materials may be designed to have a large workload. In some embodiments, fermenters constructed of steel or other metal alloys with a volume of 50,000 liters or more are utilized. In some embodiments, bioreactors that can include upper space positive pressure exceeding ambient pressure are utilized. In some embodiments, oval or cylindrical digesters or vertical shaft bioreactors with a capacity of 3,000,000 liters or more are utilized. In some embodiments, in bioreactors containing microorganisms, light cannot pass through some, most or all of the liquid volume it contains. 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 restrict the culture to a thin layer, as is generally required in photosynthesis, or has transparent walls to allow light to be available in all parts. In some embodiments, microorganisms are cultured without significant or any exposure to light. In certain such embodiments, net CO2 consumption still occurs even in the absence of light due to chemosynthetic autotrophic metabolism and state. In certain embodiments, the conversion of electricity to artificial light is not required in the biological system for CO2 capture and conversion.

[0190] In certain embodiments, the non-light-dependent nature facilitates continuous CO2 capture operations year-round, day and night, under all weather conditions, without the need for any artificial lighting.

[0191] In some embodiments, microorganisms are grown and maintained in the absence of light in a culture medium containing a gaseous carbon source, such as, but not limited to, synthesis gas, generator gas, or a gas mixture containing H2 and CO2. Such growth is known as chemosynthetic autotrophic growth.

[0192] In some embodiments, for example, synthesis gas produced from the gasification of organic matter is utilized by microorganisms for chemosynthetic autotrophic growth. The organic matter may be, for example, from agricultural resources (e.g., corn stover, bagasse).

[0193] In some embodiments, air passing through a food-grade CO2 and / or direct air recovery system is utilized by microorganisms for chemosynthetic autotrophic growth. Non-limiting examples of direct air recovery can be found in U.S. Patent Publication No. 2017 / 0106330 and Keith, D., et al. (2018) Joule 2(8):1573-1594, which are incorporated herein by reference in their entirety. In some embodiments, CO2 is supplied from an industrial source and may optionally be concentrated via a gas separation procedure, thereby yielding high concentrations of food-grade CO2.

[0194] In certain embodiments, the increase in system capacity is achieved not only through horizontal expansion but also through vertical expansion. This is in contrast to photosynthetic approaches that use algae, cyanobacteria, or higher plants for CO2 capture. While various vertical farming schemes have been proposed for photosynthetic systems, practically and economically speaking, photosynthetic systems need to be expanded horizontally, for example, in shallow ponds or photobioreactors in the case of algae. As a result, the geographical footprint becomes large, and there are many negative impacts on the environment.

[0195] Algae or higher plant systems grown under artificial lighting present problems with inefficient use of light energy and inefficient conversion of electrical energy to light energy. In certain embodiments, equivalent algae or higher plant cultures grown under artificial lighting require more power with respect to CO2 capture and / or biomass production than the CO2 capture and / or biomass production systems described herein. In certain embodiments, equivalent algae or higher plant cultures grown under artificial lighting require at least 10 times more power with respect to power per unit of CO2 capture and / or biomass production than the CO2 capture and / or biomass production systems described herein. For algae or higher plants grown under artificial lighting, the heat removal requirement is approximately directly proportional to the electrical input. In certain embodiments of the methods described herein, the heat removal requirement is lower with respect to CO2 capture and / or biomass production than equivalent algae or higher plant systems grown under artificial lighting. In certain embodiments, the heat removal requirements are at least 10 times lower than those of comparable algae or higher plant systems with respect to CO2 capture and / or biomass production when grown under artificial lighting.

[0196] In exemplary but non-limiting embodiments, producing cells are inoculated into a bioreactor containing a nutrient medium. Generally, a delay phase follows before the cells begin to double. After the delay phase, the cell doubling time shortens, and the culture enters the logarithmic phase. Eventually, the doubling time lengthens after the logarithmic phase, which is not intended to be limited by theory, but is thought to be due to either limited mass transfer, depletion of nutrients such as nitrogen or mineral sources, increased concentrations of inhibitory chemicals, or quorum sensing by microorganisms. Once the culture enters the stationary phase, growth slows and then stops. In certain embodiments, there is a equivalent growth phase preceding the stationary phase. To harvest cell aggregates, cultures in certain embodiments are harvested during the logarithmic phase and / or the arithmetic phase and / or stationary phase.

[0197] Bioreactors or fermenters are used to culture cells through various stages of the physiological cycle. Bioreactors are utilized for cell culture and can be maintained at specific stages of the cell growth curve. The use of bioreactors is advantageous in many ways for culturing chemosynthetic autotrophic growth. In certain embodiments, protein-rich cell masses used to produce proteins or protein hydrolysates are grown in a liquid suspension until dense. Generally, control of growth conditions, such as other gases like dissolved carbon dioxide, oxygen, and hydrogen, as well as other dissolved nutrients, trace elements, temperature, and pH, is facilitated within the bioreactor. In certain embodiments, protein-rich cell masses used to produce amino acids, peptides, proteins, hydrolysates, extracts, or whole cell products are grown in a liquid suspension within a bioreactor until dense and / or grown with high productivity.

[0198] Nutrient media and gases can be added to the bioreactor as batch additions, periodically, in response to detected depletion or programmed setpoints, or continuously over the period during which the culture grows and / or is maintained. In certain embodiments, the bioreactor at inoculation is filled with an initial batch of nutrient media and / or one or more gases at the start of growth, and no additional nutrient media and / or one or more gases are added after inoculation. In certain embodiments, nutrient media and / or one or more gases are added periodically after inoculation. In certain embodiments, nutrient media and / or one or more gases are added after inoculation in response to detected depletion of nutrients and / or gases. In certain embodiments, nutrient media and / or one or more gases are added continuously after inoculation.

[0199] In certain embodiments, the added nutrient medium does not contain any organic compounds.

[0200] In certain embodiments, a small amount of microbial cells (i.e., an inoculum) is added to a set amount of culture medium. The culture is then incubated, and the cell mass goes through a slowing phase, an exponential phase, a slowing phase, and a stationary phase of growth.

[0201] In batch culture systems, the conditions under which microorganisms are cultured (e.g., nutrient concentration, pH, etc.) generally change continuously throughout the entire growth period. In certain non-limiting embodiments, to avoid the variability inherent in batch culture and to improve the overall productivity of the culture system, microorganisms used for protein and / or vitamin and / or other nutrient production are grown in a continuous culture system called a chemostat. In such a system, the culture can be maintained in a persistent exponential growth phase by maintaining a constant culture volume [V] while simultaneously supplying fresh medium at a constant rate [F]. In certain embodiments, the continuous culture system ensures that cells are cultured under environmental conditions that remain nearly constant. In certain embodiments, cells are maintained in a persistent exponential phase by using a chemostat system. In some cases, the culture is maintained in a steady state, and a nearly constant amount of existing biomass is maintained in a bioreactor for a long period. In such cases, the dilution ratio (D) of the culture is equal to the growth rate of the microorganism and is obtained by the following equation: D = F / V. The growth rate of microorganisms in continuous culture can be changed by changing the dilution ratio. In certain embodiments, the growth rate of microorganisms is altered by changing the dilution ratio. In certain non-limiting embodiments, cells grow at a dilution ratio of approximately 0.2h -1 It grows within the chemostat.

