Protein hydrolysates derived from microorganisms and methods of making and uses thereof

Abstract

Protein hydrolysate compositions derived from microorganisms, such as chemoautotrophic microorganisms, and methods of making and using the protein hydrolysate compositions are provided. The protein hydrolysate composition can be sustainably produced by immobilizing carbon dioxide from biological or atmospheric sources. The protein hydrolysate compositions can be used to supplement media for serum-free culture of animal cells as well as for growth of other types of cells such as probiotics and lactic acid bacteria. Thus, the present disclosure provides sustainable humanistic methods for culturing cells for pharmaceutical and nutraceutical applications and for human consumption as food ingredients or products.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202180017247.2, application date January 22, 2021, and invention name “Protein hydrolysates from microbial sources, preparation methods and uses thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 965,303, filed on January 24, 2020, which is incorporated herein by reference in its entirety. Field of the Invention

[0004] The present disclosure relates to the field of protein hydrolysates produced from biological sources and methods for making the same. In particular, the present disclosure relates to the production of protein hydrolysates from renewable sources, such as biological processes designed to capture carbon dioxide emissions and other waste carbon conversion or transfer processes. The present disclosure relates to the use of protein hydrolysates to support the growth of other microorganisms or single cells, including probiotic microorganisms, eukaryotic cells, and vitamin-producing cells. The present disclosure also relates to cell culture methods for producing meat-like products, including culturing animal cells, for example, using a culture medium that does not contain animal components to produce artificial meat. Background Art

[0005] Various cell culture systems are used to produce useful biological products, including small biomolecules, therapeutic antibodies and cell-based therapies. Industrial fermentation is usually carried out in semi-defined and complex culture media that can produce higher levels of biomass or fermentation-derived products. Crude and unidentified additives such as peptones, protein hydrolysates, yeast extracts, growth factors, etc. can be added to the fermentation medium to provide a broad spectrum of nutrients. There is a long history of including animal-derived materials in microbial culture media. Traditionally, the culture medium for culturing animal cells incorporates animal serum or animal-derived protein hydrolysates to maintain and promote cell growth. For example, animal serum provides nutrients, hormones, growth and attachment factors, trace elements (such as iron), buffering capacity, protection against reactive oxygen species and mechanical shearing, and various other beneficial factors. Animal protein hydrolysates serve as nitrogen sources and provide peptides and / or amino acids and vitamins.

[0006] The beneficial effects of protein hydrolysates on the growth of cell cultures, such as animal cell cultures or lactic acid bacteria (LAB) cultures, are well known, and they have been used for decades as useful cell culture additives. The ability of LAB to synthesize amino acids is limited and depends on exogenous sources of amino acids and peptides. Therefore, providing a biochemical nitrogen source (e.g., protein, peptides, and amino acids) is essential for the growth of LAB cultures. Typical sources of amino acids are peptides found in milk and other hydrolyzed proteins. It is known that protein hydrolysates from different sources have different effects on various LAB strains, some of which support the growth of organisms, and others induce specific types of amino acid metabolism.According to reports, different commercial protein hydrolysate products that have been successfully used in LAB fermentation include NZ-amine, Hy-Case, Hy-Soy, Edamin and NZ-Case (Misono, H., Goto, N., & Nagasaki, S. (1985). Purification, crystallization and properties of nadp+-specific glutamate dehydrogenase from lactobacillusfermentum. Agricultural and Biological Chemistry. https: / / doi.org / 10.1080 / 00021369.1985.10866676; Molskness, T. A., Lee, D. R., Sandine, W. E., and Elliker, P. R. (1973). -D-phosphogalactoside galactohydrolase of lactic streptococci. AppliedMicrobiology. https: / / doi.org / 10.1128 / aem.25.3.373-380.1973; Viniegra-Gonzalez, G., and Gomez, J. (1984). Lactic acid production by pure and mixed bacterial cultures. Bioconversion Systems. Boca Raton, FL; CRC Press. pp. 17–39; Vedamuthu, ER (1980). Method for diacetyl flavor and aroma development in creamedcottage cheese. U.S. Patent 4,191,782.; Kegel, MA, and Wallace, DL (1989). Use ofstabilizing agents in culture media for growing acid producing bacteria. U.S. Patent 4,806,479).

[0007] Tryptone (digestion of casein or a pancreatic digest of casein by the protease trypsin) is a common component of laboratory and fermentation media for the growth of wild-type and genetically modified microorganisms. The advent of recombinant DNA technology and its incorporation into the manufacture and production of biopharmaceutical products has led to an increased demand for culture media containing components of animal origin.

[0008] Protein hydrolysates providing oligopeptides and free amino acids are widely used in animal cell culture for the production of, for example, therapeutic proteins. They are reported to increase cell density and produce higher yields of protein products. Many commercially manufactured products (such as human growth hormone, antibiotics, insulin, etc.) are produced by recombinant strains grown on nutrients and culture medium components derived from bovine sources.

[0009] There are many different types of commercially available hydrolyzed proteins produced from a variety of sources ranging from vegetables to meat and milk. The example of source includes casein, whey protein, skim milk, milk and whey protein isolate and concentrate, lactalbumin, meat and animal tissue, collagen, gelatin, corn, cottonseed, soybean meal, soy protein isolate and concentrate. Some of these materials are byproducts of other processes and are highly variable. In other cases, although the protein (for example, casein and whey) of a specific source can have a fairly constant amino acid composition, the various protein hydrolysates from the same source can still have significant changes in peptides and amino acid profiles. In addition, the total amino acid composition and protein structure of different protein sources (such as casein, meat, gelatin and soybeans, etc.) are also different. Dairy hydrolysates are rich in amino acids such as Glu, Ile, Leu, Val and Met, and meat / soybean hydrolysates are relatively more abundant in Arg, Cys, Gly and Pro.

[0010] Animal-derived hydrolysates have historically been used as cell culture nutrient additives for the production of drugs such as vaccines and biologics, including lactalbumin hydrolysates, casein hydrolysates, and primatone. However, such animal-derived growth supplements have disadvantages. The composition of serum and protein hydrolysates is unclear, and the supply of serum is prone to batch-to-batch differences. Due to its animal origin, serum has the risk of being contaminated by adventitious factors and contaminants (such as prions). This is the main motivation for eliminating serum from the cell culture process. Concerns about prions in the biopharmaceutical industry and regulatory agencies (such as transmissible spongiform encephalopathy (TSE) and other adventitious factors (such as viruses and mycoplasmas) from the use of animal-derived components) have prompted the elimination of animal-derived components from the culture medium. With the emergence of bovine spongiform encephalopathy (BSE) and the resulting increase in Food and Drug Administration (FDA) regulations, efforts are being made to eliminate bovine-derived materials from fermentation media. The production cost of serum is also high. Other factors, such as serum availability, degradation of target molecules by serum proteases, impact on downstream product purification complexity, potential immunogenicity, and other serum-related artifacts, are also of concern. In addition, there is a growing interest in using humane and sustainable methods to manufacture biological products. For example, there is growing interest in methods for sustainable cultured meat products that are animal-free.

[0011] Traditionally, mammalian cells usually need to be supplemented with a nutrient mixture (basal medium) of 5%-20% serum or serum-derived components. Fetal calf serum (FCS), also known as fetal bovine serum (FBS), is a rich source of culture medium component materials, including hormones (e.g., insulin), growth factors, transport proteins (such as transferrin and albumin), nutrients and attachment factors. Serum is traditionally included in growth medium as a source of nutrients, hormones, growth factors and protease inhibitors. It promotes the attachment and diffusion of animal cells, and provides nonspecific protection against mechanical damage and shear forces, binds toxic compounds and improves the buffering capacity of culture medium. Albumin and transferrin in serum serve as carriers of lipids, fatty acids, hormones and trace elements (such as iron).

[0012] In order to avoid animal-derived components, suppliers of culture media, feeds and ingredients have responded by developing culture media that use plant-derived components or are manufactured by recombinant expression. For decades, protein hydrolysates (e.g., peptones) have been used to replace serum and serum components in various mammalian culture processes. Protein hydrolysates from plant sources such as cottonseed, soybeans or wheat, used alone or in combination, have been found to be suitable substitutes for bovine serum in certain cell culture systems. Relatively crude lysates and extracts of organs, tissues and proteins from animal sources have been eliminated in historical manufacturing processes and are being replaced by more renewable yeast and plant-derived substitutes. In many cases, milk and meat hydrolysates have also been replaced by hydrolysates of plant and yeast origin.

[0013] Plant-derived protein hydrolysates may contain oligopeptides, free amino acids, carbohydrates, vitamins, minerals (such as potassium, calcium, iron) and trace minerals, lipids, nucleosides, nucleotides, trace low molecular weight components and mixtures of undefined components. Protein hydrolysates can increase cell growth rate, recombinant protein secretion and long-term cell viability. Protein hydrolysates can provide nutrients, adhesion components and / or growth factor analogs. Protein hydrolysates are used as key culture medium additives in serum-free cell culture processes for industrial production of therapeutic recombinant proteins. The beneficial effects of plant-derived hydrolysates on industrially important cell types such as hybridoma cells, African green monkey kidney (VERO) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, baby hamster kidney (BHK) cells and baculovirus-infected fall armyworm insect cells (Sf9) are well documented. It is reported that protein hydrolysates stimulate more efficient cell metabolism by more efficiently utilizing amino acids, usually improving cell metabolism beyond the level achieved with serum.

[0014] However, there is still a continuing need for new cell culture medium supplements that do not contain animal ingredients. Some plant-derived hydrolysates show poor performance in certain applications. Previous studies have shown that wheat gluten extracts or extracts with high free amino acid content may be toxic or induce toxic effects in certain cell types in vitro, and can inhibit protein synthesis in cell-free systems of animal cells. There are concerns about pesticide and herbicide residues in some plant-derived hydrolysates. In order to be used in commercially successful fermentations, culture medium ingredients need to be cheap, easily available and of renewable quality. Unfortunately, many complex nutrients, including protein hydrolysates, extracts and serums from animal and plant sources, have great inconsistencies due to changes in biological sources and changes in these materials due to regions, climates and different seasons. These changes can significantly affect the quality of the final fermentation product, resulting in significant changes in results and production losses. More consistent and unaffected by regional, climatic or seasonal changes in complex nutrients such as lysates, protein hydrolysates and peptide compositions are needed. Protein hydrolysate products with more consistent peptide and amino acid profiles are needed. Protein hydrolysates of non-animal origin are needed as substitutes for culture medium components of animal origin (comprising culture medium nutrient components, protein hydrolysates and serum of animal origin). It is necessary to minimize the variability between batches of culture medium and / or culture medium supplement. It is also necessary to contain protein hydrolysates without any pesticide, herbicide, fungicide, hormone or antibiotic residues. Since the components of animal origin may contain infectious factors, there is interest in developing culture medium with the least animal-derived components or without animal ingredients at all. It is also a moral and environmental requirement to minimize the use of animal-derived products. Summary of the invention

[0015] Provided herein are protein hydrolysate compositions derived from microorganisms that grow by fermentation or chemoautotrophically on an organic carbon source. In certain embodiments, the microorganism grows on a C1 carbon source, such as: chemoautotrophic growth on CO2 as a carbon source; carboxydotrophic growth on CO as a carbon source; or methanotrophic or methylotrophic growth on CH4 and / or CH3OH as a carbon source.

[0016] The protein hydrolysate described herein is suitable as a substitute for all or part of an animal component supplement, for promoting the proliferation, maintenance, reproduction and / or differentiation of animal cells in culture in the absence of one or more animal-derived cell culture components (such as amino acids, protein hydrolysates or serum). For example, a protein hydrolysate may be suitable for use as a supplement that does not contain animal components in, for example, an animal-free or serum-free medium. The lysate and / or hydrolysate produced as described herein can provide growth-promoting peptides, amino acids, carbohydrates, lipids, minerals and / or vitamins. In certain embodiments, the peptides produced as described herein have one or more of the following functions: as a source of amino acids (e.g., replacing or supplementing free amino acids); as a stimulant for the growth and / or production of biomass and / or target molecules; protecting cells from shear stress. In certain embodiments, the lysate and / or hydrolysate described herein stimulates more efficient cell metabolism by including but not limited to mechanisms that more efficiently ingest and use amino acids. In certain embodiments, compared with the culture that does not comprise described lysate and / or hydrolysate, described lysate and / or hydrolysate has improved the growth rate and / or protein expression in the culture grown on the substratum that comprises described lysate and / or hydrolysate.In certain embodiments, described lysate and / or hydrolysate improves the performance of the culture grown on the substratum that comprises described lysate and / or hydrolysate by including but not limited to the effect of osmotic protectant and / or anti-apoptotic effect.In certain embodiments, described lysate and / or hydrolysate helps to protect from shearing effect.In certain embodiments, described lysate and / or hydrolysate promotes the attachment and / or subsequent spreading of cells.

[0017] The present disclosure includes protein hydrolysates without animal components that are suitable for producing consumable meat products derived from cultured animal cells. In addition, the present disclosure includes protein hydrolysates without animal components that are suitable for the growth of organisms and / or probiotic microorganisms that are "generally recognized as safe" (GRAS), and the protein hydrolysates can also be processed into meat samples and / or high-protein products, as well as other food products, ingredients, nutrients and flavorings. In addition, the present disclosure includes protein hydrolysates without animal components that are suitable for the growth of organisms that are considered to be beneficial members of plant or animal microbial groups. In certain embodiments, the plant or animal is used to produce human food or animal feed or other plant or animal-derived products used by humans. Protein hydrolysates without animal components can be sustainably produced, for example, by CO2 from sustainable and clean sources, such as CO2 waste gases from breweries or breweries fermentation and CO2 captured from the atmosphere or some other natural sources by microbial fixation.

[0018] The protein hydrolysate compositions disclosed herein can be defined by the abundance of amino acids, size distribution of peptides, abundance of different peptide sequences, protein identity and abundance, lipid composition, organic polymers, vitamins, abundance of minerals, etc. Provided herein are libraries of protein hydrolysates, wherein the different compositions in the library are characterized by one or more of the abundance of different amino acids, size distribution of peptides, abundance of peptide sequences, protein identity and abundance, lipid composition, organic polymers, vitamins, abundance of minerals. The protein hydrolysate compositions can be suitable for promoting specific aspects of animal or microbial cell culture (e.g., proliferation, reproduction, development, differentiation), depending on the hydrolysate composition.

[0019] The present disclosure includes a protein hydrolysate composition containing a biostimulant component. In certain such embodiments, the biostimulant improves the performance or characteristics of a plant or fungal crop. In some embodiments, the protein hydrolysate composition comprises a biostimulant polyester, such as a polyhydroxyalkanoate (PHA) polymer. In some embodiments, the PHA polymer may be produced by a microorganism (e.g., a chemoautotrophic microorganism) from which the protein hydrolysate composition is derived. In some embodiments, the protein hydrolysate composition comprises polyhydroxybutyrate (PHB). In some embodiments, the protein hydrolysate composition comprises polyhydroxyvalerate (PHV). In some embodiments, the protein hydrolysate composition comprises a copolymer comprising PHB and / or PHV. In other embodiments, the protein hydrolysate composition comprises an oligomer or copolymer of PHB or PHA or PHV. In certain embodiments, the oligomer is caused by the hydrolysis of a corresponding PHB or PHA or PHV polymer. In certain embodiments, the protein hydrolysate composition comprises a hydroxybutyrate (HB) or hydroxyvalerate (HV) monomer.

[0020] The present disclosure includes a protein hydrolysate composition containing one or more vitamins. The vitamins may be produced by a chemoautotrophic microorganism from which the protein hydrolysate composition is derived. In some embodiments, the protein hydrolysate composition comprises one or more B vitamins, such as vitamin B1, vitamin B2 and / or vitamin B 12. Also provided herein is a method for producing a protein hydrolysate composition from a microbial culture, such as a chemoautotrophic microbial culture. The composition of the protein hydrolysate of the present disclosure may vary according to various manufacturing parameters, such as the type and strain of the microorganism (e.g., chemoautotrophic microorganism) used in the culture, growth conditions, the source of carbon and / or energy for culturing the microorganism (e.g., chemoautotrophic microorganism), the method for preparing the protein hydrolysate, the genetic engineering of the microorganism (e.g., chemoautotrophic microorganism), and the like. In some embodiments, the microorganism is a chemoautotrophic microorganism, such as any suitable chemoautotrophic microorganism that can fix carbon from a C1 carbon source (e.g., CO2). In certain embodiments, CO2 fixation is combined with H2 oxidation. In some embodiments, the microorganism (e.g., chemoautotrophic microorganism) is genetically engineered. Or in other embodiments, the microorganism (e.g., chemoautotrophic microorganism) is not genetically engineered and is a naturally occurring strain, or is a mutant or variant produced by one or more known methods outside of genetic engineering.

[0021] The protein hydrolysate composition can be prepared by subjecting the biomass produced by the growth of a microorganism (e.g., a chemoautotrophic microorganism) to physical, chemical, and / or enzymatic treatment. Physical treatment includes subjecting the biomass to elevated pressure and / or elevated temperature. Chemical treatment includes subjecting the biomass to acidic or alkaline conditions. Enzymatic treatment includes proteolysis with exoproteases and / or sequence-specific endoproteases and / or metalloproteases. In certain embodiments, the protein hydrolysate composition retains biostimulant components and / or vitamins after subjecting the biomass to the preparation process.

[0022] Also provided is a method for culturing cells (including animal cells) using a protein hydrolysate composition of the present disclosure as a culture medium supplement. The protein hydrolysate composition can provide nutrients suitable for maintaining the growth of animal cells in culture and / or promoting the development and differentiation of the animal cells to the culture medium. The method may include providing the protein hydrolysate in the culture medium and culturing animal cells in the culture medium. The protein hydrolysate composition can replace one or more components of conventional cell culture media that are usually derived from animal sources, such as amino acids, protein hydrolysates or serum. In some embodiments, the animal cell culture medium supplemented with the protein hydrolysate composition is a serum-free culture medium. In some embodiments, the cell culture medium supplemented with the protein hydrolysate composition of the present disclosure is a vegan culture medium. In some embodiments, the cell culture medium (including but not limited to animal cell culture medium) supplemented with the protein hydrolysate composition is a culture medium that does not contain animal components. In some embodiments, the lysate, protein hydrolysate and / or amino acid composition produced by a microorganism (e.g., a chemoautotrophic microorganism) is provided to the animal cell culture together with serum. In certain embodiments, the lysate, protein hydrolysate and / or amino acid composition are used for the growth of attachment-dependent cells. In other embodiments, the lysate, protein hydrolysate and / or amino acid composition is used for the growth of attachment-independent cells.

[0023] The protein hydrolysate composition may be suitable for use in culture media for culturing cells for various purposes (including recombinant cell lines for expressing therapeutic antibodies, proteins or small molecules or immune cells cultured for cell-based therapies). In some embodiments, the protein hydrolysate composition may be suitable for use in culture media for culturing meat products (including but not limited to artificial meat or meat substitute products). In some embodiments, the protein hydrolysate composition may be suitable for use in culture media for culturing probiotics or vitamin-synthesizing microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of a method for producing and extracting nutrients from a first biological process for use in a nutrient medium for a second cell culture according to an embodiment of the present disclosure.

[0025] Figure 2 is a schematic diagram of a method for chemoautotrophically producing protein-rich biomass from CO2 and then converting the protein-rich biomass into a protein hydrolysate for use in a nutrient medium for a second cell culture according to an embodiment of the present disclosure.

[0026] Figure 3is a schematic diagram of a method according to an embodiment of the present disclosure for producing protein-rich biomass chemoautotrophically from CO2 and then converting the protein-rich biomass into a protein hydrolysate, which is used in a nutrient medium for animal cell culture, from which animal cells are grown and recovered to produce cultured meat, i.e., a meat product that does not involve killing an entire multicellular animal.

[0027] Figure 4 Schematic diagram of a method for producing protein-rich biomass from CO2 chemoautotrophically and then converting the protein-rich biomass into a protein hydrolysate according to an embodiment of the present disclosure, wherein the protein hydrolysate is used in a nutrient medium for microbial culture, wherein the nutrient medium contains probiotics and / or microorganisms beneficial to humans, plants, and animals.

[0028] Figure 5 Shown is the growth of the beneficial fungus Trichoderma atroviride on medium supplemented with protein hydrolysate (PH) produced by C. necator grown on CO2 as a carbon source, compared to control growth of Trichoderma atroviride without pH supplementation. DETAILED DESCRIPTION

[0029] Provided are protein hydrolysate compositions derived from microorganisms (e.g., chemoautotrophic microorganisms), and methods for preparing and using the protein hydrolysate compositions. Protein hydrolysate compositions derived from microorganisms can be produced by microbial biomass and can be used as culture medium supplements to provide nutrition and support for animal cells grown in culture, or can be used as other eukaryotic and / or prokaryotic cells such as, for example, lactic acid bacteria (LAB) and / or probiotic nutrition. Like other plant-based or yeast-based protein hydrolysates, protein hydrolysates derived from microorganisms of the present invention can be used for supplementary culture medium for making cells such as eukaryotic cells (e.g., animal cells) and / or prokaryotic cells (e.g., LAB and / or probiotic cells) propagate, differentiate and / or grow, and are independent of animal-derived components (e.g., serum or growth factors). In some embodiments, chemoautotrophic microorganisms can be cultured autotrophically, for example, by CO2 fixation combined with H2 oxidation, to produce biomass, for example, from a clean and sustainable source of CO2, such as waste gases from brewery or winery fermentation or CO2 captured from the atmosphere, ocean or other natural sources (such as geothermal vents or hot springs). Utilizing chemoautotrophic microorganisms provides a wide flexibility in selecting carbon sources as feedstocks for production; from fermentation waste gases to natural sources of CO2, to industrial flue gases, to gasification of biomass (e.g., agricultural or forestry waste).

[0030] Thus, the present disclosure provides a sustainable source of protein hydrolysates that can be used to support humane processes for culturing animal cells and for producing food products with animal protein and other nutrients.

[0031] The present disclosure provides a method for producing a culture medium for cultivating a microorganism or a cell, the method comprising: cultivating a first microorganism, and producing biomass thereby; processing the biomass produced by the first cultured microorganism to produce a product rich in protein-derived biomass, such as but not limited to a protein-rich lysate or protein hydrolysate composition; and adding the protein-rich nutrient (e.g., protein-rich lysate or protein hydrolysate) composition to a culture medium for a second culture comprising a second microorganism or a cell, wherein the protein-rich nutrient (e.g., protein-rich lysate or protein hydrolysate) composition serves as a nutrient source for the growth of the second microorganism or the cell. In certain non-limiting embodiments, the second culture comprises an animal cell.

[0032] In some embodiments, the protein hydrolysate composition of the present invention can be used as a culture medium supplement for cultured food products (such as meat products), and thereby support sustainable and humane methods of producing food products (such as cultured meat).

[0033] For example, PCT application number PCT / US20 / 67555, filed on December 30, 2020 and entitled HIGH PROTEIN FOOD COMPOSITIONS, describes the production of novel protein compositions suitable for human consumption that closely simulate the properties of meat and are used as meat substitutes or artificial meat products, which is incorporated herein by reference in its entirety.

[0034] In some embodiments, the protein hydrolysate composition of the present disclosure may include a biostimulant component, such as a polyhydroxyalkanoate (PHA) polymer, that promotes cell proliferation, maintenance, development and / or differentiation compared to plants that do not normally produce PHA. The PHA, PHA oligomers and monomers produced in the present invention can serve as nutrients and bioactive substances.

[0035] In some embodiments, the protein hydrolysate composition of the present invention comprises one or more vitamins, such as but not limited to vitamin B1, B2 and / or vitamin B 12 When the culture medium is supplemented with the protein hydrolysate, the vitamins can promote cell culture growth, development and / or differentiation, and / or improve the nutritional value of the cultured food product. The hydrolysate prepared as described herein can contain unique nutrients that cannot be synthesized by plants and are not usually present in plants in appreciable amounts, such as but not limited to vitamin B 12 .

[0036] In some embodiments, when provided in culture medium, the protein hydrolysate composition of the present invention can improve or enhance the flavor of cultured food products. In some embodiments, the protein hydrolysate composition of the present invention has nutritional benefits comparable to or superior to animal, plant and yeast hydrolysates and extracts.

[0037] The present disclosure provides methods for making protein hydrolysates, lysates and / or extracts having many benefits, including but not limited to: consistent and well-defined initial inputs starting from chemoautotrophic conversion of CO2 and / or other C1 inputs to protein-rich biomass; compositional consistency; large-scale production capabilities with a small footprint; no agricultural land displacement; lower water usage compared to hydrolysates of plant or animal origin. In certain embodiments of the present disclosure, no arable land is required in the production of protein hydrolysates, lysates and / or extracts produced as described herein.

[0038] The present disclosure provides acid, alkaline and enzymatic hydrolysis methods for producing hydrolysates enriched in desired fractions, including but not limited to: peptides that serve as a source of available amino acids or that play a specific role in plant or cell health; amino acid cleavage sequence-specific endoproteases or combinations of proteases for protein digestion; controlling peptide composition to produce hydrolysates enriched in peptides of a desired length; peptides having a residue distribution of about 2 to about 10 amino acid residues, which improve the growth and / or yield of some cultured animal cells.

[0039] Certain peptides can act as external molecular signals that affect plants, animals, and microbiomes. Protein hydrolysates can provide nutrients to plants at the root layer, or stimulate beneficial microbial strains in the soil rhizosphere microbiome, thereby indirectly promoting plant growth and productivity. Certain protein hydrolysates, lysates, and / or extracts disclosed herein can stimulate organisms in the plant or animal microbiome, thereby improving disease resistance and / or nutrient uptake.

[0040] The protein hydrolysates, lysates and / or extracts disclosed herein can provide nutrients and bioactive substances to animals through the intestine or stimulate host beneficial strains in the intestinal microbiome.

[0041] Unless otherwise defined in this article, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. The methods and techniques disclosed herein are usually carried out according to conventional methods well known in the art. In general, the nomenclature used in conjunction with the biochemistry, enzymology, molecule and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and its technology are those well known in the art and commonly used. Unless otherwise indicated, the methods and techniques disclosed herein are usually carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification.

[0042] Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, John Wiley and Sons, New York (1994) and Hale and Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in the present invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods, systems, and compositions described herein.

[0043] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the capabilities of those skilled in the art. Such techniques are fully described in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, 1984; Current Protocols in Molecular Biology (FM Ausubel et al., 1994); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); and Gene Transfer and Expression: A Laboratory Manual (Kriegler, 1990).

[0044] Numerical ranges provided herein are inclusive of the numbers defining the range.

[0045] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

[0046] definition

[0047] Unless the context clearly dictates otherwise, "a / an" and "the" include plural referents, so in the specification and claims, the indefinite articles "a / an" and "the" as used herein should be understood to mean "at least one", unless clearly indicated to the contrary.

[0048] As used herein, the term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass deviations of ±5%, ±1%, or ±0.1% of the specified value, as such deviations are appropriate for performing the disclosed methods or as they relate to the disclosed compositions.

[0049] "Acetogen" refers to a microorganism that produces acetic acid and / or other short chain organic acids up to C4 chain length as a product of anaerobic respiration.

[0050] "Acidophile" refers to a type of extremophile microorganism that thrives under highly acidic conditions, typically at pH 2.0 or below.

[0051] The term "amino acid" refers to a molecule containing both an amine group and a carboxyl group bonded to a carbon, which is designated as the α-carbon. Suitable amino acids include, but are not limited to, both the 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, as available for each extended aliphatic or aromatic main chain scaffold. Unless the context clearly indicates otherwise, the term amino acid as used herein is intended to include amino acid analogs. Standard three-letter abbreviations for amino acids are used herein, for example: Cys Cysteine; Gln Glutamine; Glu Glutamic acid (Glutamate); Gly Glycine; His Histidine; Ile Isoleucine; Leu Leucine; Lys Lysine; Met Methionine; Phe Phenylalanine; Pro Proline; Ser Serine; Thr Threonine; Trp Tryptophan; Tyr Tyrosine; Val Valine;

[0052] The phrase "and / or" as used in the specification and claims should be understood to mean "one or both" of the combined elements, i.e., elements that are present in combination in some cases and separately in other cases. In addition to the elements specifically designated by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically designated, unless expressly stated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open language such as "comprising," may refer to A without B (optionally including elements other than B) in one embodiment; to B without A (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; and so on.

[0053] The term "biomass" refers to material produced by the growth and / or reproduction of cells. Biomass may contain cells and / or intracellular contents as well as extracellular material, including but not limited to compounds secreted by cells.

[0054] The term "bioreactor" or "fermenter" refers to a closed or partially closed container in which cells are grown and maintained. Cells can, but are not necessarily maintained in liquid suspension. In some embodiments, in addition to being maintained in liquid suspension, cells may alternatively be grown and / or maintained in contact with, on or within another non-liquid matrix (including but not limited to a solid growth support material).

[0055] "Biostimulant" or "bio-stimulant" refers to a compound that is capable of stimulating the growth, proliferation and / or development of cells when provided in a culture medium and / or an organism when ingested or otherwise provided to the organism in an accessible form.

[0056] The term "carbon fixation" reaction or pathway refers to an enzymatic reaction or metabolic pathway that converts a C1 carbon molecule (including carbon forms that are gaseous at ambient conditions, including but not limited to CO2, CO, and CH4) into a carbon-based biochemical (including biochemical molecules that are liquid or solid at ambient conditions or dissolved into or maintained suspended in an aqueous solution).

[0057] "Carbon source" refers to the type of molecule from which microorganisms obtain the carbon required for organic biosynthesis.

[0058] "Carbon trophic" refers to a microorganism that can tolerate or oxidize carbon monoxide. In preferred embodiments, the carbon trophic microorganism can utilize CO as a carbon source and / or as a source of reduced electrons for biosynthesis and / or respiration.

[0059] "Chemicoautotrophic" refers to the ability of an organism to obtain energy by oxidizing a chemical electron donor via a chemical electron acceptor and to synthesize from carbon dioxide all of the organic compounds required for the organism to survive and grow.

[0060] In the claims and the specification, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", etc. shall be understood as open, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0061] The term "culturing" refers to growing and maintaining a population of cells, such as microbial cells or animal cells, in a liquid or solid medium under conditions suitable for proliferation, propagation, maintenance, development and / or differentiation.

[0062] The term "derived from" encompasses the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that one specified material is derived from, or has characteristics that may be described with reference to, another specified material.

[0063] "Energy source" refers to an electron donor that is oxidized by oxygen in aerobic respiration or a combination of an oxidized electron donor and a reduced electron acceptor in anaerobic respiration.

[0064] "Extremophiles" refer to microorganisms that thrive under physical or geochemical extreme conditions (e.g., high or low temperature, pH, or high salinity) on land or in the ocean compared to those normally tolerated by most life forms found on or near the terrestrial surface.

[0065] The term "gasification" refers to the overall high temperature process of converting a carbon-based material into a gas mixture comprising hydrogen, carbon monoxide, and carbon dioxide (referred to as synthesis gas, syngas, or producer gas). The process typically involves partial combustion and / or the application of externally generated heat and the controlled addition of oxygen and / or steam such that insufficient oxygen is present to completely burn the carbon-based material.

[0066] "Halophile" refers to a type of extremophile microorganism that thrives in environments with very high salt concentrations.

[0067] "Heterotrophic" refers to a mode of growth and maintenance in which an organism ingests and metabolizes organic matter, such as plant, animal, or microbial matter. Growth is heterotrophic when the organism is unable to synthesize from carbon dioxide all of the organic compounds required for the organism to survive and grow and utilize organic compounds. During heterotrophic growth, organisms are unable to produce their own food, but instead obtain food and energy by ingesting and metabolizing organic matter, such as plant or animal matter, i.e., rather than fixing carbon from inorganic sources such as carbon dioxide.

[0068] "Hydrogen-oxidizer" refers to a microorganism that utilizes reduced H2 as an electron donor for the production of intracellular reducing equivalents and / or in respiration.

[0069] "Hyperthermophile" refers to a type of extremophile microorganism that thrives in extremely hot environments, typically about 60°C (140°F) or higher.

[0070] The term "knallgas" refers to a mixture of molecular hydrogen and oxygen. An "oxyhydrogen microorganism" is a microorganism that can use hydrogen as an electron donor and oxygen as an electron acceptor in respiration to produce intracellular energy carriers such as adenosine-5'-triphosphate (ATP). The terms "oxyhydrogen" and "oxyhydrogen microorganism" may be used synonymously with "knallgas" and "knallgas microorganism," respectively. Oxyhydrogen microorganisms typically use molecular hydrogen via hydrogenases, in which some of the electrons are used to reduce NAD + (and / or other intracellular reducing equivalents) H2 is provided, and some electrons come from H2 used for aerobic respiration. Hydrogen-oxygen mixed gas microorganisms usually fix CO2 autotrophically through pathways including but not limited to the Calvin Cycle or the reverse citric acid cycle ["Thermophilic bacteria", Jakob Kristjansson, Chapter 5, Section III, CRC Press, (1992)].

[0071] The term "lysate" refers to a liquid containing a mixture and / or solution of cell contents produced by cell lysis. In some embodiments, the lysate can be dehydrated to form a concentrated lysate, or dried to form a dry solid. In certain such embodiments, the dry lysate is in powder form. In some embodiments, the methods described herein include purifying a chemical substance or a mixture of chemical substances in a cell lysate. In some embodiments, the methods include purifying amino acids and / or proteins in a cell lysate.

[0072] The term "lysis" refers to the rupture of the plasma membrane and (if present) cell wall of a cell, allowing a large amount of intracellular material to escape to the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal or viral means. In some embodiments, the methods described herein include performing lysis of cells or microorganisms as 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 performing lysis of cells or microorganisms as described herein to separate an amino acid or a mixture of amino acids and / or proteins and / or peptides from the non-protein contents of a bioreactor or cell growth medium.

[0073] "Methanogen" refers to a microorganism that produces methane as a product of anaerobic respiration.

[0074] "Methylotroph" refers to a microorganism that can grow using reduced one-carbon compounds (including methanol or methane) as a carbon source and / or as an electron donor.

[0075] "Methanotroph" refers to a microorganism that can metabolize methane as a carbon source and / or as an electron donor for growth.

[0076] The terms "microorganism" and "microbe" refer to microscopic single-celled life forms.

[0077] The term "molecule" means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes, for example, hydrocarbons, lipids, polypeptides, and polynucleotides.

[0078] A "nutritionally fastidious" strain refers to an organism that has complex or specific nutritional requirements, for example, an organism that will only grow in the presence of a specific nutrient.

[0079] "Oligopeptide" refers to a peptide containing a relatively small number of amino acid residues, for example, about 2 to about 20 amino acids.

[0080] As used in this specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one element in a plurality of elements or a list of elements, but also including more than one element, and optionally including items not listed in addition. Only terms such as "only one of" or "just one of" are explicitly pointed out to the contrary, otherwise when "consisting of" is used in the claims, it will refer to including just one element in a plurality of elements or a list of elements. In general, when there are exclusive terms such as "any", "one of", "only one of" or "just one of" in front, the term "or" as used herein should only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both"). "Substantially consisting of" when used in the claims should have its usual meaning used in the field of patent law.

[0081] The term "organic compound" refers to any gaseous, liquid, or solid chemical compound containing carbon atoms, except the following, which are considered inorganic: carbides, carbonates, simple oxides of carbon, cyanides, and allotropes of pure carbon such as diamond and graphite.

[0082] "Peptide" refers to a compound composed of two or more amino acids linked in a chain, the carboxyl group of each acid being linked to the amino group of the next amino acid by a bond of the type R-OC-NH-R', and may contain from about 2 to about 50 amino acids.

[0083] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides having any length and any three-dimensional structure and single-stranded or multi-stranded (e.g., single-stranded, double-stranded, triple helix, etc.), which contains deoxyribonucleotides, ribonucleotides and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present invention encompasses polynucleotides encoding a specific amino acid sequence. Any type of modified nucleotides or nucleotide analogs can be used, as long as the polynucleotide retains the desired function under the conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-O-Me, thiophosphate, etc.). For the purpose of detection or capture, a label, such as a radioactive or non-radioactive label or anchor, such as biotin, may also be incorporated. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably herein. The polynucleotide may contain RNA, DNA or both, and / or modified forms and / or analogs thereof. The sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), wherein each R or R' is independently H or optionally substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl containing an ether (--O--) linkage. Not all linkages in a polynucleotide need to be identical. A polynucleotide may be linear or cyclic or comprise a combination of a linear portion and a cyclic portion.

[0084] As used herein, "polypeptide" refers to a composition composed of amino acids and recognized as a protein by those skilled in the art. Conventional single-letter or three-letter codes for amino acid residues can be used. The terms "polypeptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, one or more analogs containing amino acids (including, for example, non-natural amino acids, etc.) and other modified polypeptides known in the art.

[0085] The term "precursor to" or "precursor of" is an intermediate used to produce one or more components of a finished product.

[0086] The term "probiotic" refers to microorganisms that provide health benefits when consumed, such as beneficial intestinal flora.

[0087] "Producer gas" refers to a gas mixture containing H2, CO and CO2 in varying proportions and having a calorific value per unit volume under standard conditions generally ranging between one-half and one-tenth of that of natural gas. Producer gas can be produced from a variety of raw materials by various means, including gasification, steam reforming or autoreforming of carbon-based raw materials. In addition to H2, CO and CO2, producer gas may contain other components, including but not limited to methane, hydrogen sulfide, condensable gases, tar and ash, depending on the production process and raw materials. The proportion of N2 in the mixture can be high or low, depending on whether air is used as an oxidant in the reactor and whether the heat for the reaction is provided by direct combustion or by indirect heat exchange.

[0088] The term "producing" includes both the production of a compound inside a cell and outside of a cell, including secretion of the compound from a cell.

[0089] "Psychrophile" refers to a type of extremophile microorganism that is able to grow and reproduce at low temperatures (usually about 10°C or lower).

[0090] The term "recombinant" refers to genetic material (i.e., nucleic acids, polypeptides encoded by nucleic acids, and vectors and cells comprising such polynucleotides) that has been modified to change its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to the coding sequence of another gene, placing the gene under the control of a different promoter, expressing the gene in a heterologous organism, expressing the gene at a reduced or elevated level, expressing the gene conditionally or constitutively in a manner different from its natural expression profile, etc. Typically, recombinant nucleic acids, polypeptides, and cells based thereon have been manipulated by humans so that they are different from the naturally occurring related nucleic acids, polypeptides, and cells. Recombinant cells may also be referred to as "engineered".

[0091] As used herein, the terms "recovered," "isolated," "purified," and "separated" refer to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated. For example, these terms may refer to a material that is substantially or essentially free of components that normally accompany it as found in its native state (e.g., as an intact biological system).

[0092] For any given component, the phrase "substantially free" means that such component is present only in a functionally insignificant amount, if any, i.e., it does not significantly negatively affect the intended performance or function of any process or product. Typically, substantially free means less than about 1%, including less than about 0.5%, including less than about 0.1%, and also including 0%, by weight of such component.

[0093] "Sulfur-oxidizer" refers to a microorganism that utilizes reduced sulfur-containing compounds (including but not limited to H2S) as electron donors for the production of intracellular reducing equivalents and / or in respiration.