[0202] In certain embodiments, the continuous bioreactor is maintained as a turbidstat, a certain amount of existing biomass is maintained within the bioreactor over a long period, and all surplus biomass produced in excess of the amount necessary to maintain the certain amount of existing biomass within the bioreactor is continuously recovered from the bioreactor.

[0203] In certain embodiments, inoculation of cultures into a bioreactor is carried out by methods such as transferring cultures from existing cultures growing in another bioreactor, or incubation from seed stocks grown in an incubator, but not limited to these. In certain embodiments, seed stocks of strains may be transported and stored in forms such as powder, liquid, frozen, or freeze-dried, and any other suitable form, but not limited to these, which will be readily apparent to those skilled in the art. In certain non-limiting embodiments, preliminary bacterial cultures are maintained in a metabolically inactive freeze-dried state until reactivation is required. In certain embodiments, when establishing cultures in a very large reactor, cultures are grown and established in progressively larger medium-sized containers before inoculation into full-scale containers.

[0204] In certain embodiments, the bioreactor has a mechanism that allows for the mixing of the nutrient medium, including, but not limited to, one or more of the following: rotation of a stirring rod, blade, impeller, or turbine; spinning, shaking, or turning of the 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 medium can be mixed continuously or intermittently.

[0205] In certain embodiments, the microbial nutrient medium may 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 the exponential growth phase and / or another target growth phase (e.g., arithmetic growth), and / or to replenish depleted nutrients in the growth medium and / or to remove inhibitory waste products.

[0206] Standard ports in a bioreactor can be used to deliver or recover gases, liquids, solids, and / or slurries to and from the bioreactor vessel containing microorganisms. Many bioreactors have multiple ports for various purposes (e.g., ports for medium addition, gas addition, pH and DO probes, and sampling), and specific ports may be used for various purposes in the process of carrying out fermentation. For example, a port may be used at one point to add nutrient medium to the bioreactor and at another point to sample. Preferably, multiple uses of sampling ports can be carried out without introducing contaminants or invasive species into the growth environment. Sampling ports may be equipped with valves or other actuators that allow control of sample flow or continuous sampling. In certain embodiments, the bioreactor has at least one port suitable for culture inoculation, which may be further useful for other applications, such as the addition of medium or gas. Bioreactor ports allow control of the gas composition and flow rate to the culture environment. For example, a port can be used as a gas inlet to the bioreactor into which the gas is pumped.

[0207] In some embodiments, gases that can be pumped into the bioreactor include, but are not limited to, synthesis gas, generator gas, hydrogen gas, CO, CO2, O2, air, air / CO2 mixtures, natural gas, methane, ammonia, nitrogen, argon and other noble gases, and one or more other gases. In some embodiments, the CO2 pumped into the system may originate from, but is not limited to, CO2 from the gasification of organic matter; CO2 from the calcination of limestone CaCO3 to produce quicklime CaO; CO2 from methane steam reforming, such as CO2 byproducts from ammonia, methanol, or hydrogen production; CO2 from combustion, incineration, or flaring; CO2 byproducts from anaerobic or aerobic fermentation of sugar; CO2 byproducts from methane nutrient bioprocesses; geologically or geothermally generated or released CO2; and CO2 removed from acidic gases or natural gas. In certain non-limiting embodiments, the CO2 is removed from industrial flue gases or is isolated from geological sources that would otherwise be released naturally into the atmosphere. In certain embodiments, the carbon source is CO2 and / or bicarbonates and / or carbonates dissolved in seawater or other surface or groundwater. In certain such embodiments, inorganic carbon may be introduced into the bioreactor dissolved in liquid water and / or as a solid. In certain embodiments, the carbon source is CO2 captured from the atmosphere. In certain non-limiting embodiments, CO2 is captured from a closed cabin as part of a closed-loop life support system using equipment such as a CO2 removal assembly (CDRA), which is used, for example, on the International Space Station (ISS).

[0208] In certain non-limiting embodiments, but not limited to these, high-concentration energy sources (e.g., H2, H2S, CO gas) and / or carbon sources (e.g., CO2, HCO3) may be used. - CO3 2- Geological features such as geothermal vents and / or hydrothermal vents that release, and / or other dissolved minerals can be utilized as nutrient sources for the microorganisms described herein.

[0209] In certain embodiments, in addition to carbon dioxide, or as an alternative carbon source, one or more gases are dissolved in a solution and supplied to the culture broth, and / or dissolved directly in the culture broth. These include, but are not limited to, gaseous electron donors and / or carbon sources (e.g., hydrogen and / or CO and / or methane gas). In certain embodiments, the input gas may include other electron donors and / or electron acceptors and / or carbon sources and / or other gaseous components and synthesis gases (e.g., hydrocarbons); ammonia; hydrogen sulfide; and / or other sour gases; and / or O2; and / or mineral nutrients such as mineral-containing particulate matter and ash impurities.

[0210] In certain embodiments, one or more gases are dissolved in the culture broth, including, but not limited to, one or more gaseous electron donors such as hydrogen, carbon monoxide, methane, hydrogen sulfide, or other sour gases; a gaseous carbon source such as, but not limited to, one or more CO2, CO, CH4; and an electron acceptor such as, but not limited to, oxygen in the air (e.g., 20.9% oxygen) or as pure O2, or as an O2-rich gas. In some embodiments, the dissolution of these and other gases into the solution is achieved using a system of compressors, flow meters, and flow valves known to those skilled in the art of fermentation engineering, which supplies the gases into the solution to one or more of the widely used systems (dispersing devices; diffusers having dome, tubular, disc, or donut shapes, but not limited to these; coarse-bubble or fine-bubble aeration devices; venturi devices). In certain embodiments, surface aeration and / or gaseous mass transfer may also be carried out using paddle aeration devices, etc. In certain embodiments, gas dissolution is enhanced by mechanical mixing with an impeller or turbine, as well as by a fluid shear device to reduce bubble size. After passing through a reactor system that holds the gas-taking microorganisms, in certain embodiments, the residual gas may be recirculated back to a bioreactor, burned for process heat, flared, injected underground, or released into the atmosphere. In certain embodiments of this specification that utilize H2 as an electron donor, H2 may 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 is in contact with the liquid culture medium.

[0211] In certain embodiments, microorganisms grow and proliferate under microaerophilic conditions on H2 and CO2 and other dissolved nutrients. In certain embodiments, mixtures containing C1 chemicals such as carbon monoxide, methane, methanol, formate, or formic acid, and / or not limited to, various synthesis gas compositions produced from fixed carbon feedstocks that have been gasified, pyrolyzed, or steam-reformed, are biochemically converted into long-chain organic chemicals (i.e., carbon chain molecules of C2 or greater, and in some embodiments, C5 or greater) under one or more of the following conditions: aerobic, microaerophilic, anaerobic, anaerobic, and / or facultative conditions.

[0212] In some embodiments of the culture broth, a controlled amount of oxygen can also be maintained, and in certain embodiments, oxygen is actively dissolved in the solution supplied to the culture broth and / or directly dissolved in the culture broth. In certain aerobic or microaerophilic embodiments where it is necessary to pump air or oxygen into the culture broth to maintain a target DO level, oxygen bubbles can be injected into the broth with a diameter optimized for mixing and oxygen transport. In some embodiments, conditions suitable for the growth of oxyhydrogen microorganisms are developed, such as the use of H2 and O2 gaseous substrates (electron donors and acceptors), and optionally C1 gaseous carbon sources such as CO2 and / or CO.

[0213] In some embodiments, the microorganisms convert fuel gases such as synthesis gas, generator gas, CO, CO2, H2, natural gas, methane, and mixtures thereof, but are not limited to these. In some embodiments, the thermal content of the fuel gas is at least 100 BTU per standard cubic foot (scf). In some embodiments, the bioreactor used to house and grow the microorganisms comprises a microbubble diffuser and / or a high-shear impeller for gas delivery.

[0214] Introducing and / or increasing the gas flow rate into the bioreactor improves the mixing of the culture, and turbulence can occur if the gas inlet is located below the surface of the liquid medium and the gas bubbles or sprays through the medium. In certain embodiments, mixing is improved by turbulence caused by the gas bubbling and / or spraying and / or blocking the liquid medium with gas. In some embodiments, the bioreactor is provided with a gas outlet port for gas venting and pressure relief. In some embodiments, the gas inlet and outlet are preferably provided with check valves to prevent gas backflow.

[0215] In specific embodiments where chemosynthetic reactions occur within a bioreactor, one or more types of electron donors and one or more types of electron acceptors are added as a bolus, or periodically or continuously, by pressurization or other means to a nutrient medium containing chemosynthetic autotrophs within the reaction vessel. Chemosynthetic reactions, driven by electron transfer from electron donors to electron acceptors in cellular respiration, fix inorganic carbon dioxide and / or other dissolved carbonates and / or other carbon oxides to organic compounds and biomass.

[0216] In certain embodiments, nutrient media are used for the growth and production of cultures, comprising aqueous solutions containing appropriate minerals, salts, vitamins, cofactors, buffers, and other components necessary for microbial growth, as is 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)].

[0217] In certain embodiments, the chemicals used for maintaining and growing microbial cultures, as known in the art, are contained in the nutrient medium. In certain embodiments, these chemicals may include, but are not limited to, one or more of the following: nitrogen sources, e.g., ammonia, ammonium (e.g., ammonium chloride (NH4Cl), ammonium sulfate ((NH4)2SO4)), nitrates (e.g., potassium nitrate (KNO3)), urea, or organic nitrogen sources; phosphoric acid (e.g., disodium phosphate (Na2HPO4), potassium phosphate (KH2PO4), phosphoric acid (H3PO4), potassium dithiophosphate (K3PS2O2), potassium orthophosphate (K3PO4)), dipotassium phosphate (K2HPO4)); sulfates; yeast extracts; 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 (MgSO47H2O) or magnesium chloride (MgCl2), calcium chloride (Ca Cl2) or calcium carbonate (CaCO3), manganese sulfate (MnSO47H2O) or manganese chloride (MnCl2), ferric chloride (FeCl3), ferrous sulfate (FeSO47H2O) or ferrous chloride (FeCl24H2O), sodium bicarbonate (NaHCO3) or sodium carbonate (Na2CO3), zinc sulfate (ZnSO4) or zinc chloride (ZnCl2), ammonium molybdate (NH4MoO4) or sodium molybdate (Na2MoO42H2O) (2O), copper sulfate (CuSO4) or copper chloride (CuCl22H2O), cobalt chloride (CoCl26H2O), aluminum chloride (AlCl3.6H2O), lithium chloride (LiCl), boric acid (H3BO3), nickel chloride (NiCl26H2O), tin chloride (SnCl2H2O), barium chloride (BaCl22H2O), copper selenite (CuSeO45H2O) or sodium selenite (Na2SeO3), sodium metavanadate (NaVO3), chromium salts).In certain embodiments, mineral salt medium (MSM) formulated by Schlegel et al. can be used ["Thermophilic bacteria", Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0218] The microorganisms described herein can be cultured in any type (rich or minimal) of medium, including fermentation medium, and in any composition, in some embodiments. As will be understood by those skilled in the art, routine optimization will allow for the use of various types of medium. Various additional components can be supplemented to the selected medium. Some non-limiting examples of supplement components include glucose, fructose, sucrose, starch, polysaccharides, protein hydrolysates, antibiotics, IPTG for gene induction, and ATCC trace mineral supplements. Similarly, other aspects of the medium and growth conditions for 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 the selection of medium, medium supplements, and temperature may affect the production level of the desired molecule. In some embodiments, the concentration and amount of supplement components can be optimized. In some embodiments, the frequency with which one or more supplement components are added to the medium and the amount of time the medium is cultured before obtaining the desired molecule can be optimized.

[0219] In certain embodiments, the concentrations of nutritional chemicals (e.g., electron donors, electron acceptors, carbon sources, and / or various mineral nutrients) are maintained in the bioreactor at or near the optimal levels of carbon uptake and / or fixation and / or conversion and / or production of biomass and / or organic compounds, particularly proteins, which vary depending on the microorganisms utilized but can be routinely determined and / or optimized by one of the techniques of those skilled in the art for culturing microorganisms.

[0220] In certain embodiments, one or more of the following parameters: pH; temperature; salinity; dissolved oxygen; dissolved carbon dioxide gas; liquid flow rate; stirring speed; and gas pressure are monitored and / or controlled in the bioreactor. In certain embodiments, operating parameters affecting chemoautotrophic growth and / or other types of growth (e.g., heterotrophic growth) are monitored by sensors (e.g., dissolved oxygen probes or redox probes for measuring electron donor / receptor concentrations) and / or controlled manually or automatically based on feedback from the sensors via the use of equipment such as operating valves, pumps, and stirrers. In certain embodiments, the temperature of the incoming broth and incoming gas is regulated by systems such as coolers, heaters, and / or heat exchangers, but is not limited to these.

[0221] In certain embodiments, microbial culture and biological reactions are maintained over time at a constant level, targeting a steady state of cell population and environmental parameters (e.g., cell density, pH, DO, chemical concentration) using continuous inflow and removal of nutrient medium and / or biomass. In certain embodiments, the constant level is the optimal level for raw material conversion and / or production of target organic compounds. In certain embodiments, the targeted organic compounds include proteins and / or amino acids. In certain embodiments, cell density can be monitored by direct sampling, by correlation between optical density and cell density, and / or using a particle size analyzer. In certain embodiments, fluid retention time and biomass retention time can be separated to allow independent control of both broth chemistry and cell density. In certain embodiments, the dilution ratio can be kept sufficiently high so that the fluid retention time is relatively short compared to the biomass retention time, resulting in a highly replenished broth for cell growth and / or raw material conversion and / or production of organic compounds. In certain embodiments, the dilution ratio is set to the optimal technoeconomic trade-off between supplementing culture broth and nutrients and / or removing waste, and to accommodate the increased process costs due to pumping, increased input, and other demands that rise with the dilution ratio.