[0094] "Syngas" or "Synthesis gas" refers to a type of gas mixture that contains H2 and CO like producer gas, but is more specifically tailored in terms of the ratios and levels of H2 and CO content and impurities for the synthesis of a particular type of chemical product, such as, but not limited to, methanol or Fischer-Tropsch diesel. Syngas typically contains H2, CO, CO2 as major components, and it can be produced by established methods, including: steam reforming of methane, liquefied petroleum gas, or biogas; or by gasification of any organic, flammable, carbon-based material, including, but not limited to, biomass, organic waste, various polymers, peat, and coal. The hydrogen component of the syngas can be increased by the reaction of CO with steam in the water-gas shift reaction, while increasing the CO2 in the syngas mixture.

[0095] "Thermophile" refers to a type of extremophile microorganism that thrives throughout its life at relatively high temperatures (usually about 45°C to about 122°C).

[0096] "Titer" refers to the amount of a substance produced by a microorganism per unit volume in a microbial culture. For example, biomass titer can be expressed as grams of biomass produced per liter of solution (eg, culture medium).

[0097] A "vitamin" is a compound, eg, an organic compound, essential for the growth and / or nutrition of an organism, usually required in small amounts in the diet or in a growth or culture medium.

[0098] As used herein, "vitamin equivalents" refer to chemical analogs of a particular vitamin that effectively replace one another in function and / or effectively alleviate the deficiency of the vitamin.

[0099] "Wild type" refers to a microorganism that occurs in nature.

[0100] "Yield" refers to the amount of product produced from a feed material relative to the total amount of material that would be produced if all of the feed material was converted to product. For example, if 100% of the feed material is converted to amino acids, the amino acid yield can be expressed as the % of amino acids produced relative to the theoretical yield.

[0101] Method for producing a protein hydrolysate composition

[0102] Cultivation of microorganisms

[0103] In certain embodiments, the method of the present disclosure includes cultivating microorganisms (e.g., chemoautotrophic microorganisms) under conditions suitable for microbial growth and biomass production in a bioreactor or fermentor, and the biomass can then be converted into a protein hydrolyzate composition. Any suitable method can be used to cultivate microorganisms. Microorganisms can grow under any suitable conditions in an environment suitable for the growth and production of biomass. In some embodiments, microorganisms can grow in autotrophic culture conditions, heterotrophic culture conditions, or a combination of autotrophic and heterotrophic culture conditions. Heterotrophic cultures can include suitable carbon sources and energy sources, such as one or more sugars (e.g., glucose, fructose, sucrose, etc.). The autotrophic culture may contain C1 chemicals, such as carbon monoxide, carbon dioxide, methane, methanol, formate and / or formic acid, and / or mixtures containing C1 chemicals, including but not limited to, for example, various syngas compositions or various producer gas compositions produced by gasification, partial oxidation, pyrolysis or steam reforming of low-value carbon sources and energy sources, such as but not limited to lignocellulosic energy crops, crop residues, bagasse, sawdust, forestry residues or food, which can be used as carbon sources and energy sources by hydrogen-oxidizing microorganisms or hydrogen-oxidizing microorganisms or carbon monoxide oxidizing microorganisms. Suitable means for cultivating microorganisms and producing biomass for use in the methods of the present invention are described in, for example, PCT Application Nos. PCT / US2010 / 001402, PCT / US2011 / 034218, PCT / US2013 / 032362, PCT / US2014 / 029916, PCT / US2017 / 023110, PCT / US2018 / 016779, and U.S. Patent No. 9,157,058, each of which is hereby incorporated by reference in its entirety. In some embodiments, the organism can be grown photosynthetically in a bioreactor, a hydroponic system, a greenhouse, or cultivated land, or can be collected from waste or natural resources.

[0104] Any suitable bioreactor or fermenter may be used to culture the microorganisms. Suitable bioreactors include, but are not limited to, one or more of the following: an airlift reactor; a bioscrubber; a bubble column; a stirred tank reactor; a continuous stirred tank reactor; a countercurrent, upflow, expanded bed reactor; a digester as known in the prior art of sewage and wastewater treatment or bioremediation, and in particular a digester system; a filter, including but not limited to a trickling filter, a rotating biocontactor filter, a rotating disk, a soil filter; a fluidized bed reactor; an airlift fermenter; an immobilized cell reactor; a loop reactor; a membrane biofilm reactor; a pachuca tank; a packed bed reactor; a plug flow reactor; a static mixer; a trickle bed reactor; and / or a vertical axis bioreactor.

[0105] In some embodiments, the microorganisms are grown and maintained in a medium suitable for chemoautotrophic growth, the medium containing a gaseous carbon and energy source, such as, but not limited to, syngas, producer gas, tail gas, pyrolysis gas, or a gas mixture of H2 and CO2 and / or CO. Light is not required in chemoautotrophic CO2 fixation, and in certain embodiments, there is little or no light in the growth environment.

[0106] In an exemplary but non-limiting embodiment, a bioreactor containing a nutrient medium is inoculated with production cells. Generally speaking, there will be a lag phase before the cells begin to double. After the lag phase, the cell doubling time decreases, and the culture enters the logarithmic phase. After the logarithmic phase, it is ultimately an increase in doubling time, although it is not intended to be limited by theory, the increase in doubling time is considered to be caused by mass transfer limitations, the depletion of nutrients including nitrogen or mineral sources, or the increased concentration of inhibitory chemicals or the quorum sensing of microorganisms. When the culture enters the stationary phase, growth slows down and then stops. In certain embodiments, there is an arithmetic growth period before the stationary phase. In order to harvest cell masses, in certain embodiments, the culture is harvested in the logarithmic phase and / or in the arithmetic phase and / or in the stationary phase.

[0107] The growth conditions in the bioreactor can be controlled, including control of dissolved gases such as carbon dioxide, oxygen and / or other gases such as hydrogen and other dissolved nutrients, trace elements, temperature and pH. For certain embodiments, the protein-rich cell mass is grown to high density and / or at high productivity in a liquid suspension within a bioreactor.

[0108] Nutrient medium and gas can be added to the bioreactor as batch addition or regularly, or added in response to detected depletion or programmed set point addition, or added continuously during culture growth and maintenance. For some embodiments, the bioreactor at the time of inoculation is filled with a starting batch of nutrient medium and / or one or more gases at the beginning of growth, and no additional nutrient medium and / or one or more gases are added after inoculation. For some embodiments, nutrient medium and / or one or more gases are added regularly after inoculation. For some embodiments, nutrient medium and / or one or more gases are added after inoculation in response to detected depletion of nutrients and / or gases. For some embodiments, nutrient medium and / or one or more gases are added continuously after inoculation.

[0109] For certain embodiments, the added nutrient medium does not contain any organic compounds, for example, does not contain an organic carbon source such as a sugar molecule or other organic molecule that can be metabolized by a microorganism as a carbon source.

[0110] In certain embodiments, a small number of microbial cells (ie, inoculum) is added to a set volume of culture medium; the culture is then incubated; and the cell mass undergoes a lag phase, an exponential phase, a deceleration phase, and a stationary phase of growth.

[0111] In batch culture systems, the conditions (e.g., nutrient concentration, pH, etc.) for cultivating microorganisms are usually constantly changing throughout the growth period. In certain non-limiting embodiments, in order to avoid inherent fluctuating conditions in batch cultures, and in order to improve the overall productivity of the culture system, the microorganisms used to produce proteins and / or vitamins and / or other nutrients are grown in a continuous culture system called a chemostat (e.g., a bioreactor or other culture container, fresh culture medium is continuously added to the bioreactor or other culture container, while the culture fluid containing residual nutrients, metabolic end products and microorganisms is continuously removed at the same rate to keep the culture volume constant). In certain embodiments, the microorganisms used to produce proteins and / or vitamins and / or other nutrients are grown in a continuous culture system called a turbidostat (e.g., a continuous microbial culture device, which has feedback between the turbidity of the culture container and the dilution rate).

[0112] For certain embodiments, the bioreactor has a mechanism capable of mixing the nutrient medium, including, but not limited to, one or more of: rotating agitator bars, blades, impellers, or turbines; rotating, shaking, or turning the container; gas lift, sparging; recirculation of broth from the bottom of the container to the top through a recirculation line so that the broth flows through a loop and / or a static mixer. The medium can be mixed continuously or intermittently.

[0113] In certain embodiments, the nutrient medium containing the microorganisms may be partially or completely, periodically or continuously removed from the bioreactor and, in certain embodiments, replaced with fresh cell-free medium to maintain the cell culture in exponential growth phase and / or arithmetic growth phase, and / or to replenish nutrients depleted in the growth medium, and / or to remove inhibitory waste products.

[0114] The port of standard in bioreactor can be used for gas, liquid, solid and / or slurry delivery to the bioreactor container of enclosed microorganism and / or take out from the bioreactor container of enclosed microorganism.Many bioreactors have multiple ports for different purposes (for example, for culture medium addition, gas addition, for pH and dissolved oxygen (DO) probe and sampling port), and during the fermentation operation process, a given port can be used for various purposes.For example, the port can be used to add nutrient medium to the bioreactor at a certain time point, and can be used for sampling at another time point.In some embodiments, the multiple use of sampling port can be performed when pollution or invasive species are not introduced into the growth environment.Valve or other actuators that can control sample flow or continuous sampling can be provided to the sampling port.For some embodiments, the bioreactor is equipped with at least one port suitable for culture inoculation, and the port can be used for other purposes in addition, including adding culture medium or gas.The bioreactor port can control the gas composition and flow rate entering the culture environment.For example, the port can be used as the gas inlet entering the bioreactor, and the gas is pumped by the gas inlet.

[0115] For some embodiments, the gases that can be pumped into the bioreactor include, but are not limited to, one or more of the following: syngas, producer gas, pyrolysis gas, hydrogen, CO, CO2, O2, air, air / CO2 mixtures, natural gas, biogas, methane, ammonia, nitrogen, inert gases (such as argon), and other gases. In some embodiments, the CO2 pumped into the system may come from sources including, but not limited to, CO2 from gasification of organic matter; CO2 from 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 of anaerobic or aerobic fermentation of sugars and / or any other organic carbon substrate for fermentation; CO2 byproducts of methanotrophic biological processes; CO2 byproducts of carbon-trophic biological processes; CO2 byproducts from heterotrophic metabolism; CO2 from wastewater treatment; CO2 byproducts from sodium phosphate production; CO2 produced or emitted geologically or geothermally; CO2 removed from acid gases or natural gas. In certain non-limiting embodiments, CO2 has been removed from industrial flue gases, or captured from geological sources that would otherwise be naturally emitted to the atmosphere. In certain embodiments, the carbon source is CO2 and / or bicarbonate and / or carbonate dissolved in seawater or other surface water or groundwater bodies. In certain such embodiments, inorganic carbon can be introduced into a 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 has been captured from a closed cabin as a part of a closed loop life support system using equipment such as, but not limited to, a CO2 removal assembly (CDRA), which is used, for example, on the International Space Station (ISS).

[0116] In certain non-limiting embodiments, geological features such as, but not limited to, emissions of high concentrations of energy sources (e.g., H2, H2S, CO gases) and / or carbon sources (e.g., CO2, HCO3 - 、CO3 2- ) and / or other dissolved minerals can be used as a source of nutrients for the microorganisms herein.

[0117] In certain embodiments, one or more gases in addition to or in place of carbon dioxide as an alternative carbon source are dissolved into solution and supplied to the culture broth and / or dissolved directly into the culture broth, including but 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 contain other electron donors and / or electron acceptors and / or carbon sources and / or mineral nutrients, such as but not limited to other gaseous components and impurities of syngas (e.g., hydrocarbons); ammonia; hydrogen sulfide; and / or other acid gases; and / or O2; and / or mineral-containing particulates and ash.

[0118] After passing through the reactor system housing the gas-ingesting microorganisms, the residual gas may, in certain embodiments, be recycled back to the bioreactor, or burned to obtain process heat, or flared, or injected underground, or released to the atmosphere. In certain embodiments herein using H2 as an electron donor, it may be supplied to the culture vessel by bubbling H2 through the culture medium or by diffusing it through a hydrogen permeable-water impermeable membrane interfacing with the liquid culture medium as known in the art.

[0119] In certain embodiments, C1 molecules such as but not limited to carbon dioxide, carbon monoxide, methane, methanol, formaldehyde, formate or formic acid, and / or mixtures containing C1 molecules, including but not limited to various syngas compositions produced by various gasified, pyrolyzed or steam reformed fixed carbon raw materials, are used as carbon sources by microorganisms and biochemically converted to longer chain organic molecules (i.e., C2 or longer, and in some embodiments, C5 or longer carbon chain molecules) under one or more of the following conditions: aerobic, microaerobic, anoxic, anaerobic and / or facultative conditions. In some embodiments, gaseous CO2 is used as a carbon source by microorganisms and is chemoautotrophically converted to longer chain organic molecules (i.e., C2 or longer, and in some embodiments, C5 or longer carbon chain molecules) under aerobic, microaerobic, anoxic, anaerobic and / or facultative conditions. In some embodiments, H2 is used as an electron donor and O2 is used as an electron acceptor for carbon fixation and conversion of C1 carbon molecules to longer chain organic molecules. In some embodiments, H2 is used as an electron donor and O2 is used as an electron acceptor for chemoautotrophic carbon fixation and conversion of CO2 to longer chain organic molecules.

[0120] In certain embodiments, the organic carbon source is used as a carbon source and / or reduced electrons in cell metabolism. In certain embodiments, the growth and metabolism are heterotrophic or mixotrophic.

[0121] In certain embodiments, one or more of the following parameters in the bioreactor are monitored and / or controlled: waste levels; pH; temperature; salinity; dissolved oxygen; dissolved carbon dioxide gas; liquid flow rate; agitation rate; gas pressure. In certain embodiments, operating parameters that affect chemoautotrophic growth are monitored with sensors (e.g., dissolved oxygen probes or oxidation-reduction probes to measure electron donor / acceptor concentrations) and / or controlled manually or automatically based on feedback from sensors using equipment including, but not limited to, actuated valves, pumps, and agitators. In certain embodiments, the temperature of the incoming broth and the incoming gas is regulated by a system such as, but not limited to, a cooler, a heater, and / or a heat exchanger.

[0122] In some embodiments, the protein production and distribution of the amino acid molecules produced by microorganisms are optimized by one or more of the following: control of bioreactor conditions, control of nutrient levels and / or genetic modification of cells. In certain embodiments, by maintaining specific growth conditions (for example, nitrogen, oxygen, phosphorus, sulfur, trace micronutrients such as inorganic ions and (if present) the level of any regulatory molecules that may not usually be considered as nutrients or energy sources) to control and optimize the approach of amino acids, or proteins, or other nutrients or whole cell products, to produce chemical products. In certain embodiments, dissolved oxygen (DO) can be optimized by maintaining broth under aerobic, microaerobic, anoxic, anaerobic or facultative conditions, depending on the needs of microorganisms. Facultative environments are considered to include aerobic upper layers and anaerobic lower layers caused by spatial separation of water column stratification or aerobic or microaerobic regions, and anaerobic regions caused by spatial separation of regions exposed to O2 gas and regions not exposed to O2 gas.

[0123] In some embodiments, microorganisms (e.g., chemoautotrophic microorganisms) are grown under conditions that favor microbial accumulation of polyhydroxyalkanoates (PHAs), such as polyhydroxybutyrate (PHB) and / or polyhydroxyvalerate (PHV). In some embodiments, microorganisms (e.g., chemoautotrophic microorganisms) are grown under the limitation of one or more nutrients (e.g., nitrogen or phosphorus limitation) to cause accumulation of PHAs (e.g., PHB; PHV). In some embodiments, microorganisms (e.g., chemoautotrophic microorganisms grown on H2 / CO2 and / or syngas) accumulate PHAs, such as PHB and / or PHV, in cell biomass. In some embodiments, PHAs (e.g., PHB; PHV) are accumulated to about 50% by weight or more, about 60% by weight or more, or about 70% by weight or more of the microbial biomass.

[0124] In certain embodiments, a microorganism (e.g., a chemoautotrophic microorganism) is grown under conditions that promote the production of vitamins by the microorganism, such as, but not limited to, B vitamins, e.g., vitamin B1, vitamin B2, and / or vitamin B 12 In some embodiments, the microorganism can grow chemoautotrophically to produce one or more vitamins, such as vitamin B1, vitamin B2 and / or vitamin B 12 .

[0125] The biomass produced by the cultured microorganisms (e.g., chemoautotrophic microorganisms) can be harvested using any suitable method, and a protein hydrolysate can then be prepared from the harvested biomass. In some embodiments, the biomass is separated from the liquid culture medium using a suitable method. Suitable methods include, but are not limited to, centrifugation; flocculation; flotation; filtration using a membrane, hollow fiber, spiral wound, or ceramic filtration system; vacuum filtration; tangential flow filtration; clarification; sedimentation; hydrocyclone. In certain embodiments in which the microbial cell mass can be fixed on a substrate, it can be harvested by methods including, but not limited to, gravity sedimentation or filtration, and separated from the growth substrate by scraping or liquid shear forces.

[0126] In certain embodiments, the harvested microbial cells can be broken open to prepare lysates using well-known methods including, but not limited to, one or more of the following: ball milling, cavitation pressure, ultrasonic treatment, homogenization, or mechanical shearing. In some embodiments, the cells in the biomass can be lysed by one or more freeze-thaw cycles, lysing enzymes, detergents, solvents, or antibiotics.

[0127] In some embodiments, the harvested biomass can be dried in one or more process steps. In certain embodiments, the biomass drying can be carried out using any suitable method, including but not limited to one or more of the following: centrifugation, drum drying, evaporation, freeze drying, heating, spray drying, vacuum drying and / or vacuum filtration. In certain embodiments, waste heat can be used for drying biomass. In certain embodiments, waste heat from the industrial flue gas source used as a carbon source can be used for drying biomass. In certain embodiments, the hot byproducts from the generation of electron donors and / or C1 carbon sources can be used for drying biomass. In certain embodiments, the hot byproducts from gasification, methane stream reforming, autoreforming or partial oxidation can be used for drying biomass.

[0128] In certain embodiments, biomass is further processed after drying or in the absence of previous drying step, to help separate and produce useful biochemicals. In certain embodiments, this extra processing relates to separation of protein or lipid content or vitamins or nucleic acid or other targeted biochemicals from microbial biomass. In certain embodiments, the separation of lipid can be carried out by using non-polar or polar solvent to extract lipid, and the solvent is such as but not limited to one or more of the following: hexane, cyclohexane, dodecane, ether, alcohol (methanol, isopropanol, ethanol etc.), tributyl phosphate, supercritical carbon dioxide, trioctyl phosphine oxide, ammonia, secondary amine and tertiary amine, propane, acetone, propylene carbonate, methylene dichloride or chloroform. In certain embodiments, solvent can be used to extract other useful biochemicals, and the solvent includes but not limited to one or more of the following: chloroform, methylene dichloride, acetone, ethyl acetate, propylene carbonate and tetrachloroethylene. In certain embodiments, cell lysis is carried out to separate and produce useful biochemicals.

[0129] In some embodiments, at least a portion of the microbial biomass is not subjected to proteolysis, and the product is or comprises a lysate of microbial cells. In some embodiments, there is no separation or drying step applied to lysate and / or hydrolysate, and the lysate and / or hydrolysate is a crude mixture of soluble components and insoluble components. In some such embodiments, the lysate and / or hydrolysate is unclarified and / or turbid. In some embodiments, there is a solid-liquid separation step applied to the lysate and / or hydrolysate, thereby producing soluble products and insoluble by-products. In some embodiments, the soluble lysate or hydrolysate product is clarified and / or not turbid. In some embodiments, the lysate or hydrolysate is passed through ultrafiltration. In some such embodiments, ultrafiltration has a molecular weight cutoff of about 10,000 or less. In some embodiments, the lysate and / or hydrolysate is subjected to one or more of the following downstream processes: centrifugation; plate and frame filtration; microfiltration; ultrafiltration; nanofiltration; ion exchange chromatography. In certain embodiments, the lysate and / or hydrolysate is passed through a filter comprising one or more carbon grades. In certain such embodiments, carbon removes color from the lysate and / or hydrolysate. In certain embodiments, the lysate and / or hydrolysate and / or the filtrate of the lysate and / or hydrolysate is passed through ion exchange chromatography, for example, to reduce salt content.