[0222] In certain embodiments, the pH of the microbial culture is controlled. In certain embodiments, the pH is controlled to be within an optimal range for the maintenance and / or growth of the microorganisms and / or the conversion of raw materials and / or the production and / or survival of organic compounds. To address a decrease in pH, in certain embodiments, the neutralization step may be performed directly in the bioreactor environment or before recirculating the medium back into the culture vessel via a recirculation loop. Neutralization of acids in the broth in certain embodiments can be achieved by adding a base, including, but is not limited to, limestone, lime, sodium hydroxide, ammonia, ammonium hydroxide, potassium hydroxide, magnesium oxide, iron oxide, and alkali ash, one or more of these.

[0223] In certain embodiments, an aqueous suspension of chemosynthetic autotrophic microorganisms converts one or more electron donors and CO2 into protoplasm. In certain embodiments, the protoplasm includes proteins, peptides, and / or amino acids. 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, 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 chemosynthetic autotrophic microorganisms converts H2 and CO2, along with several other dissolved mineral nutrients, into biochemicals and protoplasm. In certain embodiments, the biochemicals and / or protoplasm include proteins, peptides, and / or amino acids. In certain embodiments, other dissolved mineral nutrients include, but are not limited to, sources of nitrogen, phosphorus, and potassium. In certain embodiments, the protoplasm produced is of food value to 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 which have nutritional value and / or value in various organic chemical or fuel applications. In certain embodiments, the intracellular energy to drive this production of protoplasm derives from the oxidation of electron donors by electron acceptors. In certain non-limiting embodiments, electron donors include, but are not limited to, one or more of H2;CO;CH4. In certain non-limiting embodiments, electron acceptors include, but are not limited to, O2 and / or CO2. In certain non-limiting embodiments, the products of the energy production reaction or respiration include, but are not limited to, water. In certain embodiments, the intracellular energy derived from respiration used to drive this synthesis of biochemicals and protoplasm from CO2 is stored and transported in, but is not limited to, biochemical molecules such as ATP.In the case of the hydrogen-oxidizing microorganisms used in the specific embodiments of this specification, the electron acceptor is O2 and the product of respiration is water.

[0224] In some embodiments, protein production and / or the distribution of the produced amino acid molecules are optimized through one or more of the following: control of bioreactor conditions, control of nutrient levels, and / or genetic modification of cells. In certain embodiments, pathways to amino acids, or proteins, or other nutrients, or whole cell products are controlled and optimized to produce chemical products by maintaining specific growth conditions (e.g., levels of electron donors, 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) may be optimized by maintaining the broth under aerobic, microaerobic, anaerobic, or facultative conditions, depending on the requirements of the microorganism. A facultative environment is considered to be an environment having an aerobic upper layer and an anaerobic lower layer resulting from stratification of the water column. The biosynthesis of amino acids, proteins, other nutrients, or whole cell products by microorganisms disclosed herein may occur during the logarithmic phase, the arithmetic phase, or the subsequent stationary phase when cell doubling has ceased, provided that there is a sufficient supply of carbon, energy, and other nutrients.

[0225] In some embodiments, the microbial growth medium described herein may include proteins and / or nutrients from another microorganism (e.g., cell lysates, protein hydrolysates, peptides, oligopeptides, and / or amino acids, and / or organic molecules and / or other nutrients from different microorganisms). In some embodiments, the microorganisms in the growth medium are GRAS microorganisms. In one embodiment, the growth medium for lactic acid bacteria, such as, but not limited to, Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus bacteria (e.g., GRAS lactic acid bacteria such as GRAS Lactococcus, Lactobacillus, Enterococcus, Streptococcus, or Pediococcus bacteria), may include cell lysates, protein hydrolysates, peptides, oligopeptides, and / or amino acids, and / or organic molecules, and / or other nutrients of different microorganisms (but not limited to, Cupriavidus microorganisms, e.g., Cupriavidus necata, e.g., Cupriavidus necata DSM531 or DSM541). In another embodiment, the growth medium for fungal microorganisms, for example, Fusarium or Rhizopus or Aspergillus fungal microorganisms (e.g., GRAS fungal microorganisms, for example, GRAS Fusarium or Rhizopus or Aspergillus fungal microorganisms), for example, but not limited to Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae, may include whole cellular biomass, cell lysates, protein hydrolysates, peptides, oligopeptides, and / or amino acids, and / or organic molecules, and / or other nutrients from different microorganisms, for example, but not limited to Cupriavidus microorganisms, for example, Cupriavidus necata, for example, Cupriavidus necata DSM531 or DSM541.

[0226] In some embodiments, fungal microorganisms capable of lysing bacterial cells and / or hydrolyzing bacterial proteins are cultured in the presence of such bacterial cells or nutrients derived from such bacterial cells. For example, bacterial biomass may be isolated, optionally dehydrated, or optionally inactivated, and then fungal microorganisms or fungal microorganisms inoculated into the bacterial biomass may be cultured in the presence of bacterial biomass and / or bacterial-derived nutrients in the growth media described herein. In certain non-limiting embodiments, fungal microorganisms include Fusarium, Rhizopus, or Aspergillus microorganisms, for example, but not limited to, Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae. In certain non-limiting embodiments, the edible fungal species Agaricus bisporus is cultured on a medium containing cells and / or nutrients rich in the proteins produced according to the present invention. In certain embodiments, Agaricus bisporus is lysed of the cells produced according to the present invention. In certain such embodiments, Agaricus bisporus utilizes the proteins, amino acids, and / or other nutrients released by the lysing of the cells for nutrition and growth.

[0227] Bioreactors, culture conditions, heterotrophic and chemotrophic growth, maintenance, and specific examples of methods for producing amino acids, or proteins, or other nutrients, or whole cell products as described herein can be combined in any appropriate manner to improve the efficiency of microbial growth and amino acid, or protein, or other nutrient, or whole cell production.

[0228] Electron donors and receptors In certain non-limiting embodiments, the microorganisms described herein grow chemosynthetically and autotrophically. For example, microbial growth can utilize the biosynthesis and reduction of CO2 using an O2 electron acceptor and / or an H2 electron donor. In certain embodiments, O2 and H2 are produced by the electrolysis of water. In certain non-limiting embodiments, some of the O2 and all of the H2 produced by the electrolysis of water are supplied to an aqueous suspension of the microorganisms described herein. In certain non-limiting embodiments, the molar ratio of H2 to O2 supplied to the aqueous suspension of the microorganisms is greater than 2:1. In certain non-limiting embodiments where the O2 electron acceptor and H2 electron donor are produced by the electrolysis of water, there is a surplus of O2 remaining after all of the H2 and O2 metabolic requirements of the microorganisms described herein have been met. In certain such embodiments, the surplus O2 may be supplied to a hydroponic system for aeration of humans and / or other aerobic organisms and / or roots, and / or used in gasification, partial oxidation, or combustion processes, and / or stored and sold as a chemical byproduct.