[0130] In certain embodiments, a lysate and / or hydrolysate as disclosed herein is sterile filtered prior to use in the growth of another cell culture.

[0131] In certain embodiments, the lysate and / or hydrolysate and / or filtrate thereof is concentrated using one or more of: falling film evaporator; rising film evaporator; membrane distillation, nanofiltration; reverse osmosis.

[0132] In certain embodiments, one or more lysates and / or hydrolysates and / or extracts and / or concentrates and / or isolates as described herein are used in industrial fermentation and / or dehydrated culture medium and / or cell culture applications. In certain embodiments, lysates and / or hydrolysates and / or peptide compositions and / or amino acid compositions as described herein are subjected to ultrafiltration to remove higher molecular weight materials. In certain embodiments, cell cultures grown on a culture medium comprising this ultrafiltration product are superior to cell cultures grown on an unfiltered equivalent. In certain embodiments, one or more lysates and / or hydrolysates as described herein are used for the growth of animal cells in culture. In certain such embodiments, animal cells are mammalian cells. In certain embodiments, cell cultures are cultivated using lysates and / or hydrolysates as described herein, which produce proteins and / or tissues for forming meat products. In certain such embodiments, meat products are produced for human consumption.

[0133] In certain embodiments, a cell culture is grown using a lysate and / or hydrolysate as described herein, which produces one or more pharmaceutical products. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are substituted for various natural or recombinant cells (such as prokaryotic cells) for growing one or more animal-derived components in the culture medium for producing nutrients and / or biopharmaceuticals. In certain embodiments, such natural or recombinant prokaryotes include but are not limited to one or more of the following: Bacillus subtilis (Bacillus subtilis); Corynebacterium ammoniagenes (Corynebacterium ammoniagenes); Pseudomonas species (Pseudomonas sp.); Streptomyces lividans (Streptomyces lividans). In certain embodiments, pharmaceutical products include, but are not limited to, one or more of the following: antibiotics, such as, but not limited to, cephalosporins and cephamycins; anticoagulants; blood factors; vaccines; polysaccharide vaccines; recombinant vaccines; recombinant proteins; antibodies; cytokines, such as, but not limited to, interleukin-11, human granulocyte colony stimulating factor (hG-CSF); fusion proteins; growth factors; interferons; coagulation factors; hormones, such as, but not limited to, human growth hormone, insulin, gonadotropin-releasing hormone, human parathyroid hormone; monoclonal antibodies; nucleic acids; therapeutic enzymes, such as, but not limited to, human tissue plasminogen activator; plasmin; therapeutic proteins, such as, but not limited to, transforming growth factor-α-pseudomonas exotoxin fusion protein (TGF-α-PE40), human epidermal growth factor (hEGF). In certain embodiments, cell cultures are grown using lysates and / or hydrolysates as described herein, which produce recombinant proteins. In certain embodiments, monoclonal antibodies produced using culture medium components (e.g., microbial lysates and / or hydrolysates) as described herein include, but are not limited to, one or more of the following: Herceptin, Remicade, Rituxan, Synagis. In certain embodiments, lysates or hydrolysates as described herein are used to replace serum or serum-derived components, including fetal calf serum (FCS).

[0134] In certain embodiments, whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors produced as described herein are supplied to one or more other organisms or cells (e.g., one or more organisms or cells different from the microorganism from which the whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors are derived), including but not limited to one or more of the following: Actinomycetes, Aspergillus awamori, Aspergillus fumigates, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus foetidus, Bacillus alkaliphilic alkalophilus), Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilis, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Escherichia coli (E.coli), Escherichia coli strain B, Escherichia coli strain C, Escherichia coli strain K, Escherichia coli strain, Streptomyces lividans, Streptomyces murinus, Trichoderma atroviride, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Humicola insolens, Humicola lanuginose, Mucor miehei, Rhizomucor miehei, Rhodococcus opacus, 293 cells, 3T3 cells, BHK cells, CHO cells, COS cells, Cvl cells, HeLa cells, MDCK cells, P12 cells, VERO cells.

[0135] In certain embodiments, whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors produced as described herein are supplied to one or more other organisms or cells (e.g., one or more organisms or cells different from the microorganism from which the whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors are derived), including but not limited to one or more members of the following genera: Aspergillus, Bacillus, Chrysosporium, Escherichia, Fusarium, Humicola, Kluyveromyces. veromyces, Lactobacillus, Mucor, Myceliophtora, Neurospora, Penicillium, Phanerochaete, Pichia, Pleurotus, Pseudomonas, Rhizomucor, Rhodococcus, Saccharomyces, Schizosaccharomyces, Stenotrophamonas, Streptomyces, Trametes, Trichoderma, and Yarrowia.

[0136] In certain embodiments, whole-cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors produced as described herein are supplied to one or more other organisms or cells (e.g., one or more organisms or cells different from the microorganism from which the whole-cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors were derived) that are beneficial to the plant and / or are beneficial members of the plant microbiome and / or rhizosphere and / or are beneficial soil organisms, including, but not limited to, one or more of: Trichoderma atroviride, Azospirillum brasilense, Bradyrhizobium japonicum, and / or mycorrhizal fungi, including arbuscular mycorrhizal fungi.

[0137] In certain embodiments, whole-cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors produced as described herein are supplied to one or more other organisms or cells (e.g., one or more organisms or cells different from the microorganism from which the whole-cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors are derived), including but not limited to one or more of the following: archaeal cells, bacterial cells (including Gram-negative bacteria and / or Gram-positive bacteria), filamentous fungal cells, fungal cells, insect cells, mammalian cells, animal cells, plant cells, yeast cells.

[0138] In certain embodiments, whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or PHB and / or hydroxybutyrate and / or vitamins and / or other nutrients or cofactors produced as described herein are provided to a cell culture as a nutrient source for producing one or more microbial or chemical products, such as, but not limited to, one or more of the following: polysaccharides, lipids, biodiesel, butanol, ethanol, propanol, isopropanol, propane, alkanes, olefins, aromatics, fatty alcohols, fatty acid esters, alcohols, 1,3-propanediol, 1,3-butadiene, 1,3-butanediol, 1,4-butanediol, 3-hydroxypropionate, 7-ADCA / cephalosporin, ε-caprolactone, γ-valerolactone, acrylates, acrylic acid, adipic acid, ascorbate esters, aspartate esters, ascorbic acid, aspartic acid, caprolactams, carotenoids, citrates, citric acid , DHA, docetaxel, erythromycin, ethylene, gamma butyrolactone, glutamate, glutamic acid, HPA, hydroxybutyrate, isopentenol, isoprene, isoprenoid, itaconate, itaconic acid, lactate, lactic acid, lanosterol, levulinic acid, lycopene, lysine, malate, malonic acid, peptides, ω-3 DHA, ω-3 EPA, ω-3 ALA, ω fatty acids, ω-7 fatty acids, ω-7 rich oils, paclitaxel, PHA, PHB, polyketides, polyols, propylene, pyrrolidone, serine, sorbitol, statins, steroids, succinates, terephthalates, terpenes, THF, rubber, wax esters, polymers, commodity chemicals, industrial chemicals, specialty chemicals, paraffin substitutes, additives, nutritional supplements, nutraceuticals, pharmaceuticals, pharmaceutical intermediates, personal care products, commercial enzymes, antibiotics, amino acids, vitamins, bioplastics, glycerol, jet fuel, diesel, gasoline, octane.

[0139] The use of chemoautotrophic microorganisms for producing proteins, amino acids and other nutrients from gaseous feedstocks comprising H2 and / or CO2 and / or CO and / or CH4 is described in, for example, international patent application number PCT / US17 / 23110 received on March 18, 2017 and entitled MICROORGANISMS AND ARTIFICIAL ECOSYSTEMS FOR THE PRODUCTION OF PROTEIN, FOOD, AND USEFUL CO-PRODUCTS FROM C1 SUBSTRATES. This application is incorporated herein by reference in its entirety for all purposes.

[0140] The use of chemoautotrophic microorganisms for producing plant, animal and human nutrients from gaseous feedstocks comprising H2 and / or CO2 and / or CO and / or CH4 is described, for example, in PCT Application No. PCT / US2018 / 016779, filed on February 4, 2018 and entitled VEGAN NUTRIENTS, FERTILIZERS, BIOSTIMULANTS, AND SYSTEMS FOR ACCELERATED SOIL CARBON SEQUESTRATION, which is incorporated herein by reference in its entirety.

[0141] The production of protein hydrolysates from microbial sources is described in PCT Application No. PCT / US20 / 50902, filed on September 15, 2020, and entitled MICROBIALPROTEIN HYDROLYSATE COMPOSITIONS AND METHODS OF MAKING SAME, which is incorporated herein by reference in its entirety.

[0142] In certain non-limiting embodiments, lysate and / or hydrolysate as described herein are concentrated to about 25% to about 50% solids. In certain such embodiments, the drying step is after being concentrated to about 25% to about 50% solids. In certain embodiments, lysate and / or hydrolysate are in the form of pumpable concentrates. In certain such embodiments, pumpable lysate and / or hydrolysate are about 25% to about 60% solids. In other such embodiments, pumpable lysate and / or hydrolysate are about 60% or more solids. In certain embodiments, concentrated lysate and / or hydrolysate have sufficiently low water activity to make it stable on microorganisms. In certain embodiments, lysate and / or hydrolysate and / or their filtrate and / or supernatant and / or concentrate are pumped through a cartridge filter to remove any larger particles. In certain embodiments, the lysate and / or hydrolysate and / or their filtrate and / or supernatant and / or concentrate are dried by any suitable method, including but not limited to one or more of the following: spray dryer; drum dryer; freeze drying.

[0143] In certain embodiments, one or more defatting steps are performed to biomass and / or lysate and / or hydrolysate. In certain embodiments, one or more defatting steps remove or reduce the content of lipopolysaccharide (LPS) in the product. In certain embodiments, one or more filtration or ultrafiltration steps are performed to lysate and / or hydrolysate. In certain embodiments, one or more filtration or ultrafiltration steps remove or reduce the content of LPS in the product. In certain embodiments, the ultrafiltration step has a molecular weight cutoff of 100 kilodaltons (kD) or less, or 50kD or less, or 25kD or less, or 20kD or less, or 10kD or less, or 5kD or less. In certain embodiments, the LPS removed or reduced in one or more defatting steps and / or one or more filtration or ultrafiltration steps is endotoxin.

[0144] Lysates and / or hydrolysates and / or peptide compositions and / or amino acid compositions produced as described herein can provide one or more of the following: peptides, including but not limited to growth-promoting peptides; amino acids; nucleosides, nucleotides and / or nucleic acids; carbohydrates; lipids; minerals, such as but not limited to potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn) and trace minerals; vitamins; and / or other trace low molecular weight components. In certain embodiments, peptides can be used as growth and / or survival factors in cell culture. In certain embodiments, lysates and / or hydrolysates and / or peptide compositions and / or amino acid compositions and / or extracts produced as described herein are used as a source of nucleotides (RNA and DNA precursors) for another culture and / or consortium and / or microbial group, and / or the nucleotides are recycled back to the bioreactor and / or to the microorganisms for producing proteins and / or biomass as disclosed herein. In certain embodiments, nucleotides are used for mixotrophic growth and / or production together with CO2 carbon sources.

[0145] In certain embodiments, compared with a culture medium not comprising a lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition, the lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition improves the growth rate and / or protein expression in a cell culture grown on a culture medium comprising the lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition. In certain embodiments, a lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition as described herein improves one or more of the following parameters in a culture that has received the composition: growth rate; productivity; and / or long-term viability. In certain embodiments, a lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition delivers or promotes adhesion, fibronectin-like activity by promoting the attachment and spreading of cells on a matrix. In certain embodiments, a lysate and / or a hydrolysate and / or a peptide composition and / or an amino acid composition improves the performance of a culture grown on a culture medium comprising the composition by including but not limited to the effects of an osmotic protectant and / or an anti-apoptotic effect. In certain embodiments, protein hydrolysate and / or peptide composition and / or amino acid composition as described herein are used in the freezing preservation solution of animal cells. In certain embodiments, protein hydrolysate and / or peptide composition and / or amino acid composition as described herein are used in embryo freezing culture medium. In certain described embodiments, protein hydrolysate and / or peptide composition and / or amino acid composition improve cell and / or embryo survival during freezing and / or thawing step.

[0146] In some embodiments, biomass may be processed before, during, or after the production of a protein hydrolysate composition (including but not limited to polyhydroxyalkanoate (PHA) polymer) to extract and / or purify biodegradable polyesters. In some embodiments, biomass may be processed to extract a polymer product comprising polyhydroxybutyrate (PHB). In some embodiments, biomass may be processed to extract a polymer product comprising polyhydroxyvalerate (PHV). PHA or PHB or PHV polymers may be extracted from biomass using any suitable method. In some embodiments, PHA or PHB or PHV polymers may be first extracted by mixing biomass with a solvent, such as one or more of chloroform, methanol, methylenechloride, 1,2-dichloroethane, dichloromethane, diethyl succinate, acetone, hexane, propylene carbonate, isopropanol, and ethanol. In some embodiments, biomass is cracked (e.g., by homogenization) before mixing with a solvent. The extraction may be performed at any suitable temperature, and may be performed at a temperature ranging from room temperature to 150°C or higher. In some embodiments, extraction includes separating the aqueous phase and the organic phase after mixing the biomass with the solvent. Phase separation can be performed using any suitable method (such as but not limited to centrifugation). In some embodiments, extraction includes precipitating PHA or PHB or PHV by, for example, cooling the mixture and / or adding an antisolvent (such as hexane) to the mixture. Extraction can include removing the solvent from the biomass-solvent mixture. In some embodiments, after extraction, the extracted material is further purified by mixing the extracted material with a second solvent such as hexane, wherein non-polar lipids are soluble, but PHA or PHB or PHV is insoluble. The second solvent can be removed after mixing.Suitable methods for extracting PHA or PHB or PHV polymers are described, for example, in Fei et al. (2016) "Effective recovery of poly-β-hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system" Biotechnol Prog. 32 (3): 678-85; Ujang et al. (2009) "Recovery of Polyhydroxyalkanoates (PHAs) from Mixed Microbial Cultures by Simple Digestion and Saponification" Malaysia: University Teknology, Institute of Environmental and Water Resource Management, 8-15, which are incorporated herein by reference in their entirety.

[0147] Preparation of protein hydrolysate compositions

[0148] Biomass produced from a culture of a microorganism (e.g., a chemoautotrophic microorganism) can be hydrolyzed to produce a protein hydrolyzate according to aspects of the present disclosure. One or more of the physical, chemical, and enzymatic treatments applied to microbial biomass can be used for hydrolysis. The quality of the resulting protein hydrolyzate (e.g., peptone, peptide, hydrolyzed protein) is limited by factors including starting materials, the hydrolyzing agent used, process parameters, and the degree of hydrolysis. The degree of hydrolysis is usually limited by the amino nitrogen / total nitrogen (AN / TN) ratio. Different methods for protein digestion result in the release of amino acids and peptides to different degrees of hydrolysis (DH%). The size and amino acid composition of these released peptides can affect the growth rate and biomass yield of the organism supplied with the protein hydrolyzate. The biological value of a specific protein hydrolyzate depends not only on the total level of amino acids, but also on their form (i.e., oligopeptides, dipeptides, and tripeptides, or free amino acids). Different commercial protein hydrolyzates have different DH% and total protein contents.

[0149] In certain non-limiting embodiments, the microbial biomass is hydrolyzed with at least one enzyme that can hydrolyze microorganism (for example bacteria) protein into free amino acids and / or short peptides. In certain embodiments, the controlled digestion of the protein produced as described herein is carried out using a mixture of protease and / or protease and peptidase added. In certain embodiments, several commercially available proteolytic enzymes with various specificities are used to digest the protein produced as described herein. In other non-limiting embodiments, exogenous enzymes are not used in the hydrolysis process. In certain embodiments, enzymatic hydrolysis comprises enzymatic hydrolysis with purification. In certain embodiments, enzymatic hydrolysis comprises hydrolysis with a mixture of enzyme and the culture medium for preparing enzyme.

[0150] In certain embodiments, enzymatic hydrolysis includes hydrolysis with enzymes of plant and / or animal and / or bacterial and / or archaebacterial and / or fungal origin. In certain embodiments, enzymatic hydrolysis includes hydrolysis with a mixture of one or more enzymes of plant, animal, bacterial, archaebacterial and / or fungal origin. In certain embodiments, non-animal enzymes are used. In certain embodiments, the enzyme used for enzymatic hydrolysis is not derived from animal sources (e.g., trypsin derived from porcine pancreas). In certain embodiments, proteases and non-proteolytic hydrolytic enzymes are used, which can release the main components of the polymerized non-protein fractions of the raw materials, such as microbial biomass, such as chemoautotrophic biomass. In exemplary embodiments, bacterial cells can be hydrolyzed with one or more proteases, lipases and / or amylases. In certain embodiments, enzymatic hydrolysis includes one or more proteolytic enzymes of bacteria, plants, fungi, archaebacteria and / or animal origin.

[0151] In certain embodiments, the method comprises using one or more alkaline proteases and / or acidic proteases and / or neutral proteases to hydrolyze. In some embodiments, enzymatic hydrolysis comprises using one or more exoproteinases. In some embodiments, enzymatic hydrolysis comprises using one or more endoproteinases, including sequence-specific endoproteinases. Suitable endoproteinases include but are not limited to serine proteases, aspartic proteases, metalloproteinases and cysteine ​​proteases. In certain embodiments, enzymatic hydrolysis comprises hydrolysis with at least one enzyme selected from the group consisting of pancreatin, papain, bromelain, ficin, bacterial proteases, fungal proteases, neutral proteases produced by Bacillus species, alkaline protease 2.4L and / or subtilisin carlesberg from Bacillus B. licheniformis, Esperase from B. lentus, Nutrase from B. amyloliquifacus, Protamex from Bacillus species, Therolysin / therolase from B. thermoproteolyticus, Protease from Aspergillus oryzae oryzae), Aspergillus protease 2A, protease N from B. subtilis, B. subtilis neutral enzyme, trypsin, chymotrypsin, keratinase, pepsin, subtilisin and / or chymosin. In some embodiments, enzymatic hydrolysis comprises using multiple proteases to hydrolyze proteins in the biomass sequentially or simultaneously.

[0152] In certain embodiments, peptone or tryptone are made by being separated from the biomass and / or protein of one or more microorganisms as described herein.In certain embodiments, pancreatic digest or tryptic digest are made by being separated from the biomass and / or protein of one or more microorganisms of the present invention.In certain embodiments, recombinant protease is used for hydrolysis processing.In certain embodiments, recombinant protease provides unique cleavage specificity and / or more uniform hydrolyzate.