[0229] In certain embodiments utilizing hydrogen molecules as electron donors, chemical byproducts may be formed in the production of hydrogen molecules using renewable and / or CO2-free energy input. In certain embodiments, the oxyhydrogen reaction used in respiration is enzymatically related to oxidative phosphorylation. In certain embodiments, the ATP and / or other intracellular energy carriers thus formed are utilized for the assimilation and synthesis of amino acids and / or proteins. In certain embodiments, oxygen produced by water splitting in excess of what is required for respiration to maintain optimal conditions for carbon fixation and hydrogen-oxidizing microbial organic compound production can be processed into a form suitable for sale by process steps known in the field for commercial oxygen gas production.

[0230] Certain embodiments apply to hydrogen-oxidizing and / or CO-oxidizing and / or CH4-oxidizing microorganisms, which, while not limited to these, use more electronegative electron acceptors than CO2 in energy conservation reactions for ATP production (e.g., respiration), such as O2. For example, hydrogen-oxidizing microorganisms that combine the hydrogenotrophic oxyhydrogen or oxyhydrogen reaction 2H2 + O2 -> 2H2O for ATP production can produce more ATP per H2 and / or other electron donors consumed for respiration than acetic acid production or methane production, which use CO2 as the electron acceptor in respiration. For example, hydrogen-oxidizing microorganisms can produce at least two ATP for every H2 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 as a whole], which is about eight times the amount of ATP produced per H2 consumed in respiration than can be produced in microorganisms undergoing methane production or acetic acid production when H2 is used in respiration as an electron donor and CO2 as an electron acceptor. Therefore, using microorganisms that can utilize more electronegative electron acceptors in respiration and ATP production (such as hydrogen-oxidizing microorganisms, but not limited to these) for anabolic biosynthesis, such as the biosynthesis of amino acids, proteins, or fatty acids from synthesis gas or H2, may be more efficient than using acetic acid production or methane production, as is currently used in biological gas-to-chemical (GTC) techniques for producing short-chain acids or alcohols (such as acetic acid or ethanol). In certain embodiments, the oxyhydrogen reaction used in respiration is enzymatically related to oxidative phosphorylation. In certain embodiments, aerobic respiration is utilized by microbial cells described herein for ATP production. In certain embodiments, the ATP and / or other intracellular energy carriers thus formed are utilized for anabolic biosynthesis of amino acids and / or proteins.In some embodiments, oxyhydrogen and / or carboxydotrophic microorganisms and / or methanetrophic microorganisms and / or heterotrophic microorganisms, or compositions or consortia containing these microorganisms are utilized, the microorganisms expressing one or more enzymes that enable the biosynthesis of a useful carbon-based product of interest from carbon-containing gas raw materials such as synthesis gas or generator gas or natural gas or biogas or CO2 combined with renewable H2 or CO or methane-containing gas, but not limited to these. In some embodiments, these carbon-based products of interest can be heterotrophically biosynthesized from organic polycarbonate raw materials such as glucose, fructose, sucrose, and other sugars, but not limited to these. In some non-limiting embodiments, microorganisms or compositions containing microorganisms are utilized, the microorganisms require less than 4H2 or NADH to produce one ATP through respiration. In other non-limiting embodiments, microorganisms are utilized that produce two or more ATP for every H2 or NADH consumed by respiration. In other non-limiting embodiments, microorganisms are used that produce at least 2 ATP molecules per H2 or NADH consumed by respiration, or at least 2.5 ATP molecules per H2 or NADH consumed by respiration.

[0231] Additional features of certain non-limiting embodiments relate to the source, production, or recycling of electron donors used by chemosynthetic autotrophic microorganisms to fix carbon dioxide and / or other C1 raw materials into organic compounds. Electron donors used for carbon capture and carbon fixation can, in certain embodiments, be generated or recycled electrochemically or thermochemically using power from multiple different renewable and / or low-carbon emission energy technologies, such as photovoltaic, solar, wind, hydro, nuclear, geothermal, enhanced geothermal, ocean thermal, ocean wave, and tidal energy. Many of the reducing inorganic chemicals on which chemoautotrophs can grow (e.g., H2, CO, H2S, ferrous, ammonium, Mn) 2+Carbon dioxide can be readily produced using electrochemical and / or thermochemical processes well known in chemical engineering techniques and sciences that can power electricity with no or low carbon emissions, and / or using renewable power sources, such as, but not limited to, photovoltaic, solar, wind, hydro, nuclear, geothermal, enhanced geothermal, ocean thermal, ocean wave, or tidal power.

[0232] Hydrogen production from renewable energy sources is gradually replacing production from fossil fuel systems, and technological advancements in the energy sector are expected to lower the cost 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 electrolytic cells, and research on novel materials and electrolytic cell configurations has shown that efficiencies as high as 96% are possible. In certain embodiments, commercially available electrolytic technologies with electrical energy efficiencies exceeding 70% are used for the production of H2 electron donors and / or O2 electron acceptors. In certain embodiments, electrolytic technologies with energy efficiencies of 73% or more, and / or up to 96% or more, are used.

[0233] In certain embodiments using hydrogen molecules as electron donors, H2 is produced by methods well known to those skilled in the art of chemistry and process engineering, including but not limited to: electrolysis of water, approaches using proton exchange membranes (PEM), liquid electrolysis such as KOH, alkaline electrolysis, solid polymer electrolyte electrolysis, high-pressure electrolysis, high-temperature electrolysis of steam (HTES), two-stage electrochemical cycles such as those utilizing nickel oxide and nickel hydroxide electrodes, and / or thermochemical decomposition of water by methods such as the iron oxide cycle, cerium(IV) oxide-cerium(III) oxide cycle, zinc oxide cycle, sulfur-iodine cycle, copper-chlorine cycle, calcium-bromine-iron cycle, hybrid sulfur cycle, etc.; 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 methane reforming or carbon capture and sequestration (CCS) which enables the gasification of biomass. In certain embodiments, approaches for generating H2 include, but are not limited to, electrolysis powered by renewable electrical energy and / or electricity from low GHG sources. In certain embodiments, electrolysis may be powered by, but are not limited to, one or more of the following: sunlight, photovoltaic and / or solar thermal, wind, hydroelectric; nuclear; geothermal; enhanced geothermal; ocean thermal; ocean wave; or tidal.

[0234] Across the globe, there are vast wind energy resources, but only a fraction of them are being utilized. Low current utilization is primarily due to the intermittent nature of wind resources, resulting in fluctuating power generation over time and underutilization of capacity to meet energy demand for most of the time. The common mismatch between wind power and grid demand is evident in examples around the world, such as Scotland, where wind farms are paid to shut down their 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 parts of Texas, where wind demand is high and grid demand is low, with electricity being supplied free of charge 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 solved by utilizing wind power generated during off-peak demand hours to produce H2 feedstock for the process in certain embodiments of this specification.

[0235] Currently, hydrogen is increasingly seen as a viable energy storage system in the so-called "power-to-gas" approach. The inherent instability of renewable energy production (particularly solar and wind energy), and excess grid power (off-peak energy), can be mitigated by producing hydrogen through the electrolysis of water. According to most current schemes, the generated hydrogen gas can be converted back into electricity by fuel cells and / or gas turbines during periods of peak demand. Alternatively, H2 can be supplied to the gas grid or converted to methane by methanation. Furthermore, hydrogen can be used as a raw material in the chemical, petrochemical, metallurgical, and food industries. Certain embodiments offer new options within the power-to-gas framework by enabling H2 to be used in a wider range of products, particularly biochemicals such as proteins, amino acids, fertilizers, and biostimulants. In certain embodiments, hydrogen produced using excess grid power and / or off-peak energy is used as an electron donor in 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 are produced by electrolysis of water using renewable energy, particularly off-peak electricity, i.e., electricity available when energy supply exceeds demand (which, in current circumstances, is often wasted).

[0236] In certain embodiments, on-site storage of H2 and CO2 gases allows for the diversion of electricity from the grid only during periods when renewable power generation exceeds electricity demand. In certain embodiments, the electricity can flow into the grid as usual during periods of higher demand. In certain embodiments, this process promotes the full utilization of renewable power generation capacity, such as wind and solar, but not limited to these, rather than hindering the supply of renewable electricity. In certain embodiments, continuous renewable operation and generation are possible even during periods when generation exceeds grid demand (e.g., off-peak wind or solar power generation).

[0237] In certain embodiments, the hydrogen electron donor is not necessarily produced in a manner that results in low or no carbon dioxide emissions. However, in certain such embodiments, hydrogen is produced from sustainable or low-value energy sources and / or carbon sources using methods known in the fields of chemistry and process engineering. Such methods include, but are not limited to, gasification, pyrolysis, steam reforming, or autothermal reforming of one or more raw materials from agricultural materials, wood, methane hydrate, straw, seaweed, and kelp, and generally low-value, high-lignocellulosic biomass. In certain embodiments, synthesis gas or generator gas containing H2 and / or CO and / or CO2 is used as the electron donor and / or carbon source. In certain embodiments, the H2 and / or CO and / or CO2 contained in the synthesis gas or generator gas is supplemented by H2 produced using conversion processes such as renewable and / or low-GHG energy sources and one or more of those described herein.

[0238] In certain non-limiting embodiments, reduction of CO2 occurs and / or synthesis of cellular material that can be used as food or a nutrient source occurs. In certain embodiments, the ratio of hydrogen to carbon monoxide in the synthesis gas or generator gas can be adjusted by a water-gas shift reaction and / or carbon capture before the gas is delivered to the microbial culture. In certain embodiments, the C1 compound is produced by methane or natural gas, particularly residual natural gas or natural gas flared or released into the atmosphere in other ways, or by methane steam reforming of biogas or landfill gas, and is supplied to the microbial culture as synthesis gas and / or generator gas or liquid stream of the C1 compound, and in certain embodiments, the ratio of hydrogen to carbon monoxide in the synthesis gas or generator gas can be adjusted by a water-gas shift reaction and / or carbon capture before the gas is supplied to the microbial culture.

[0239] The following examples are intended to illustrate the present invention, but are not limited thereto. Examples Example 1 - Processing of single-cell proteins The microbial cells are washed to remove the culture medium contained in the bioprocess. The cells are lysed and inactivated (confirming that the viability is zero), and nucleic acids are removed. Optionally, the cell wall is removed, and optionally, the protein isolate is hydrolyzed to minimize the risk of allergic reactions.

[0240] To assess nutritional value, the Protein Digestibility Corrected Amino Acid Score (PDCAAS) and the Digestible Essential Amino Acid Score (DIAAS) are determined.

[0241] The taste is evaluated to confirm that the isolate has a neutral or non-offensive flavor. If necessary, the flavor can be masked by adding compounds or materials.

[0242] Functional properties such as solubility, emulsification, water retention, fat retention, and gelation are evaluated.

[0243] Allergy-inducing properties are assessed to ensure that the isolate is reliably hypoallergenic.

[0244] Example 2 - Analysis of microbial protein isolates Protein isolates were prepared from the whole cell biomass of the Cupriavidus necatar microorganism as described in Example 1. The preparation of the protein isolates involved concentrating the amino acid content and reducing the nucleic acid content of the biomass. Functional properties were measured, and high water and oil absorption was observed in the protein isolate (PI) samples. The PI samples exhibited high PDCAAS values, demonstrating excellent digestibility and nutritional value.

[0245] Table 1 shows the percentage of N, total amino acids, and nucleic acids of whole cell biomass (WCB) of sugar-grown cells compared to the PI sample. [Table 1]

[0246] Table 2 shows the density and water and oil absorption of sugar-grown WCB, sugar-grown PI samples (PI#1, 2, and 3), and gas-grown PI (PI#4) compared to pea isolate (Nutrassuma pea protein). [Table 2]

[0247] Table 3 shows the digestibility of sugar-grown whole cell backbone (WCB) compared to other protein sources (data from Miller, et al., Nutritional factors in SCP, pp. 79-89, Single Cell Protein, MIT Press, Eds. Mattales and Tannenbaum). [Table 3]

[0248] Example 3 - Preparation of food compositions from microbial protein isolates Several meat substitutes, such as hamburgers, carne asada bits, and bacon bits, were prepared from Cupria vidus nekatar whole cell biomass (WCB) or protein isolates (PI). PI exhibited high fat absorption capacity. Therefore, when boiled or fried, it absorbs oil, resulting in a greasy aftertaste. PI samples also showed high water retention capacity. Thus, boiling can prevent the problem of high fat absorption. Cooking by boiling promotes water absorption, ensuring that the food composition is cooked thoroughly and completely. Frying after boiling gives the food composition texture and a crisp appearance. This can mimic a meat-like structure both externally and internally.

[0249] Two hamburgers weighing a total of 400g were prepared from whole-body chocolate (WCB). The ingredients are shown in Table 4. [Table 4]

[0250] Common spices: Apple cider vinegar (1 teaspoon); garlic powder (1 / 4 teaspoon); onion powder (1 / 4 teaspoon); turmeric (1 / 8 teaspoon); baking soda (1 / 4 teaspoon); paprika (1 / 4 teaspoon); maple syrup (1 teaspoon); oil (2 tsp)

[0251] B1: All ingredients were first ground in a Nutri bullet and then blended in a food processor. The appearance was very smooth. The hamburger was consistently sticky. Using 100% walnuts resulted in a darker color for the hamburger.

[0252] B2: All ingredients were blended in a food processor. This looked thick and good in appearance. It contains essential wheat gluten, which may contribute to the thick texture. Using a 60:40 walnut:cashew ratio resulted in a lighter color for the hamburger.

[0253] Coloring ingredients: Carrot fiber binder; beet powder and tomato powder

[0254] A hamburger weighing 464g was prepared from protein extract (PI). The protein content was 6.5% by weight (wet). The components are shown in Table 5. [Table 5]

[0255] The hamburger was dark in color. A change in color occurred after cooking. This was due to lipid oxidation in the walnuts. The taste was good, and there was no aftertaste.