[0153] Enzymatic hydrolysis of microbial biomass can include merging enzyme and microbial biomass with suitable amount under suitable conditions.In certain embodiments, the conditions of pressure, temperature, pH and time of enzymatic hydrolysis are conditions that reach maximum or suitable level of enzymatic activity.In certain embodiments, carrying out enzymatic hydrolysis includes merging enzyme and microbial biomass with a weight ratio within the range of about 0.1 to about 10g enzyme of nitrogen content of every 100g biomass.In another specific embodiment, enzymatic hydrolysis is to use the enzyme stock solution that concentration is 0.05 volume %-0.5 volume % to carry out, and the activity of the enzyme stock solution measured using azocasein is about 70,000 units.In various exemplary embodiments, any suitable method can be adopted to improve the enzymatic hydrolysis efficiency of microbial biomass.In certain embodiments, enzymatic hydrolysis includes merging enzyme and microbial biomass, and stirring the enzyme and microbial biomass of described merging by any suitable method.Enzyme treatment can be carried out in any suitable device, such as for example in a reactor with temperature control and stirring.

[0154] In certain embodiments, enzymes are used to cleave proteins at specific peptide bonds. For example, pepsin will digest amide bonds between amino acids in a protein, where at least one amino acid is an aromatic amino acid (e.g., phenylalanine; tryptophan; or tyrosine). In certain embodiments, pepsin is used to cleave amide bonds involving aromatic amino acids.

[0155] As described above, hydrolysis can be carried out under any suitable conditions, however, in various exemplary embodiments, hydrolysis can be carried out at a pH in the range of about 2 to about 10. In certain embodiments, enzymatic hydrolysis is carried out under pH conditions falling within the range of pH about 4 to about 12. In other embodiments, hydrolysis is carried out in the pH range of about 5 to about 9.5 or pH about 6 and about 8 or at about pH 7. In certain specific embodiments, the pH value is kept constant during enzymatic hydrolysis by adding a base (such as, for example, ammonia, ammonium hydroxide, potassium hydroxide, calcium oxide or potassium phosphate), and in other embodiments, pH is maintained by adding an acid such as phosphoric acid, sulfuric acid or carbonic acid (i.e., CO2). In various exemplary embodiments, hydrolysis can be carried out at a temperature of about 15.5°C to about 55°C for a period of time in the range of about 2 to about 120 hours. In certain embodiments, enzymatic hydrolysis is carried out under temperature conditions in the range of about 10°C to about 80°C, and in other embodiments, at a temperature in the range of about 10°C to about 65°C or about 10°C to about 55°C. The pH, temperature, and duration for hydrolysis may depend on the specific enzyme used and the extent of hydrolysis desired (eg, size distribution of the peptides).

[0156] In certain embodiments, enzymatic hydrolysis is included in the presence of a catalyst and enzyme is contacted with microbial biomass. Any catalyst that improves the efficiency of one or more enzymes can be used, such as heterogeneous catalysts, homogeneous catalysts and / or electrocatalysts. In some such embodiments, catalyst includes at least one of iron, copper, cobalt, nickel, boron, magnesium, calcium and rare earth metals, such as but not limited to lanthanum. In certain embodiments, enzymatic hydrolysis is included in contacting enzyme with microbial biomass while applying electric current. In certain embodiments, enzymatic hydrolysis is included in the process of treating microbial biomass with electric current before and / or during the enzymatic hydrolysis microbial biomass. Electric current can be applied to microbial biomass by any suitable method and under any suitable conditions. In various exemplary embodiments, the amount of about 2V to about 120V applies electric current for a period of about 1 to about 60 minutes.

[0157] Various exemplary embodiments of the methods herein may also include pretreatment before enzymatic hydrolysis of microbial biomass. The efficiency of microbial protein hydrolysis using enzymes can be improved by using various pretreatment methods. In certain embodiments, enzymatic hydrolysis includes treating microbial biomass with acid or alkali before enzymatic hydrolysis. In various exemplary embodiments, acid pretreatment is performed by adjusting the pH of a suspension containing biomass to a range of about 3 to about 5 using an acid such as, but not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, or carbonic acid (i.e., CO2). In certain embodiments, the acid pretreatment may be performed for a period of about 0.25 hours to about 10 hours at a temperature of about 100°C to about 130°C. In various exemplary embodiments, alkaline pretreatment is performed by adjusting the pH of a biomass suspension to about 9 to about 14, for example, about 10 to about 13, including about 11 to about 13 using an alkali such as, but not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium oxide, or ammonia. In certain embodiments, alkaline pretreatment may be performed for a period of about 0.25 hours to about 10 hours at a temperature of about 100°C to about 130°C. In certain embodiments, the enzymatic hydrolysis comprises treating the microbial biomass with ultrasonic vibrations prior to and / or during the enzymatic hydrolysis. In certain embodiments, the enzymatic hydrolysis comprises treating the microbial biomass with supercritical water and / or supercritical carbon dioxide prior to the enzymatic hydrolysis.

[0158] Certain embodiments include methods for obtaining hydrolyzates or organic enzyme extracts by a "one-pot" method. In the context of the present invention, the expression "one-pot" means that the procedure is carried out without an intermediate separation step. In certain embodiments, the physical treatment of the cell broth containing microbial biomass occurs at superatmospheric pressure and high temperature without phase separation, wherein the cell broth is treated with concentrated alkali before enzymatic hydrolysis. In certain embodiments, the biomass suspension is subjected to a type of "one-pot" method, and in certain non-limiting embodiments, the method comprises the following steps: (a) adding concentrated alkali to the microbial cell suspension to adjust its pH; (b) subjecting the mixture obtained in step (a) to superatmospheric pressure and high temperature; and (c) subjecting the mixture obtained in step (b) to enzymatic hydrolysis to obtain an organic enzyme extract. In certain embodiments, the alkali treatment of step (a) uses a suitable alkali, such as one or more alkalis selected from ammonium hydroxide, ammonia, potassium hydroxide and / or calcium hydroxide. In certain non-limiting embodiments, about 28% by weight of ammonium hydroxide is used, and in other embodiments, about 10M potassium hydroxide is used.

[0159] In certain embodiments, after the broth is subjected to alkali treatment, the physical treatment of the mixture is carried out at superatmospheric pressure and / or high temperature. In a specific non-limiting embodiment, a pressure of about 102 kPa to about 141 kPa is applied in step (b) at an elevated temperature. In some specific embodiments, a temperature of about 90 ° C to about 140 ° C is used in the physical treatment. In some specific embodiments, a pressure of about 102 kPa to about 141 kPa is applied at a temperature of about 90 ° C to about 140 ° C in step (b). This physical treatment is carried out in any suitable device (e.g., autoclave) selected by those skilled in the art.

[0160] In certain embodiments, enzymatic treatment is performed after physical treatment. In certain embodiments, after physical treatment and before enzymatic treatment, concentrated alkali and / or acid are added so that the mixture to be treated has the optimal pH value of the enzyme to be used. In certain embodiments, one or more enzymes used for the enzymatic hydrolysis of step (c) are proteolytic enzymes of bacterial, plant, fungal, archaeal or animal origin. In a specific embodiment, the enzymatic hydrolysis of step (c) is performed at a temperature of about 40°C to about 70°C and a pH of about 8 to about 11 for a period of about 2 hours to about 48 hours. In certain embodiments, the one-pot method increases the simplicity of the method and / or reduces costs and / or pollution compared to comparable multi-pot methods.

[0161] With or without the help of enzymes, the hydrolysis of microbial biomass can be accomplished by applying a combination of acid or alkaline hydrolysis and sufficient heat and pressure conditions. In certain non-limiting embodiments, hydrolyzing microbial biomass includes performing acid hydrolysis. In certain such embodiments, acid hydrolysis includes adjusting the pH of a composition containing microbial cells with an acid, the acid being at least one agent such as, for example, selected from sulfuric acid, hydrochloric acid, phosphoric acid, carbonic acid, boric acid, acetic acid, propionic acid and citric acid. In various exemplary embodiments, acid hydrolysis is performed by adjusting the pH of a microbial biomass cream to a pH of about 0.5 to about 5. In certain such embodiments, acid hydrolysis includes adjusting the pH of a suspension containing microbial biomass and heating the pH-adjusted suspension to a suitable temperature, such as a temperature of about 30°C to about 200°C for a suitable period of time, such as about 10 minutes to about 48 hours. In certain such embodiments, acid hydrolysis includes adjusting the pH of a suspension containing microbial biomass and heating the pH-adjusted suspension under pressure. In certain non-limiting embodiments, hydrolyzing microbial biomass includes performing alkaline hydrolysis. In some such embodiments, performing alkaline hydrolysis includes adjusting the pH of the suspension containing microbial biomass to a pH of about 8 to about 14. In some such embodiments, alkaline hydrolysis includes adjusting the pH of the composition containing microbial biomass with at least one base, such as a base selected from potassium hydroxide, sodium hydroxide, calcium oxide, magnesium oxide, ammonium hydroxide, ammonia and tripotassium phosphate. In some such embodiments, alkaline hydrolysis includes adjusting the pH of the composition containing microbial biomass and heating the pH-adjusted composition to a temperature of about 30°C to about 200°C for a period of about 10 minutes to about 48 hours. In some such embodiments, alkaline hydrolysis includes adjusting the pH of the composition containing microbial biomass and heating the pH-adjusted composition under pressure. In some embodiments, alkaline or acid hydrolysis is performed under elevated pressure. In some embodiments, the suspension containing microbial biomass is subjected to a pressure of about 5 to about 40 psi during alkaline or acid hydrolysis. Such acid or alkaline hydrolysis techniques as described herein generally produce a hydrolyzate composition comprising soluble materials and cell wall fragments. In certain embodiments, the cell wall fragments can be separated from the protein hydrolysate by solid-liquid separation, such as centrifugation.

[0162] In certain non-limiting embodiments, hydrolysis of microbial biomass involves the addition of acid or alkali. In certain embodiments, the acid or alkali is a strong acid or alkali, respectively. In certain embodiments, the acid includes one or more of the following: sulfuric acid, phosphoric acid, hydrochloric acid and / or carbonic acid. In certain embodiments, the alkali includes one or more of the following: potassium hydroxide, ammonia, ammonium hydroxide, potassium phosphate, calcium oxide, magnesium oxide and / or sodium hydroxide. Acid or base hydrolysis is not site-specific, and therefore generally produces more uniform peptide sizes and higher levels of low molecular weight (MW) peptides and / or free amino acids. In certain embodiments, acid or base hydrolysis is used to obtain a more uniform peptide size distribution and / or a higher level of low MW peptides. Under sufficient processing time and / or the intensity of the conditions applied (e.g., pH, temperature (T), pressure (P)), acid or base hydrolysis can completely release all or substantially all of the free amino acids (e.g., casamino acids). In certain embodiments, the input protein is mostly or completely converted into free amino acids by acid and / or base hydrolysis. Proteins that are hydrolyzed to free amino acids from beginning to end by acid or base usually contain up to 40% salt, such as NaCl. In certain embodiments, a free amino acid composition that is mostly or completely free of salt is produced, for example, wherein the NaCl content is less than about 2%. In certain such embodiments, the total salt content may be less than about 20%, about 10%, about 5% or about 2%. In acidic hydrolysis, some amino acids (e.g., cysteine ​​(Cys), tryptophan (Trp)) may be destroyed. In certain embodiments, enzymes and / or pancreatic digests are used to preserve unstable amino acids, such as cysteine ​​and / or tryptophan.

[0163] In certain embodiments, cell autolysis is carried out using controlled temperature and / or osmotic pressure changes, thereby producing cell lysates. In certain embodiments, one or more of the following can be used to trigger autolysis: acid, alkali and / or heat. In certain embodiments, cell autolysis is followed by and / or accompanied by protein digestion carried out by endogenous enzymes, thereby producing cell lysates with hydrolyzed proteins and / or protein hydrolysates and / or oligopeptides and / or free amino acids.

[0164] In certain embodiments, starting biomass, for example, chemoautotrophic biomass or lysate include some proteins of MW ≥ 10,000Da. In some such embodiments, the product of one or more protein hydrolysis steps as described herein produces less or no protein or peptide of MW ≥ 10,000Da. In certain embodiments of the present invention, the peptide in the protein hydrolysate produced by one or more protein hydrolysis steps as described herein and amino acid whose average MW fall into about 9,000 to about 10,000Da, or about 8,000 to about 9,000Da, or about 7,000 to about 8,000Da, or about 6,000 to about 7,000Da, or about 5,000 to about 6,000Da, or about 4,000 to about 5,000Da, or about 3,000 to about 4,000Da, or about 2,000 to about 3,000Da, or about 1,000 to about 2,000Da or less than about 1,000Da in the scope. In certain embodiments, the average MW of the peptide and amino acid content of the protein hydrolysate (PH) is about 700 Da.

[0165] In certain embodiments, various hydrolyzing agents are used to design a pH with a desired peptide fraction range and a ratio of peptides to free amino acids and an average MW. It is known that hydrolyzates with low molecular weight peptides can stimulate the growth of dairy lactic acid bacteria (LAB). In certain embodiments, a pH comprising low MW peptides is produced. In certain embodiments, a pH comprising low MW peptides produced as described herein is provided to another culture, such as but not limited to a culture comprising LAB. In certain embodiments, the LAB culture comprises one or more Lactococcus lactis (L.lactis) strains. In certain embodiments, the pH produced as described herein comprises a greater proportion of small peptides (3 to 7 amino acid residues) and free essential amino acids compared to one or more of the following types of protein hydrolyzates: papain digest of casein; soy peptone; and / or whey.

[0166] In certain embodiments, pH and / or nitrogen source are designed to include a combination of oligopeptides and dipeptides and tripeptides and essential amino acids that support initial growth, which can be transported across cell membranes and used for biomass synthesis with minimal free energy (ATP) consumption. In certain embodiments, the composition of amino acids, dipeptides and tripeptides is designed to transport amino acids, dipeptides and tripeptides to the minimum ATP requirement in another cell and / or organism.

[0167] In enzyme and acid hydrolysis, it is known that certain amino acids, such as cysteine ​​(Cys) in casein hydrolysate and tryptophan (Trp) in acid-digested casein peptone, are destroyed. In certain embodiments, hydrolyzing agents and methods are selected to preserve sensitive amino acids, such as, but not limited to, Cys and / or Trp. In certain embodiments, pancreatic digestion of proteins is performed to preserve Trp.

[0168] When designing pH and culture medium, several enzyme specificities should be considered. It is reported that the biosynthesis of lactobacillus PrtB / PrtH ​​proteases is regulated by the peptide collection of the culture medium and is inhibited by peptide-rich culture medium (Kenny, O., FitzGerald, RJ, O'Cuinn, G., Beresford, T., and Jordan, K. (2003) Growth phase and growth medium effects on the peptidase activities of Lactobacillus helveticus. International Dairy Journal. https: / / doi.org / 10.1016 / S0958-6946(03)00073-6). Similarly, it is reported that pepX and pepN of lactococci are regulated by the peptide content in the growth medium. On the other hand, it was reported that the pepN aminopeptidase activity of Lactobacillus helveticus CRL 1062 and the pepX, pepI and pepN activities in Lactobacillus bulgaricus (Morel, F., Gilbert, C., Geourjon, C., Frot-Coutaz, J., Portalier, R., and Atlan, D. (1999) The prolylaminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus belongs to the α / βhydrolase fold family. Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology. https: / / doi.org / 10.1016 / S0167-4838(98)00264-7) were independent of the peptide content of the growth medium. It is reported that in Lactobacillus helveticus, enzymes such as aminopeptidase, dipeptidase, tripeptidase and endopeptidase activities are strain and species dependent. Therefore, in the case of using lactobacillus strains as cheese starters or adjunct cultures, culture medium components that induce and maintain important metabolic pathways such as extracellular and intracellular proteolytic systems should be used. These microbial properties can acidify milk and / or release enzymes required for flavor development during cheese ripening in a predictable time.

[0169] In certain embodiments of the peptide-rich culture medium, such as extracellular and intracellular proteolytic systems, whole cell biomass and / or lysate and / or whole protein (i.e., unhydrolyzed protein) produced as described herein (for example, chemoautotrophic) are used as culture medium components. In certain such embodiments, this culture medium can be used for the growth of starter and / or adjunct culture. Compared with the situation of some starters and adjunct culture, the main priority of producing probiotic strains (for example, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus reuteri) is high yield and viable cell number, which will enable the culture to survive after freeze drying and exposure to extended shelf life at room temperature. In certain embodiments, nutrients such as whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactor extracts and / or preparations comprising one or more of the foregoing components are selected to maximize one or more of the following characteristics of the culture supplying the nutrients: yield; number of viable cells; cells that survive freeze drying; and / or shelf life at room temperature. In certain such embodiments, the culture comprises a probiotic strain such as, but not limited to, one or more of Lactobacillus acidophilus, Lactobacillus johnsonii and / or Lactobacillus reuteri.

[0170] In certain embodiments, an undetermined or determined starter culture consisting of several species / strains is produced. In certain embodiments, protein hydrolysates and / or other nutrients, and formulated into complex culture medium, are designed to maintain reproducible performance and / or maintain appropriate and / or required ratios between the strain / species during whole subculture and fermentation. In order to achieve this, various factors are optimized in the culture medium formulation, such as but not limited to correctly balancing the carbon source and nitrogen source in the fermentation growth medium.

[0171] In some embodiments, the biomass of hydrolysis can undergo one or more post-hydrolysis processes, such as clarification, concentration, drying, pasteurization and / or separation. In certain embodiments, the method described herein comprises one or more further steps after the hydrolysis, wherein the hydrolyzate is concentrated to obtain a concentrated hydrolyzate. In certain embodiments, the concentrated hydrolyzate has a dry matter of at least 40 % by weight, at least 50 % by weight in other embodiments, and about 50 % by weight to about 55 % by weight or higher in other embodiments. This concentration can be achieved by any conventional method known in the art, such as, for example, by heating and using a rotary evaporator with a thermostatic bath or reverse osmosis, or any other suitable device. In some embodiments, the biomass of hydrolysis is lyophilized to remove most or substantially all of the water content.

[0172] In certain embodiments, by ultrafiltration and / or by chemical means such as but not limited to acetone precipitation, lysate and / or protein hydrolysate and / or peptide and / or amino acid composition are further purified, for example to remove non-protein components. In certain embodiments, lysate and / or protein hydrolysate and / or peptide and / or amino acid composition are fractionated by size exclusion chromatography. In certain embodiments, the fraction produced by this fractionation is used as a separate product and / or by-product. In certain embodiments, lysate and / or protein hydrolysate and / or peptide and / or amino acid composition are subjected to fine fractionation. In certain such embodiments, one or more peptides with the highest activity are recovered and purified. In certain embodiments, high performance fractionation procedures are applied to lysate and / or protein hydrolysate and / or peptide and / or amino acid composition produced as described herein, for example to obtain homogeneous peptides. In certain embodiments, homogeneous peptides are sequenced.

[0173] In another specific embodiment, the method comprises a further step after the protein hydrolysis, wherein the protein hydrolysate obtained after the hydrolysis is separated to obtain: (i) a soluble hydrolysate fraction, and (ii) a solid phase insoluble hydrolysate fraction. This separation can be carried out by any suitable solid-liquid separation method, such as, for example, by filtration or centrifugation using a decanter or other suitable industrial equipment.

[0174] In certain non-limiting embodiments, the lysate and / or hydrolyzate undergo one or more solid-liquid separation steps, including but not limited to one or more of the following: centrifugation; and / or filtration. In certain non-limiting embodiments, the insoluble cell membrane component is separated from the soluble fraction. In certain embodiments, the soluble and / or insoluble fraction is subjected to a drying method, including but not limited to spray drying and / or freeze drying. In certain embodiments, the soluble fraction is used in cell culture applications. In certain embodiments, the insoluble fraction is used for nutritional applications of plants, animals, fungi or other organisms.

[0175] In certain embodiments, in addition to proteins, peptides and amino acids, the lysate and / or hydrolysate and / or amino acid composition further comprises one or more of the following: vitamins; lipids; carbohydrates; nucleic acids; minerals; and / or other micronutrients.