[0256] Desired characteristics for a hamburger: For a lighter color, use rice flour instead of chickpeas, use cashews instead of walnuts, and / or beet juice may be included to add a reddish hue. Eggs or egg substitutes may be included for a less cracked texture. Fat such as olive oil or margarine may be added for extra moisture.

[0257] Bacon-like products were prepared using the ingredients shown in Table 6. Recipe 1 contained 25% WCB dry base (uncooked), while Recipe 2 contained 48% WCB dry base and 16% wet base (uncooked). [Table 6]

[0258] Carne Asada / bacon-like product was prepared using the ingredients shown in Table 7. The total weight was 402 g. The protein percentage was 15% wet and 28.5% dry. [Table 7]

[0259] The protein dissolved well into the wheat gluten, forming a pliable dough. This dough may be processed for texture by rolling and re-rolling. The dough, once baked or boiled, had a texture similar to meat. After boiling, the meat-like texture became more pronounced. Muscular texture was visible on the surface of the product. At taste testing, the flavor was vague and bland. The flavor of the product inherited the flavor of the added flavor components.

[0260] Carne Asada / bacon-like products were prepared using the ingredients shown in Table 8. [Table 8]

[0261] Example 4 - Production of protein concentrates from Cupria viidas necatar grown in H2 and CO2. C·Nekata DSM541 biomass was grown in a 2-liter continuous stirring tank reactor (CSTR) on H2 and CO2 substrates and aqueous minimal salt medium. The culture broth was continuously collected from the CSTR and temporarily stored at 4°C. Next, the biomass was separated from the liquid broth by centrifugation, the supernatant was poured out, and the dehydrated but moist biomass (approximately 20% solids / 80% water content) was collected and stored at -80°C. Subsequently, the moist frozen biomass was thawed and resuspended in water using a Turrax stick and hand blender until the solids content was 8% to produce a smooth, homogeneous slurry. This slurry was then placed in an autoclave. The autoclave was heated to 110°C and held at that temperature for 30 minutes. After heat treatment, the autoclaved slurry was cooled in a water bath and centrifuged at 13,000 g for 40 minutes. The supernatant was drained and separated for analysis, and the moist solid obtained by centrifugation was collected. Next, the moist solid produced by centrifugation was freeze-dried and ground to obtain a fine beige powdered protein concentrate.

[0262] Next, the protein concentrate was analyzed. The total amino acid content was determined to be 80.8% on a dry weight basis using the AOAC method AOAC 994.12. The moisture content was determined to be 2.88% using the NFTA 2.2.2.5 test method. The ash content was determined to be 6.4% on a dry weight basis using the AOAC 942.05 test method. The total lipid content was determined by Bligh-Dyer extraction (Bligh, E.G. and Dyer W.J. (1959) J Biochem Physiol 37(8):911-917), and gravimetric analysis showed it to be 9.15% on a dry weight basis. Vitamins B1 and B2 were measured using the AOAC methods AOAC 942.23 and AOAC 970.65, respectively, and it was found that vitamin B1 was 1.39 mg and vitamin B2 was 20.6 mg per 100 grams of dry weight.

[0263] While the aforementioned inventions have been described in some detail as examples and illustrations for the purpose of clarifying understanding, it will be apparent to those skilled in the art that certain changes and modifications can be implemented without departing from the spirit and scope of the invention. Therefore, this description should not be construed as limiting the scope of the invention.

[0264] All publications, patents, and patent applications cited herein are incorporated herein in whole, by reference, for all purposes, and to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference.

Claims

1. A method for manufacturing food products, (a) Processing microbial cells collected from a culture medium to produce a protein product, wherein the protein product comprises one or more of the following: single-cell protein, cell lysate, protein concentrate, protein isolate, protein extract, protein hydrolysate, free amino acid, peptide, and oligopeptide. (b) A method comprising processing the protein product to produce a food product.

2. The method according to claim 1, wherein the food product is a meat-like product.

3. The method according to claim 2, wherein the meat-like product includes one or more physical characteristics and / or functional properties of meat, including texture, flavor, aroma, and / or appearance.

4. The method according to claim 2, wherein the meat-like product comprises at least about 10% by weight of the protein product.

5. The method according to claim 2, wherein the meat-like product contains a heme compound.

6. The method according to claim 5, wherein the heme compound is a heme-containing polypeptide.

7. The method according to claim 5 or 6, wherein the heme compound is produced by the same microorganism that produces the protein product.

8. The method according to claim 2, wherein a hydrogel, lipogel, and / or emulsion are included in the meat-like product as an agent release system.

9. The method according to claim 8, wherein the agent release system facilitates the release of colorants, flavorants, fatty acids, leavening agents, and / or gelling agents during the cooking of the meat-like product.

10. The method according to claim 2, wherein the meat-like product comprises about 5% to about 30% by weight of lipids, about 0.5% to about 10% by weight of carbohydrates, and / or about 0.5% to about 5% by weight of dietary fiber.

11. The method according to claim 10, wherein the meat-like product contains at least about 30% by weight of moisture.

12. The method according to claim 1, wherein the food product is a food product, a food ingredient, a nutritional product, an animal feed product, or a pet food product.

13. The method according to claim 1, wherein the food product is a vegetarian or vegan food product.

14. The method according to claim 1, wherein the food product is an organic food product, a pesticide-free food product, a herbicide-free food product, a fungicide-free food product, an antibiotic-free food product, or a non-genetically modified (non-GMO) food product.

15. The method according to claim 1, wherein the food product is a probiotic food product or a prebiotic food product.

16. The method according to claim 1, wherein the food product does not contain animal protein or fat.

17. The method according to claim 1, wherein the food product includes dairy products, dairy substitutes, bakery products, confectionery, health or protein bars, protein powders, sports and / or energy drinks, protein shakes, or smoothies.

18. The method according to claim 1, wherein the food product comprises one or more plant protein sources.

19. The method according to claim 18, wherein the plant protein source comprises one or more of peas, rice, glutinous rice, wheat, gluten, soybeans, hemp, canola, and buckwheat.

20. The method according to claim 1, wherein the food product comprises a protein source of insects or algae.

21. The method according to claim 1, wherein the food product is a meat product, and the protein product is incorporated into the meat product as a meat filler.

22. The method according to claim 21, wherein the protein product replaces at least about 10% of the meat in the meat product.

23. The method according to claim 1, wherein the protein product is processed to improve texture for incorporation into meat or a meat-like product.

24. The method according to claim 1, wherein the protein product is formed into fibers, thermally extruded, and / or coagulated to form a gel.

25. The method according to claim 1, wherein the microorganism is a chemosynthetic autotrophic microorganism.

26. The method according to claim 25, wherein the chemosynthetic autotrophic microorganism is an acid-hydrogen microorganism.

27. The method according to claim 26, wherein the oxyhydrogen microorganism is a Cupriavidus microorganism.

28. The method according to claim 27, wherein the oxyhydrogen microorganism comprises Cupriavidus necatar DSM531 or DSM541.

29. The method according to claim 1, wherein the microorganism is a lactic acid bacterium.

30. The method according to claim 29, wherein the lactic acid bacteria include one or more of Lactococcus, Lactobacillus, Enterococcus, Streptococcus, and Pediococcus bacteria.

31. The method according to claim 29 or 30, wherein the lactic acid bacteria are generally recognized as safe (GRAS) bacteria.

32. The method according to claim 1, wherein the microorganism is a Fusarium, Rhizopus, or Aspergillus fungal microorganism.

33. The method according to claim 32, wherein the fungal microorganism comprises one or more of Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae.

34. The method according to claim 32 or 33, wherein the fungal microorganism is a GRAS fungal microorganism.

35. The method according to claim 1, comprising: (a) releasing an organic molecule from the microbial cell by excretion, secretion, cytolysis, or a combination thereof, wherein the organic molecule includes a protein; and (ii) processing the released organic molecule to hydrolyze peptide bonds between at least some amino acids in at least some of the protein, thereby producing a polypeptide containing 20 to 50 amino acids, an oligopeptide containing 2 to 20 amino acids, and / or a hydrolyzed protein product containing free amino acids.

36. The method according to claim 1, wherein step (b) comprises combining the protein product with other edible ingredients to form the food product.

37. The other edible ingredients mentioned above include apple cider, apple cider vinegar, baking powder, baking soda, beans, beef, beet juice, beet powder, black pepper, brown sugar, butter, canola oil, caramel, carrot fiber, carrots, cashews, cheese, chicken, chocolate, citrus fruits, citrus extracts, coconut oil, condensed milk, dairy products, eggs, egg substitutes, fish, wheat flour, chickpeas, garlic powder, honey, smoke flavoring, maple syrup, margarine, monosodium glutamate, and trout. The method according to claim 36, comprising one or more of the following: tard powder, oil, olive oil, onion powder, paprika, pork, potato, potato starch, rice flour, salt, sodium benzoate, soy protein, soybean oil, soy sauce, spices, spirulina, sugar, sunflower oil, tomato juice, tomato powder, tomato sauce, tomato, turmeric, vanilla, vinegar, vitamins and minerals, walnuts, water, wheat, wheat flour, wheat gluten, xanthan gum, yeast, and yeast extract.

38. A food product prepared by the method described in any one of claims 1 to 37.

39. A food product comprising a protein product produced from a microorganism, wherein the protein product comprises one or more of the following: single-cell protein, cell lysate, protein concentrate, protein isolate, protein extract, protein hydrolysate, free amino acid, peptide, and oligopeptide.

40. A food product according to claim 39, which is a meat-like product.

41. The food product according to claim 40, wherein the meat-like product includes one or more physical characteristics and / or functional properties of meat, including texture, flavor, aroma, and / or appearance.

42. The food product according to claim 40, wherein the meat-like product comprises at least about 10% by weight of protein product.

43. The food product according to claim 40, wherein the meat-like product contains a heme compound.

44. The food product according to claim 43, wherein the heme compound is a heme-containing polypeptide.

45. The food product according to claim 43 or 44, wherein the heme compound is produced by the same microorganism that produces the protein product.

46. The food product according to claim 40, wherein a hydrogel, lipogel, and / or emulsion are included in the meat-like product as an agent release system.

47. The food product according to claim 46, wherein the agent release system facilitates the release of colorants, flavoring agents, fatty acids, leavening agents, and / or gelling agents during the cooking of the meat-like product.

48. The food product according to claim 40, wherein the meat-like product comprises about 5% to about 30% by weight of lipids, about 0.5% to about 10% by weight of carbohydrates, and / or about 0.5% to about 5% by weight of dietary fiber.

49. The food product according to claim 48, wherein the meat-like product contains at least about 30% by weight of moisture.

50. The food product according to claim 49, wherein the food product is a food product, a food ingredient, a nutritional product, an animal feed product, or a pet food product.

51. The food product according to claim 39, wherein the food product is a vegetarian or vegan food product.

52. The food product according to claim 39, wherein the food product is an organic food product, a pesticide-free food product, a herbicide-free food product, a fungicide-free food product, an antibiotic-free food product, or a non-genetically modified (non-GMO) food product.

53. The food product according to claim 39, which is a probiotic food product or a prebiotic food product.

54. The food product according to claim 39, which does not contain animal protein or fat.

55. The food product according to claim 39, wherein the food product comprises dairy products, dairy substitutes, bakery products, confectionery, health or protein bars, protein powders, sports and / or energy drinks, protein shakes, or smoothies.

56. The food product according to claim 39, comprising one or more plant protein sources.

57. The food product according to claim 56, wherein the plant protein source comprises one or more of peas, rice, glutinous rice, wheat, gluten, soybeans, hemp, canola, and buckwheat.

58. The food product according to claim 39, comprising a protein source of insects or algae.

59. The food product according to claim 39, wherein the food product is a meat product, and the protein product is incorporated into the meat product as a meat filler.

60. The food product according to claim 59, wherein the protein product contains at least about 10% meat in the meat product.

61. The food product according to claim 39, wherein the microorganisms include chemosynthetic autotrophic microorganisms.

62. The food product according to claim 61, wherein the chemically synthesized autotrophic microorganism includes an oxyhydrogen microorganism.

63. The food product according to claim 62, wherein the oxyhydrogen microorganism includes the microorganism *Cupriavidus*.

64. The food product according to claim 63, wherein the oxyhydrogen microorganism comprises Cupriavidas necatar DSM531 or DSM541.

65. The food product according to claim 39, wherein the microorganisms include lactic acid bacteria.

66. The food product according to claim 65, wherein the lactic acid bacteria include one or more of Lactococcus, Lactobacillus, Enterococcus, Streptococcus, and Pediococcus bacteria.

67. The food product according to claim 65 or 66, wherein the lactic acid bacteria are generally recognized as safe (GRAS) bacteria.

68. The food product according to claim 39, wherein the microorganism is a Fusarium, Rhizopus, or Aspergillus fungal microorganism.

69. The food product according to claim 68, wherein the fungal microorganism comprises one or more of Fusarium venenatum, Rhizopus oligosporus, Rhizopus oryzae, Aspergillus oryzae, or Aspergillus sojae.

70. The food product according to claim 68 or 69, wherein the fungal microorganism is a GRAS fungal microorganism.

71. The aforementioned food products include apple cider, apple cider vinegar, baking powder, baking soda, beans, beef, beet juice, beet powder, black pepper, brown sugar, butter, canola oil, caramel, carrot fiber, carrots, cashews, cheese, chicken, chocolate, citrus fruits, citrus extracts, coconut oil, condensed milk, dairy products, eggs, egg substitutes, fish, wheat flour, chickpeas, garlic powder, honey, smoke flavoring, maple syrup, margarine, monosodium glutamate, and mustard powder. The food product according to claim 39, further comprising one or more of the following: udon, oil, olive oil, onion powder, paprika, pork, potato, potato starch, rice flour, salt, sodium benzoate, soy protein, soybean oil, soy sauce, spices, spirulina, sugar, sunflower oil, tomato juice, tomato powder, tomato sauce, tomato, turmeric, vanilla, vinegar, vitamins and minerals, walnuts, water, wheat, wheat flour, wheat gluten, xanthan gum, yeast, and yeast extract.

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