[0176] Various embodiments of hydrolysis methods as described herein produce protein hydrolysates and / or peptide compositions and / or amino acid compositions of different purity levels. In certain embodiments, the protein hydrolysates and / or peptide compositions and / or amino acid compositions produced as described herein are one or more of the following grades: industrial grade; food grade; and / or non-food grade. In certain embodiments, only food grade materials are used to produce fermentation cultures. In certain embodiments, only food grade materials are used to produce LAB cultures and / or probiotic cultures and / or single cell proteins and / or animal cell cultures, such as but not limited to mammalian cell cultures and / or another nutrient (including but not limited to one or more vitamins). In certain embodiments, the protein hydrolysates and / or peptide compositions and / or amino acid compositions produced as described herein are used as substitutes for protein hydrolysates and / or peptide compositions and / or amino acid compositions produced by meat and / or soy and / or wheat and / or dairy protein.

[0177] In some embodiments, one or more batches of biomass from one or more different microorganisms or co-cultures or consortia of microorganisms can be processed using a variety of hydrolysis protocols to produce a hydrolysate library with discrete compositions. In some embodiments, one or more different organisms; one or more different hydrolyzing agents; one or more sets of different process parameters; one or more different degrees of hydrolysis; and one or more different post-hydrolysis processing and / or purification steps can be used to generate the library. In the hydrolysate library, a specific hydrolysate composition can be suitable for culturing a specific cell type and / or growth stage of a cell, such as, but not limited to, an animal cell type and / or growth stage of an animal cell.

[0178] In some embodiments, the biomass obtained from the culture of microorganisms (e.g., chemoautotrophic microorganisms) can be processed to extract the organic polymers accumulated by the microorganisms during growth. In some embodiments, microorganisms as described herein can grow on H2 / CO2 and / or syngas, and the microorganisms can accumulate polyhydroxyalkanoates (PHA), such as polyhydroxybutyrate (PHB) and / or polyhydroxyvalerate (PHV) to about 50% by weight or more of cell biomass. In some embodiments, microorganisms have the natural ability to guide high-flux carbon through acetyl-CoA metabolic intermediates, which can lead to fatty acid biosynthesis and many other synthetic pathways (including PHA and / or PHB and / or PHV synthesis) and amino acids. In some embodiments, the microorganisms showing these characteristics are Cupriavidus necator (e.g., Cupriavidus necator DSM 531 or DSM 541) and / or Cupriavidus metallidurans (e.g., Cupriavidus metallidurans DSM 2839) to resist heavy metals.

[0179] In certain embodiments, processing the biomass obtained from the chemoautotrophic microorganisms comprises extracting PHA and / or PHB and / or PHV from the insoluble hydrolysate fraction. In other embodiments, processing the biomass obtained from the chemoautotrophic microorganisms comprises recovering solids rich in PHA or PHB or PHV from the soluble hydrolysate fraction. Any suitable method for extracting PHA or PHB or PHV may be used, as discussed above with respect to processing microbial biomass to extract PHA or PHB or PHV.

[0180] In some embodiments, one or more lysates and / or hydrolysates and / or amino acid compositions as described herein are merged with other factors and / or peptides to enrich the culture medium nutritionally and / or increase the stability of glutamine and / or enhance viable cell density. It is reported that for many different cell types, small oligopeptides, low molecular weight substances and free amino acids have positive contributions to the nutritional value of the culture medium and / or the productivity of the culture grown on this culture medium. In certain embodiments, the oligopeptides, low molecular weight substances and free amino acids produced as described herein contribute to the nutritional value of the culture medium and / or increase the productivity of the culture grown on this culture medium. In certain embodiments, oligopeptides, low molecular weight substances and / or free amino acids can be added to the culture medium. In certain embodiments, oligopeptides, low molecular weight substances and / or free amino acids can be secreted and / or released into the culture medium by one or more microorganisms as described herein. In some embodiments, microorganisms include hookworm greedy copper bacteria (for example, hookworm greedy copper bacteria DSM 531 or DSM 541) and / or heavy metal-resistant greedy copper bacteria (for example, heavy metal-resistant greedy copper bacteria DSM 2839). In one embodiment, the microorganism comprises Cupriavidus necrotus DSM 541.

[0181] In certain embodiments, the peptides produced as described herein mitigate the presence of additional toxic levels of inorganic ions by providing a protective effect to the cell culture. In certain embodiments, whole cell biomass and / or lysate and / or hydrolyzate and / or peptide compositions and / or amino acid compositions as described herein are one or more of the following: vitamins; fatty acids, including but not limited to oleic acid, arachidonic acid and / or linoleic acid; phospholipids; and / or sterols.

[0182] In certain embodiments, whole cell biomass and / or lysates and / or hydrolysates and / or peptide compositions and / or amino acid compositions as described herein affect cellular function and physiology in a cell culture or organism to which they are applied.

[0183] microorganism

[0184] In some embodiments, the microorganisms of the present disclosure are chemoautotrophic microorganisms. In some embodiments, the microorganisms of the present disclosure are capable of mixotrophic growth and / or are heterotrophic microorganisms. In some embodiments, the microorganisms of the present disclosure are photosynthetic microorganisms. In some embodiments, the microorganisms of the present disclosure are oxyhydrinos or oxyhydrinos strains, i.e., microorganisms that use hydrogen as an electron donor and oxygen as an electron acceptor in respiration to produce intracellular energy carriers such as adenosine-5'-triphosphate (ATP). Oxyhydrinos microorganisms typically use molecular hydrogen via hydrogenases, some of which are used to reduce NAD +(and / or other intracellular reducing equivalents), and some electrons come from H2 used for aerobic respiration. Hydrogen-oxygen mixed gas microorganisms typically chemoautotrophically fix CO2 through pathways including, but not limited to, the Calvin cycle or the reverse citric acid cycle.

[0185] In some embodiments, the microorganism or composition comprising the microorganism comprises one or more of the following hydrogen and oxygen mixed gas microorganisms: Aquifex pyrophilus, Aquifex aeolicus, or other Aquifex sp.; Cupriavidus or heavy metal-tolerant Cupriavidus or other Cupriavidus sp.; Corynebacterium autotrophicum or other Corynebacterium sp.; Gordonia desulfuricans, Gordonia polyisoprenivorans, Gordonia rubripertincta, Gordonia hydrophobica, Gordonia westfalica, or other Gordonia sp.; Nocardia autotrophicum. autotrophica, Nocardia opaca or other Nocardia sp.; purple non-sulfur photosynthetic bacteria, including but not limited to Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophila or other Rhodopseudomonas sp.; Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas capsulata, Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas spheroides, Rhodopseudomonas acidophila or other Rhodopseudomonas sp.; sp.); Rhodospirillum rubrum or other Rhodospirillum sp.; Rhodococcus opacus or other Rhodococcus sp.; Rhizobium japonicum or other Rhizobium sp.); Thiocapsa roseopersicina or other Thiocapsa sp.; Pseudomonas facilis, Pseudomonas flava, Pseudomonas putida, Pseudomonas hydrogenovora, Pseudomonas hydrogenothermophila, Pseudomonas palleronii, Pseudomonas pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophile or other Pseudomonas sp.; Hydrogenomonas pantotropha, Hydrogenomonas eutropha eutropha, Hydrogenomonas facilis or other Hydrogenomonas sp.; Hydrogenobacter thermophilus, Hydrogenobacter halophilus, Hydrogenobacter hydrogenophilus, Hydrogenobacter sp.; Hydrogenophilus islandicus or other Hydrogenophilus sp.; Hydrogenovibrio marinus or other Hydrogenovibrio sp.; Hydrogenothermus marinus or other Hydrogenothermus sp.; Helicobacter pylori or other Helicobacter sp. sp.; Xanthobacter autotrophicus, Xanthobacter flavus or other Xanthobacter sp.); Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga pseudoflava or other Hydrogenophaga sp.; Bradyrhizobium japonicum or other Bradyrhizobium sp.; Ralstonia eutropha or other Ralstonia sp.; Alcaligenes eutrophus, Alcaligenes facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Alcaligenes paradoxus, Alcaligenes ruhlandii or other Alcaligenes sp. sp.); Amycolata sp.; Aquaspirillum autotrophicum or other Aquaspirillum sp.; Arthrobacter strain 11 / X, Arthrobacter methylotrophus or other Arthrobacter sp.; Azospirillum lipoferum or other Azospirillum sp.; Variovorax paradoxus or other Variovorax sp.; Acidovorax facilis or other Acidovorax sp.; Bacillus schlegelii, Bacillus tusciae, other Bacillus sp. sp.); Calderobactenum hydrogenophilum or other Calderobacterium species; Derxia gummosa or other Derxia sp.); Flavobacterium autothermophilum or other Flavobacterium sp.; Microcyclus aquaticus or other Microcyclus sp.; Mycobacterium gordoniae or other Mycobacterium sp.; Paracoccus denitrificans or other Paracoccus sp.; Persephonella marina, Persephonella guaymasensis or other Persephonella sp.; Renobacter vacuolatum or other Renobacter sp.; Seliberia carboxydohydrogena or other Seliberia sp., Streptomycetes coelicoflavus, Streptomycetes griseus, Streptomycetes xanthochromogenes, Streptomycetesthermocarboxydus, and other Streptomycetes sp.; Thermocrinis ruber or other Thermocrinis sp.; Wautersia sp.; cyanobacteria, including but not limited to Anabaena oscillarioides, Anabaena spiroides, Anabaenacylindrica, or other Anabaena sp.; and Arthrospira platensis, Arthrospira maxima, or other Arthrospira sp. sp.); green algae, including but not limited to Scenedesmus obliquus or other Scenedesmus sp.); Chlamydomonas reinhardii or other Chlamydomonas sp.; Ankistrodesmus sp.; and Rhaphidium polymorphium or other Rhaphidium sp.; and microbial consortia including hydrogen and oxygen mixed gas microorganisms.

[0186] In some embodiments, the microorganism or composition comprising the microorganism comprises an obligate and / or facultative chemoautotrophic microorganism, including one or more of the following: anaerobic acetic acid bacteria species (Acetoanaerobium sp.); Acetobacterium species (Acetogenium sp.); Achromobacter sp.; Acidianus sp.; Acinetobacter sp.; Actinomadura sp.; Aeromonas sp.; Alcaligenes sp.; Alcaliqenes sp.; Aquaspirillum sp.; Arcobacter sp.; Aureobacterium sp.; Bacillus sp.; sp.; Beggiatoa sp.; Butyribacterium sp.; Carboxydothermus sp.; Clostridium sp.; Comamonas sp.; Dehalobacter sp.; Dehalococcoide sp.; Dehalospirillum sp.; Desulfobacterium sp.; Desulfomonile sp.; Desulfotomaculum sp.; Desulfovibrio sp.; Desulfurosarcina sp.; Ectothiorhodospira sp. sp.); Enterobacter sp.; Eubacterium sp.; Ferroplasma sp.; Halothibacillus sp.; Hydrogenobacter sp.; Hydrogenomonas sp.; Leptospirillum sp.; Metallosphaera sp.; Methanobacterium sp.); Methanobrevibacter sp.; Methanococcus sp.; Methanococcoides sp.; Methanogenium sp.; Methanolobus sp.; Methanomicrobium sp.; Methanoplanus sp.; Methanosarcina sp.; Methanospirillum sp.; Methanothermus sp.; Methanothrix sp.; Micrococcus sp.; Nitrobacter sp.; Nitrobacteraceae sp.; Nitrococcus sp. sp.), Nitrosococcus sp.; Nitrospina sp., Nitrospira sp., Nitrosolobus sp., Nitrosomonas sp.; Nitrosospira sp.; Nitrosovibrio sp.; Nitrospina sp.; Oleomonas sp.; Paracoccus sp.; Peptostreptococcus sp.; Planctomycetes sp.; Pseudomonas sp.; Ralstonia sp.; Rhodobacter sp. sp.); Rhodococcus sp.; Rhodocyclus sp.; Rhodomicrobium sp.; Rhodopseudomonas sp.; Rhodospirillum sp.; Shewanella sp.; Siderococcus sp.; Streptomyces sp.; Sulfobacillus sp.); Sulfolobus sp.; Thermothrix sp., Thiobacillus sp.; Thiomicrospira sp.; Thioploca sp.; Thiosphaera sp.; Thiothrix sp.; Thiovulum sp.; sulfur-oxidizers; hydrogen-oxidizers; iron-oxidizers; acetogens; and methanogens; microbial consortia comprising chemoautotrophs; chemoautotrophs originating from at least one of: hydrothermal vents, geothermal vents, hot springs, cold seeps (cold seeps), underground aquifers, salt lakes, salt formations, mines, acid mine drainage, tailings, oil wells, refinery wastewater, coal seams, deep underground, wastewater and sewage treatment plants, geothermal power plants, sulfur fields and soil; and extremophiles selected from one or more of the following: thermophiles, hyperthermophiles, acidophiles, halophiles and psychrophiles. .

[0187] In some embodiments, the microorganism or the composition comprising the microorganism comprises a methanotroph and / or a methylotroph. In some embodiments, the microorganism belongs to the genus Methylococcus. In some embodiments, the microorganism is Methylococcus capsulatus. In some embodiments, the microorganism is a methylotroph. In some embodiments, the microorganism belongs to the genus Methylobacterium. In some embodiments, the microorganism is taken from one or more of the following species: Methylobacteriumzatmanii; Methylobacterium extorquens; Methylobacterium chloromethanicum. In some embodiments, a composition is provided, wherein the microorganism is a hydrogen-oxidizing chemoautotroph and / or a carbon monoxide troph and / or a methylotroph and / or a methanotroph.

[0188] Many different microorganisms have been characterized that can grow on carbon monoxide as an electron donor and / or carbon source (i.e., carbon monoxide nutrient microorganisms). In some cases, carbon monoxide nutrient microorganisms may also use H2 as an electron donor and / or grow mixotrophically. In some cases, carbon monoxide nutrient microorganisms are facultative chemolithoautotrophs [Biology of the Prokaryotes, edited by J Lengeler, G.Drews, H.Schlegel, John Wiley & Sons, July 10, 2009, incorporated herein by reference in its entirety.]. In some embodiments, the microorganism or composition comprising the microorganism comprises one or more of the following carboxydotrophic microorganisms: Acinetobacter sp.; Alcaligenes carboxydus or other Alcaligenes sp.; Arthrobacter sp.; Azomonas sp.; Azotobacter sp.; Bacillus schlegelii or other Bacillus sp.; Hydrogenophaga pseudoflava or other Hydrogenophaga sp.; Pseudomonas carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas compransoris, Pseudomonas In some embodiments, carbon monoxide trophic microorganisms are used. In some embodiments, carbon monoxide trophic microorganisms capable of chemolithoautotrophy are used.In certain embodiments, carboxydotrophic microorganisms are used that are capable of utilizing H2 as an electron donor in respiration and / or biosynthesis.

[0189] The microorganisms (e.g., chemoautotrophic microorganisms) of the present disclosure may be naturally occurring strains or may be genetically engineered. When a hydrolyzate containing protein is provided to cells in culture, for example as a medium supplement, the microorganism may be genetically modified to express one or more proteins that have high nutritional value for animal cells. In some embodiments, the chemoautotrophic microorganism may be genetically modified to express a polypeptide sequence containing multiple peptide subsequences interspersed with protease cleavage sites. Such a polypeptide sequence may be schematically represented as: H2N-XC-[AC] n -Y-COOH, wherein "H2N" and "COOH" represent the N- and C-termini of the polypeptide, respectively; "A" is a peptide subsequence; "C" is a protease cleavage site; "X" and "Y" are linker sequences that may or may not be present; and n is an integer of 1 or greater, such as 2 or greater, 5 or greater, 10 or greater, including 20 or greater. The peptide subsequence (A) can be designed to include a peptide sequence (A') that has a beneficial effect on the growth of animal cells in culture when provided in the culture medium. When the biomass obtained from the genetically modified microorganism is hydrolyzed using an appropriate protease to cut the cleavage site, the resulting protein hydrolysate can be enriched with beneficial peptides.

[0190] In some embodiments, microorganisms (e.g., chemoautotrophic microorganisms) can be genetically modified to disrupt the expression of one or more endogenous genes involved in a biosynthetic pathway. In some embodiments, microorganisms can be genetically modified to disrupt the expression of one or more genes involved in the biosynthesis of polyhydroxyalkanoates (e.g., polyhydroxybutyrate). Suitable genes involved in the biosynthesis of polyhydroxyalkanoates and whose expression can be disrupted include, but are not limited to, genes encoding 3-ketothiolase, acetoacetyl-CoA reductase, and / or PHB synthase. In some embodiments, microorganisms can be genetically modified to disrupt or increase the expression of genes involved in the synthesis of vitamins (e.g., but not limited to, vitamin B1, vitamin B2, and / or vitamin B 12 ) is involved in the biosynthesis of one or more genes. In some embodiments, 12 The expression of the biosynthetic gene of the microorganism is destroyed or increased. The expression of one or more genes can be destroyed or increased by any suitable method, for example, by deleting or mutating all or part of the coding region or regulatory region of the gene in the microorganism genome, or duplicating the coding region or regulatory region of the gene in the microorganism genome.

[0191] Any suitable method can be used to genetically engineer the microorganism. For example, genetic engineering of oxyhydride microorganisms is described in U.S. Pat. No. 9,879,290 B2, which is incorporated herein by reference in its entirety.

[0192] Protein hydrolysate composition

[0193] Provided herein are protein hydrolysate compositions derived from microorganisms, such as the chemolithoautotrophic microorganisms described herein. The protein hydrolysate compositions are suitable, for example, for use as a medium supplement in animal cell culture media, for example, as a replacement for all or part of the serum, for example, to provide a cell culture medium free of serum or animal components, for propagating, developing and / or differentiating animal cells, or replacing one or more animal-derived components of an animal cell culture medium.

[0194] In certain embodiments, the protein hydrolysate composition has an organic content rich in protein, peptides and / or free amino acids. In some embodiments, the organic content of the protein hydrolysate includes about 10% or more by weight / weight of the organic content, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, including about 90% or more of the amount of amino acids (in protein, peptides or as free amino acids). In some embodiments, the organic content of the protein hydrolysate includes about 10% to about 98% by weight / weight of the organic content, for example, about 20% to about 98%, about 30% to about 98%, about 40% to about 98%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, including about 75% to about 95% of the amount of amino acids (in protein, peptides or as free amino acids). Any suitable method can be used to measure the total organic content.

[0195] Protein hydrolysate compositions of the present disclosure may include a distribution of polypeptide sizes. In some embodiments, the average size of polypeptides in the hydrolysate composition is about 25 kDa or less, such as about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 5 kDa or less, about 2 kDa or less, about 1 kDa or less, including about 0.5 kDa or less. In some embodiments, the average size of polypeptides in the hydrolysate composition is about 0.1 kDa to about 100 kDa, such as about 0.5 kDa to about 50 kDa, about 0.1 kDa to about 10 kDa, about 0.5 kDa to about 20 kDa, about 1 kDa to about 10 kDa, including about 1 kDa to about 5 kDa. In some embodiments, about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, including about 95% or more of the polypeptides by number are about 25 kDa or less, such as about 20 kDa or less, about 15 kDa or less, about 10 kDa or less, about 5 kDa or less, about 2 kDa or less, about 1 kDa or less, including about 0.5 kDa or less, such as about 0.1 kDa to about 100 kDa, such as about 0.5 kDa to about 50 kDa, about 0.1 kDa to about 10 kDa, about 0.5 kDa to about 20 kDa, about 1 kDa to about 10 kDa, including about 1 kDa to about 5 kDa. In certain embodiments, the average MW of the peptides falls within the range of about 600 to about 1,100 Da.

[0196] In certain embodiments, the lysate, protein hydrolysate, peptide composition and / or amino acid composition produced as described herein contains peptides whose MW falls within the range of about 600 to about 1,100 Da. In some embodiments, the molecular weight present in the lysate, protein hydrolysate, peptide composition or amino acid composition ranges from about 0.1 kDa to about 100 kDa, or from about 0.1 kDa to about 10 kDa, or from about 1 kDa to about 10 kDa. In some embodiments, a small fraction or no molecular weight is greater than about 10 kDa.

[0197] In some embodiments, the lysate and / or protein hydrolysate comprises about 1% free amino acids, or about 5% free amino acids, or about 30% free amino acids, or about 40% or more free amino acids.

[0198] In certain embodiments, the lysates, protein hydrolysates, extracts, peptide compositions and / or amino acid compositions produced as described herein have one or more of the following characteristics: at least about 60% total amino acid content (i.e., amino acids polymerized within peptides and / or proteins and / or other biochemical substances), or ≥ about wt%, or ≥ about 73 wt%, or ≥ about 75 wt%, or about ≥ 80 wt% total amino acids; of which about 35% to about 40%, or about 30% to about 45%, or about 20% to about 50% are free amino acids; and about 10% to about 15%, or about 5% to about 20% are dipeptides and tripeptides; and about 40% to about 45%, or about 30% to about 50%, or about 20% to about 60% are oligopeptides having a MW of about 2,000 Da to about 3,000 Da.

[0199] In certain embodiments, the culture medium of chemical composition determination is provided with low MW peptides produced as described herein and / or protein hydrolysates comprising low MW peptides as described herein.In certain embodiments, low MW peptides are mostly or entirely MW≤about 1,000Da.In certain embodiments, low MW peptides and / or protein hydrolysates are provided to culture, and the culture includes but is not limited to one or more LAB, such as lactobacillus, for example lactococcus lactis strain.In certain embodiments, the protein hydrolysate produced as described herein contains low MW peptides, and its average MW is about 700Da, less than about 700Da or about 700Da to about 1000Da.In certain embodiments, protein hydrolysate, for example protein hydrolysate with low MW peptides as described herein is provided to culture comprising LAB.

[0200] In certain embodiments, the lysate, protein hydrolysate, extract, peptide composition and / or amino acid composition produced as described herein contain hydrophobic peptides and / or contain a basic peptide fraction with a molecular weight in the range of about 600Da to about 1,100Da. In certain embodiments, the lysate, protein hydrolysate, extract, peptide composition and / or amino acid composition produced as described herein contain a higher proportion and / or weight percentage of hydrophobic peptides and / or contain a basic peptide fraction with a molecular weight in the range of about 600Da to about 1,100Da compared to milk or milk-derived proteins or protein hydrolysates. In certain embodiments, such hydrophobic and / or basic peptides with a molecular weight in the range of about 600Da to about 1,100Da are provided to LAB cultures, including but not limited to cultures containing Lactobacillus strains, such as Lactococcus lactis.

[0201] In certain embodiments, a nutrient source such as a lysate, protein hydrolysate, extract, peptide composition, and / or amino acid composition produced as described herein contains a lower proportion and / or weight percentage of acidic phosphopeptides having a MW of about 1,400 Da to about 3,200 Da compared to milk or milk-derived proteins or protein hydrolysates. In certain embodiments, a nutrient source comprising a relatively low content of acidic phosphopeptides having a MW of about 1,400 Da to about 3,200 Da is provided to a LAB culture, including, but not limited to, a culture comprising a Lactobacillus strain, such as Lactococcus lactis.

[0202] In certain embodiments, the protein hydrolysate produced as described herein is provided to one or more strains that utilize the protein hydrolysate better for growth and / or production than they can utilize whole protein (i.e., unhydrolyzed protein). In certain such embodiments, the strain comprises one or more LAB, such as lactobacillus.

[0203] In certain embodiments, protein hydrolysates as described herein are better utilized by the culture than soy protein hydrolysates and / or whole soy protein.

[0204] In certain embodiments, the protein hydrolysate produced as described herein is supplemented in the culture medium with a concentration of about 3% (w / v). In certain embodiments, the protein hydrolysate provided with a concentration of about 3% (w / v) has an average peptide MW of about 700Da or about 700Da to about 1000Da. In certain embodiments, the nitrogen source (for example, protein biomass, lysate, protein hydrolysate, peptide composition and / or amino acid composition) produced as described herein is supplied to the culture with an amount of about 0.5% to about 2.5% (w / v).

[0205] In certain embodiments, the protein hydrolysate produced as described herein causes the growth and / or titer of one or more products produced by cell culture to increase.In certain embodiments, the growth and / or titer (g / L) of increase is observed in the LAB culture of biomass and / or lactic acid.In certain embodiments, the protein hydrolysate produced as described herein is supplemented so that the growth and / or titer of product are higher than supplementing soybean peptides, soy protein hydrolysate and / or whole soybean protein.In certain embodiments, compared with the same culture grown in the same medium without protein hydrolysate supplement producing < lactic acid titer of about 50g / L, supplementing protein hydrolysate as described herein produces the lactic acid titer of ≥ about 50g / L.In certain embodiments, compared with the same culture grown in the same medium without protein hydrolysate supplement, supplementing the protein hydrolysate produced as described herein makes the fermentation time that LAB culture reaches pH 4.50 reduce.

[0206] In certain embodiments, lysate as described herein and / or hydrolyzate and / or peptide composition and / or amino acid composition and / or extract comprise peptides with two to ten amino acid residues.In certain embodiments, lysate as described herein and / or protein hydrolyzate and / or peptide composition are mainly composed of peptides or comprise peptides, and the peptides are composed of 2 to 10 amino acid residues.In certain embodiments, purified product is mainly composed of peptides or comprises peptides, and the peptides are composed of 2 to 10 amino acid residues.In certain embodiments, lysate as described herein and / or hydrolyzate and / or peptide composition and / or amino acid composition and / or extract comprise two to five peptides (two to five amino acid residues).In certain described embodiments, lysate and / or hydrolyzate and / or peptide and / or amino acid composition and / or extract are mainly composed of peptides or comprise peptides, and the peptides are composed of two to five peptides.

[0207] In some embodiments, the protein hydrolysate comprises peptides having a degree of polymerization (DP) of about 50 or less, such as about 40 or less, about 30 or less, about 20 or less, about 10 or less, including about 5 or less, such as any of about 50, 40, 30, 20, 10, or 5 to about 1. In some embodiments, the protein hydrolysate comprises peptides having a DP ranging from about 1 to about 50, such as about 1 to about 30, about 1 to about 20, including about 2 to about 10.

[0208] In some embodiments, the protein hydrolysate produced as described herein has an AN / TN ratio of about 10% to about 30%. In some of the embodiments, an enzyme is used to produce a protein hydrolysate with an AN / TN of about 10% to about 30%. In some embodiments, the protein hydrolysate produced as described herein has an AN / TN ratio of about 20% to about 50%. In some embodiments, a protein hydrolysate with an AN / TN of about 20% to about 50% is produced as a pancreatic digest. In some embodiments, the protein hydrolysate produced as described herein has an AN / TN ratio of about 50% to about 80%. In some of the embodiments, an acid or base is used to produce a protein hydrolysate with an AN / TN of about 50% to about 80%. In some embodiments, the whole cell product or lysate and / or protein hydrolysate produced as described herein has an AN / TN ratio of less than about 10%. In some embodiments, the protein hydrolysate and / or amino acid composition produced as described herein has an AN / TN ratio of greater than about 80%.

[0209] In some embodiments, the protein hydrolysate is substantially free of full-length proteins. In some embodiments, the protein hydrolysate is depleted of proteins and polypeptides and enriched in free amino acids. In some embodiments, the protein hydrolysate is substantially free of proteins and polypeptides. As described herein, the size distribution of polypeptides may depend on the degree of hydrolysis achieved using one or more enzymes, chemical and physical means.

[0210] In some embodiments, the protein hydrolysate composition is enriched for one or more peptides having a specific amino acid sequence that may have a beneficial effect on cell growth and / or development. In certain embodiments, the enriched sequence may be represented by the total number of polypeptides in the hydrolysate of about 0.001% or more, such as about 0.01% or more, about 0.1% or more, about 1% or more, including about 5% or more. In some embodiments, the enriched sequence may be represented by the total number of polypeptides in the hydrolysate of about 0.001% to about 5%, such as about 0.001% to about 1%, including about 0.01% to about 1%.

[0211] In some embodiments, the protein hydrolysate composition has an ash content of about 5% to about 50%, such as about 10% to about 40%, including about 10% to about 35%. In some embodiments, the protein hydrolysate composition has an ash content of less than or equal to about 5%. In some embodiments, the protein hydrolysate composition has a total nitrogen content of about 3% to about 20%, such as about 5% to about 15%, including about 5% and about 15%.

[0212] In certain embodiments, the protein hydrolysate composition comprises one or more vitamins. In some embodiments, the protein hydrolysate composition comprises vitamin B1, vitamin B2 and / or vitamin B 12 In some embodiments, the protein hydrolysate composition comprises vitamin B 12 and / or one or more vitamin equivalents thereof (e.g., cyanocobalamin, hydroxocobalamin, adenosylcobalamin, methylcobalamin). In certain embodiments, relative to the volatile organic matter in the protein hydrolysate, vitamin B 12 and / or its equivalent vitamins in a concentration of about 2 μg vitamin B 12 and / or its equivalent vitamins / 100 g dry volatile organic matter to about 6.5 μg / 100 g volatile organic matter, or about 6.5 μg / 100 g volatile organic matter to about 13 μg / 100 g volatile organic matter, or about 13 μg / 100 g volatile organic matter or higher. In some embodiments, vitamin B 12 and / or one or more of its vitamin equivalents are derived from microorganisms (eg, chemoautotrophic microorganisms).

[0213] In certain embodiments, vitamins such as, but not limited to, riboflavin and / or vitamin B 12 (and / or its equivalent vitamins) are produced by microorganisms grown on protein hydrolysates and / or other nutrients produced as described herein. In certain embodiments, one or more flavoring agents or aromas such as, but not limited to, vanillin are produced by microorganisms grown on protein hydrolysates and / or other nutrients produced as described herein.

[0214] In certain embodiments, the lysate and / or protein hydrolysate produced as described herein provides one or more of the following to another cell culture: nutrients; adhesion components; and / or growth factor analogs. In certain embodiments, the lysate and / or protein hydrolysate produced as described herein is used in a culture medium that does not contain amino acids or protein hydrolysates of animal origin. In certain embodiments, the lysate and / or protein hydrolysate produced as described herein is used in a serum-free culture medium. In certain embodiments, the lysate and / or protein hydrolysate produced as described herein is used to reduce or replace amino acids, protein hydrolysates and / or fetal bovine / bovine serum commonly used in human, animal, rodent and / or insect cell cultures of animal origin. In certain embodiments, the lysate, protein hydrolysate and / or free amino acid composition as described herein is added to the cell culture together with ITS (insulin-transferrin-selenium), such as about 1% of ITS.

[0215] In certain embodiments, peptides produced as described herein are used as one or more of: directly as a source of amino acids; indirectly as a stimulator (e.g., serum replacement); and / or for protecting cultured cells from shear stress (e.g., apoptosis).

[0216] In some embodiments, the lysate and / or protein hydrolysate and / or amino acid composition produced as described herein provides a source of vitamins for growing cells in culture. The lysate and / or protein hydrolysate and / or amino acid composition can be a source of vitamins, such as but not limited to vitamin B1, vitamin B2 and / or vitamin B 12 In certain embodiments of the present invention, the protein hydrolysate and / or the amino acid composition may be a source of one or more vitamins, including but not limited to: vitamin A; β-carotene; lutein; zeaxanthin; thiamine (B1); riboflavin (B2); niacin (B3); pantothenic acid (B5); vitamin B6; folic acid (B9); vitamin B 12; choline; vitamin C; vitamin D; vitamin E; and / or vitamin K; and / or their equivalent vitamins. The protein hydrolysate and / or amino acid composition can be a source of one or more minerals, including but not limited to: calcium; iron; magnesium; manganese; phosphorus; potassium; sodium; and / or zinc.

[0217] In certain embodiments, whole-cell biomass products and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients, cofactors or components, and / or formulations comprising one or more of the same produced as described herein are used to grow one or more other organisms that produce one or more of the following vitamins: vitamin A; beta-carotene; lutein; zeaxanthin; thiamine (B1); riboflavin (B2); niacin (B3); pantothenic acid (B5); vitamin B6; folic acid (B9); vitamin B 12 ; Choline; Vitamin C; Vitamin D; Vitamin E; and / or Vitamin K; and / or their vitamin equivalents.

[0218] In certain embodiments, the whole-cell biomass product and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other extract produced as described herein is used as a source of one or more of the following for another cell culture: amino acids; peptides, including oligopeptides; lipids; carbohydrates; polysaccharides; vitamins and / or minerals, including iron.

[0219] In certain embodiments, the lysates and / or protein hydrolysates produced as described herein are water soluble.

[0220] In certain embodiments, compared to free free amino acid mixtures containing the same or similar ratios and amounts of each amino acid in free form and polymerized form without equivalent free amino acids, the lysates and / or protein hydrolysates and / or peptide compositions produced as described herein stimulate energy-efficient metabolism in cell cultures supplied with the lysates and / or protein hydrolysates and / or peptide compositions. In certain embodiments, compared to free free free amino acid mixtures containing the same or similar ratios and amounts of each amino acid in free form and polymerized form without equivalent free amino acids, cell cultures supplied with lysates and / or protein hydrolysates and / or peptide compositions produced as described herein produce less ammonia and / or lactate and / or undergo less glycolysis and / or glutaminolysis.

[0221] In certain embodiments, the lysate and / or protein hydrolysate and / or amino acid composition produced as described herein is derived from more than one microbial source. In certain embodiments, the microbial source includes one or more chemoautotrophic microorganisms. In certain embodiments, the microbial source is or includes a microbial consortium.

[0222] In some embodiments, the protein hydrolysate composition comprises a biodegradable polyester. In some embodiments, the protein hydrolysate composition comprises a polyhydroxyalkanoate (PHA) polymer, such as PHB and / or PHV. In some embodiments, the PHA polymer can be represented by the following formula: [COCH2CH(R)O] n , wherein R is a C1-C5 alkyl group, and n is an integer of 50 or greater. In some embodiments, a PHA oligomer is present, which can be represented by the formula: [COCH2CH(R)O] n , wherein R is a C1-C5 alkyl group, and n is an integer of 50 or less. In some embodiments, there are free hydroxybutyrate and / or other hydroxyalkanoate (e.g., hydroxyvalerate) monomers. In some embodiments, the average molecular weight of the PHA polymer is about 1 kDa or greater, for example, about 10 kDa or greater, about 100 kDa or greater, including about 1,000 kDa or greater. In some embodiments, the average molecular weight of the PHA polymer is in the range of about 1 kDa to about 10,000 kDa, for example, about 10 kDa to about 5,000 kDa, including about 100 kDa to about 2,000 kDa. In some embodiments, the average molecular weight of the PHA oligomer is about 1 kDa or less. In certain embodiments, the PHA polymer includes polyhydroxybutyrate (PHB), wherein R is CH3. In some embodiments, the PHA polymer is a copolymer, including a block copolymer. In some embodiments, PHA (e.g., PHB and / or PHV) is derived from one or more microorganisms, for example, including chemoautotrophic microorganisms.

[0223] The protein hydrolysate composition may comprise about 1% w / w or more, such as about 5% w / w or more, about 10% w / w or more, about 20% w / w or more, about 30% w / w or more, about 40% w / w or more, including about 50% w / w or more of a biodegradable polyester, such as a PHA (e.g., PHB and / or PHV). In some embodiments, the protein hydrolysate composition may comprise about 1% to about 90% w / w, such as about 5% to about 80% w / w, about 10% to about 70% w / w, about 20% to about 60% w / w, including about 30% to about 50% w / w of a biodegradable polyester, such as a PHA (e.g., PHB and / or PHV).

[0224] In certain embodiments, the container for making lysate and / or protein hydrolysate and / or amino acid composition as described herein is fully disinfected, and verified treatment is used to eliminate potential cross contamination from animal-derived materials that may be produced using the same processing equipment. In certain embodiments, the hydrolysis process is carried out in a facility where the equipment for processing non-animal-derived materials is separated from the equipment for processing animal materials. In certain embodiments, the hydrolysis process is carried out in a facility only for processing non-animal-derived materials and / or with equipment only for processing non-animal-derived materials. In certain embodiments, the biomass and / or lysate and / or hydrolysate and / or amino acid composition produced as described herein is not exposed to animal-derived materials for secondary exposure, such as nutritional components in bacterial fermentation broth or processing enzymes of animal origin. In certain embodiments, there is no secondary animal-derived material in the entire manufacturing process.

[0225] In certain embodiments, the biomass and / or lysate and / or protein hydrolysate and / or amino acid composition as described herein does not have any pesticide and / or herbicide and / or fungicide residues and / or is not exposed to any pesticide and / or or herbicide and / or fungicide. In certain embodiments, the biomass and / or lysate and / or protein hydrolysate and / or amino acid composition as described herein is not exposed to any antibiotics, such as but not limited to cephalosporins.

[0226] Also provided herein are cell culture medium supplements, including but not limited to animal cell culture medium supplements, comprising nutrient sources, such as lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions derived from microorganisms (e.g., chemoautotrophic microorganisms) as described herein. In certain embodiments, the supplementary nutrient sources supplement the protein hydrolysates and / or other nutrient compounds and growth factors in a given culture medium.

[0227] In certain embodiments, the protein hydrolysate composition can be merged with the basal cell culture medium to provide a complete medium suitable for culturing animal cells. The basal culture medium generally comprises amino acids, vitamins, organic and / or inorganic salts, trace elements, buffer salts and sugars. Examples of basal culture medium include but are not limited to DMEM (Dubé-Cohn's Modified Eagle's Medium), DMEM / F12, Iskoff's modified Dulbecco's Medium (IMDM), IMDM / F12, IMDM / F12 / NCTC 135, MEM (minimum essential medium Eagle's), culture medium 199, RPM-I 1640 and RPMI 1640 / DMEM / F12. The complete medium made by supplementing the basal culture medium with the protein hydrolysate composition of the present invention can support the growth and / or development of animal cells in the culture, without any additional components or without any animal-derived components, such as amino acids or protein hydrolysates derived from animals, or serum of animal origin. In some embodiments, the basal culture medium can be supplemented with the protein hydrolysate composition and one or more other components, such as but not limited to somatomedin or plant or yeast-derived protein hydrolysates, etc. In certain embodiments, the lysate and / or protein hydrolysate and / or amino acid composition of the present invention can be used to replace or substitute the nutritional components in the basal medium, including but not limited to one or more of the following: amino acids; vitamins; organic and / or inorganic salts; trace elements; buffer salts; and / or sugars. In certain embodiments, the lysate and / or protein hydrolysate and / or amino acid composition of the present invention can be used to replace the basal medium or as a substitute for the basal medium.

[0228] In certain embodiments, nutrients such as whole cell biomass products and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients, cofactors or components that have been produced as described herein (e.g., by synthesis by CO2 chemoautotrophic biosynthesis) can be combined with other typical culture medium components from other sources, such as but not limited to amino acids, vitamins, organic and / or inorganic salts, trace elements, buffer salts and / or sugars to provide a complex culture medium. In certain such embodiments, the complex culture medium is a complete culture medium suitable for culturing another cell culture. In certain embodiments, nutrients such as whole-cell biomass products and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients, cofactors or components that have been produced as described herein are used to supplement and / or replace culture medium components in commonly used complex culture media such as, but not limited to, Elliker broth (also known as Lactobacillus broth); Tryptone dextrose yeast extract (TGYE) medium; Lysogeny broth (also known as Luria-Bertani broth-LB); Seed medium; M9 medium; M9CA; Complex medium M101; YT medium; MMBL medium; Terrific broth; Super broth; Yeast extract-malt extract (YEME) medium. In certain embodiments, the lysate and / or protein hydrolysate and / or extract produced as described herein is used to replace or supplement the protein hydrolysate and / or amino acid composition of animal, dairy or plant origin used in one of the above-mentioned composite culture media, such as but not limited to one or more of the following: tryptone; casein (acid and / or enzyme) hydrolysate; peptone; casamino acids; and / or to replace or supplement yeast extract and / or malt extract. In certain embodiments, the lysate and / or protein hydrolysate and / or extract produced as described herein is used in the composite culture medium at a concentration ranging from about 5g / L to about 25g / L. In certain embodiments, the lysate and / or protein hydrolysate and / or extract produced as described herein is used in the composite culture medium at a concentration ranging from about 25g / L to about 32g / L, or from about 32g / L to about 52g / L, or greater than about 52g / L. In certain embodiments, the surface response methodology ( TV, BOUCHU, A., PERLOT, P., PARK, JW, PARK, KN, WEBER, FJ, TRAMPER, J., RINZEMA, A., D'SOUZA, F., LALI, A., and others (1999) D.LEVISAUSKAS, V.GALVANAUSKAS, R.SIMUTIS and AL 37--42. Biotechnology Techniques, 13, 937-943) to determine the optimal concentration (g / L) of nutrients produced as described herein to be included in the complex culture medium, for example, in order to maximize a desired culture metric, such as, for example, biomass or product yield.

[0229] In certain embodiments, lysate and / or hydrolysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition as described herein can be used to prepare the growth medium for another culture (including but not limited to LAB strain). In certain embodiments, nutrient modifier or supplement helps or satisfies the nitrogen demand of another culture (including but not limited to LAB culture). In certain embodiments, preparation can include carbohydrate source (such as lactose). In certain embodiments, carbohydrate can be converted into acid, such as but not limited to lactic acid, for example by reducing the LAB culture of pH, which can be used to preserve product and contribute to the taste and flavor of product.

[0230] Yeast extract (the concentrated soluble components from yeast cells) is also widely used in culture growth media. In certain embodiments, the preparations produced as described herein may include a lysate and / or hydrolysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein and yeast extract (e.g., in combination with yeast extract, or replacing a portion of the yeast extract in the culture medium). In other embodiments, the lysate and / or hydrolysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are used to replace all yeast extract in the preparation.

[0231] In certain embodiments, supplementing the basal formulation with a protein hydrolysate produced as described herein occurs in an amount ranging from about 100 mg / L to about 2.5 g / L. In certain embodiments, the culture medium is supplemented with a protein hydrolysate produced as described herein at a concentration ranging from about 0.25 to about 4 g / L, or from about 4 g / L to about 25 g / L, or from about 25 g / L to about 32 g / L, or from about 32 g / L to about 52 g / L, or greater than about 52 g / L.

[0232] In certain embodiments, milk and / or meat hydrolysate is replaced by the hydrolysate of microbial origin produced as described herein. In certain embodiments, lysate as described herein and / or protein hydrolysate and / or amino acid composition can reduce the amount of serum of animal origin or eliminate serum of animal origin, such as fetal bovine serum, thereby providing complete serum-free medium. In certain embodiments, the replacement of this milk and / or meat hydrolysate and / or the reduction or elimination of serum contribute to regulatory approval. In certain embodiments, the replacement of this milk and / or meat hydrolysate and / or the reduction or elimination of serum avoids the requirement of verifying the process of removing potential extraneous factors from suspicious raw materials.

[0233] Protein hydrolysates are generally relatively stable and can be stored dry, for example, at refrigerated temperatures. In certain embodiments, the protein hydrolysates produced as described herein are stored dry and / or stored at refrigerated temperatures.

[0234] By including certain protein hydrolysates, production of dry media by conventional ball milling processes may become complicated because elevated temperatures and physical damage can degrade unstable hydrolysate components. In certain embodiments using lysates and / or protein hydrolysates and / or amino acid compositions as described herein, media production processes that reduce residence time, thermal denaturation, and mechanical shear are used, such as hammer milling or fluidized bed granulation.

[0235] How to use

[0236] Also provided is a method for culturing cells (e.g., eukaryotic cells, such as animal cells or prokaryotic cells such as lactic acid bacteria (LAB)) in a culture medium, wherein the culture medium is supplemented with or comprises a lysate and / or a protein hydrolysate composition and / or a peptide composition and / or an amino acid composition and / or other nutrients derived from a microorganism as described herein, such as a chemoautotrophic microorganism or a microbial consortium. In certain embodiments, the lysate and / or a protein hydrolysate and / or a peptide composition and / or an amino acid composition and / or other nutrients produced as described herein provide energy and / or carbon and / or a nitrogen source for another culture. In certain embodiments, the lysate and / or a protein hydrolysate and / or a peptide composition and / or an amino acid composition and / or other nutrients produced as described herein supplement or replace the nitrogen source used in the culture medium, such as but not limited to one or more of the following: a protein hydrolysate and / or an amino acid from an animal or plant source; and / or a yeast extract.

[0237] In certain embodiments, protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactor extracts and / or preparations comprising one or more of the foregoing components are designed and / or selected based on the nutritional requirements of another microorganism and / or organism, including but not limited to one or more of the following: peptides; amino acids; carbon sources; nitrogen sources; vitamins; minerals; growth factors; and / or other nutrients. In certain embodiments, nutritional components and / or preparations are designed and selected to provide or optimize one or more of the following: predictable fermentation time; cell yield; cell viability; downstream processing; and / or shelf life storage.

[0238] In certain embodiments, protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactor extracts and / or formulations comprising one or more of the foregoing components are designed and / or selected based on one or more of the following: degree of hydrolysis; peptide spectrum; ratio between amino nitrogen (AN) and total nitrogen (TN); free amino acid level; mineral content; production cost; ease of recovery and / or downstream processing; cost-effectiveness; and / or regulatory compliance.

[0239] In certain embodiments, lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactors meet the nutritional requirements of another species / strain by providing essential elements including, but not limited to, one or more of the following: amino acids; peptides; vitamins; minerals; nucleic acid bases; and / or other growth factors. In certain embodiments, one or more peptones, protein hydrolysates, yeast extracts, growth factors and / or vitamins are replaced or substituted by one or more nutrients produced as described herein. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients, cofactors or components (including but not limited to one or more of lipids, polysaccharides, carbohydrates, PHB, PHA, PHV, nucleic acids, vitamins and / or minerals) are used to replace culture medium components, including but not limited to one or more of the following: carbohydrates such as monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose, maltose), dextrins and / or maltodextrins; protein sources such as skim milk powder, whey and / or whey protein concentrate; protein hydrolysates or lysates such as peptone, casein hydrolysate, whey protein hydrolysate, soy protein hydrolysate, meat protein hydrolysate, primatone, hydrolyzed grain solids and / or yeast extract; sources of vitamins and minerals such as yeast extract, corn steep liquor; and / or other culture medium components such as Tween / oleic acid, mineral salts, defoaming agents and / or buffers.

[0240] In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients, cofactors or components (including but not limited to one or more of the following: lipids; polysaccharides; carbohydrates; PHB; PHA; PHV; nucleic acids; vitamins and minerals) are contained in a culture medium formulation, which contains other culture medium components, including but not limited to one or more of the following: carbohydrates such as monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose, maltose), dextrins and / or maltodextrins; protein sources such as skim milk powder, whey and / or whey protein concentrate; protein hydrolysates or lysates such as peptone, casein hydrolysate, whey protein hydrolysate, soy protein hydrolysate, meat protein hydrolysate, hydrolyzed grain solids and / or yeast extract; sources of vitamins and / or minerals such as yeast extract and / or corn steep liquor; and / or other culture medium components such as Tween / oleic acid, mineral salts, defoaming agents and / or buffers.

[0241] In certain embodiments, one or more chemoautotrophic microorganisms are co-cultured with one or more heterotrophic organisms, wherein the chemoautotrophic microorganisms secrete nutrients such as, but not limited to, amino acids into the culture broth, and wherein the heterotrophic organisms ingest and / or utilize nutrients such as, but not limited to, amino acids for growth and / or production. In certain embodiments, the chemoautotrophic microorganisms include Cupriavidus microorganisms, such as Cupriavidus necator and / or Cupriavidus metallidurans. In certain such embodiments, the chemoautotrophic microorganisms include Cupriavidus necator microorganisms, including Cupriavidus necator DSM 531 and / or DSM 541, and / or Cupriavidus metallidurans, including Cupriavidus metallidurans DSM 2839. In certain embodiments, the chemoautotrophic microorganisms are lysed and / or consumed; and / or chemoautotrophically synthesized proteins are hydrolyzed by one or more other organisms in the co-culture.

[0242] Some aspects of the compositions and methods described herein relate to the nutritional requirements of cell cultures and the use of protein hydrolysates and / or other extracts in fermentation and / or cell culture growth. Some aspects relate to culture media for fermentation and / or cell culture growth. Some aspects relate to the growth of lactic acid bacteria (LAB) and the culture media used in such growth. Some aspects relate to the positive effects of specific protein hydrolysates on the growth and survival of cell cultures (including but not limited to cultures comprising LAB).

[0243] In certain embodiments, the chemically defined medium (CDM) is supplemented with low molecular weight peptides (LMWP) produced as described herein. In certain embodiments, the LMWP includes peptides with a molecular weight (MW) ≤ 3,000Da. In certain embodiments, most or all or substantially all of the LMWP has a MW ≤ 3,000Da. In certain embodiments, when a CDM supplemented with the LMWP is provided to grow another strain (e.g., a microbial strain or species different from the microorganism from which the LMWP is derived) compared to growing other strains only on CDM (i.e., without LMWP supplementation), the cell number increases by at least 1.3 times. In certain embodiments, the other strain is a Lactobacillus strain, and in certain non-limiting embodiments is Lactobacillus helveticus.

[0244] In some embodiments, the use of a protein hydrolysate composition in a culture medium allows the culture of animal cells without some or any animal-derived components, such as protein hydrolysates, amino acids or serum of animal origin, in the culture medium. Therefore, in some embodiments, the method includes culturing cells in a serum-free culture medium to which a protein hydrolysate composition of the present invention has been added. For example, cells can be cultured in a culture medium without fetal bovine serum, horse serum, goat serum or any other animal-derived serum by providing a protein hydrolysate of the present disclosure in the culture medium. In some embodiments, the method includes culturing cells in a culture medium containing a protein hydrolysate composition without any animal-derived components (such as serum-derived albumin, transferrin, insulin, growth and / or other factors).

[0245] In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients, cofactors or components produced as described herein improve the strain vigor of the culture over time. In certain embodiments, it helps to maintain the redox potential in the culture. For example, the strain vigor over time can be measured based on CFU / g (colony forming units per gram of culture).

[0246] In certain embodiments, lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactors are produced in a consistent and reproducible manner, regardless of region, season or climate. In certain embodiments, the batch-to-batch variation of lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactors is less than a comparable animal or plant-based hydrolysate.

[0247] In certain embodiments, the protein hydrolysate produced as described herein is used to produce one or more of the following: fermentation culture; auxiliary fermentation agent; and / or one or more probiotics. In certain embodiments, the protein hydrolysate produced as described herein is used to grow industrial fermentation culture, such as industrial fermentation culture fermentation. In certain embodiments, the lysate and / or protein hydrolysate produced as described herein and / or peptide composition and / or amino acid composition and / or other nutrients are incorporated into the preparation of the fermentation medium for producing the fermentation culture. In certain embodiments, the industrial fermentation culture is incorporated with LAB. In certain embodiments, the fermentation culture is recovered from the solution and converted into a concentrated frozen or freeze-dried form for storage, transportation and / or sale. In certain embodiments, one or more of the following downstream process steps are used for the fermentation culture: concentration; adding cryoprotectants; freezing; and / or freeze drying. In certain embodiments, the probiotic strains grown on the nutrients produced as described herein are used as components of the fermentation culture of fermented milk and / or as nutritious food products for which they can be prepared in the form of free and / or encapsulated freeze-dried materials. Commercial starter culture concentrates are sometimes provided in the form of "batch" (Redi) or "direct vatset" (DVS). "Batch" is used to prepare intermediate starter cultures, which are then inoculated into production tanks to prepare the final product. These cultures are obtained in frozen (about 70 mL) or lyophilized form (about 5-10 g packaging) and are designed to inoculate 100-1,000 L of material. DVS cultures serve as direct inoculants in the final production culture. They are available as frozen or lyophilized cultures, with about 500 g of frozen culture used to inoculate 2,500–5,000 L of material, depending on the culture type and application. In certain embodiments, the starter culture produced as described herein is prepared as a batch culture or a DVS culture.

[0248] In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients produced as described herein are used to grow one or more bacterial strains, and the bacterial strain supplements the primary fermentation culture and / or contributes to the value increase of the final fermented product. In certain embodiments, the lysate and / or hydrolysate of the present invention and / or peptide composition and / or amino acid composition are added in the traditional fermentation of food. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition provide nutrients for the growth and / or vitality of LAB and / or probiotic cultures. In certain embodiments, the protein hydrolysate produced as described herein is used to produce dairy products and / or meat fermentation culture. In certain embodiments, the protein hydrolysate produced as described herein meets the nitrogen demand of LAB species. In certain embodiments, the lysate and / or hydrolysate and / or peptide composition and / or amino acid composition as described herein are supplied to the culture comprising the bacterial species commonly used as fermentation agent and / or flavoring agent and / or auxiliary fermentation agent. In certain embodiments, the lysate and / or hydrolysate and / or peptide composition and / or amino acid composition as described herein is fed to a culture spiked with LAB and / or other probiotics, including but not limited to one or more of: Lactococcus; Lactobacillus; Streptococcus; Pediococcus; and / or Bifidobacterium.

[0249] An important parameter affecting biomass quality is the composition and state of the organism's cell wall, which determines the survival rate of the cell during downstream processing. Although the firmness of the cell wall may depend on the species or even the strain, it is also affected by the composition of the culture medium, the growth conditions, and the physiological state of the cell during harvest. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein affect one or more of the following: cell wall thickness; cell extension; and / or cell division.

[0250] In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are fed to a culture spiked with one or more of the following microorganisms: Lactococcus lactis; Leuconostoc spp.; Streptococcus thermophilus; Lactobacillus spp. spp.), including but not limited to Lactobacillus bulgaricus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus plantarum; Pediococcus spp., including but not limited to Pediococcus pentosaceus, Pediococcus acidilactici; and / or Bifidobacterium spp. spp.), including but not limited to Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium infantis. In certain embodiments, the microorganisms include one or more nutritionally demanding strains. In certain embodiments, the microorganisms include one or more probiotics.

[0251] In certain embodiments, whole-cell biomass and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients or cofactors produced as described herein are supplied to one or more microorganisms and / or macroorganisms that are considered "generally recognized as safe" (GRAS) and / or traditionally (e.g., historically) used to make human food and fermentation products. In certain embodiments, whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are supplied to one or more other organisms (e.g., one or more organisms different from the microorganism from which the whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors were derived), including but not limited to one or more of the following: yeast, such as Candida humilis, Candida milleri, Debaryomyces hansenii, Kazachstania exigua (Saccharomyces exiguous), Saccharomyces cerevisiae, Saccharomyces florentinus, Torulaspora delbrueckii, Trichosporon beigelli; fungi, such as Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Fusarium venenatum A3 / 5, Neurospora intermedia var.oncomensis, Rhizopus oligosporus, Rhizopus oryzae; bacteria such as Acetobacter aceti, Bacillus amyloliquefaciens, Bacillus subtilis, Bifidobacterium animalis (lactic acid), Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Gluconacetobacter xylinus (Komagataeibacter xylinus), Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus spp. delbrueckiisubsp.bulgaricus), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus hilgardii (Brevibacterium vermiforme), Lactobacillus kefiranofaciens, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus sanfranciscensis, Lactococcus lactis (Streptococcus lactis; Streptococcus lactis subsp. diacetylactis), Leuconostoc sp.), Leuconostoc carnosum, Leuconostoc cremoris, Leuconostoc mesenteroides, Pediococcus sp., Propionibacterium freudenreichii, Arthrospira (Spirulina) platensis, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus xylosus. In certain embodiments, the organism is present in a co-culture or consortium or symbiotic culture (SCOBY) of bacteria and yeast. In certain embodiments, the co-culture, consortium, or SCOBY comprises one or more chemoautotrophic microbial strains. .

[0252] In certain embodiments, a lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients produced as described herein are supplied to a mesophilic or thermophilic dairy culture.

[0253] Microorganisms are used to make a variety of fermented products. The use of microorganisms in fermented foods dates back to long before their existence was known. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients produced as described herein are supplied to a culture for the production of one or more of the following foods, beverages or feed additives: fermented dairy products, including but not limited to yogurt, kefir, buttermilk; sour cream; cheeses, including but not limited to cream cheese, soft cheese, cheddar cheese, continental cheese, semi-hard cheese, hard cheese, Cheddar cheese, Swiss cheese, Gouda cheese, Mozzarella cheese, Emmental cheese, Parmesan cheese, Romano cheese, Provolone cheese, Jarlsberg cheese, Leerdammer cheese, Maasdam cheese; fermented meats, sausages, saucisson, salami; sourdough bread; dosa; fermented vegetables and / or plant materials, including but not limited to pickled vegetables; vegetable), pickles, sauerkraut, cucumbers, kimchi, tsukemono, tempeh, soy sauce, miso, fermented bean paste, red onions, natto, olives and brine olives, cocoa; fermented beverages, including but not limited to tea, kombucha, jun, ginger beer; nutritional yeast; bread; beer; wine; mescal; colonche; tequila; cider; mirin; strawberry tree fruits juice; sugar cane juice; vinegar; nutritional supplements; probiotics, including beverages, powders, supplements and capsules; tecuitlatl; dihe; silage.

[0254] In certain embodiments, lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition provide amino acid and / or peptide that LAB microorganism synthesis ability is limited or can not synthesize.In certain embodiments, amino acid substitute or supplement usually derive from milk and / or derive from the amino acid or peptide of the hydrolyzed protein that is derived from milk, animal or plant.

[0255] In order to use available proteins, peptides and / or free amino acids as building blocks for synthesizing new proteins (including but not limited to enzymes), they must be translocated across cell membranes into cells. If proteins and peptides are too large to be processed by the uptake system of the cell, they must be further hydrolyzed into smaller peptides or free amino acids. Some LABs are capable of synthesizing and secreting extracellular proteases that decompose proteins into peptides and amino acids, thereby making them available for translocation. For example, Lactococcus lactis has a proteolytic system that includes proteases located in the cell envelope, three peptide transport systems, a set of intracellular peptidases, and nine different amino acid transport systems that work synergistically to digest milk proteins and provide the cells with essential and growth stimulating peptides and amino acids (Poolman, B., Juillard, V., Kunji, ERS, Hagting, A., and Konings, WN (1996) Case in-breakdown by Lactococcus lactis. In Lactic Acid Bacteria (pp. 303-326). Springer.; Kunji, ERS (1996) The proteolytic systems of lactic acid bacteria. In Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology. https: / / doi.org / 10.1007 / BF00395933; Mierau, I., Venema, G., Kok, J., and Kunji, ERS (1997) Case in and peptide degradation in lactic acid bacteria. Biotechnology and Genetic Engineering Reviews. https: / / doi.org / 10.1080 / 02648725.1997.10647945). In certain embodiments, a biomass or whole cell product or cell lysate or other composition comprising a whole protein or large peptide produced as described herein is provided to a microorganism capable of synthesizing and secreting one or more extracellular proteases and / or comprising one or more proteolytic systems capable of hydrolyzing proteins into peptides and / or amino acids. In certain embodiments, the microorganism is a Lactococcus microorganism, e.g., Lactococcus lactis.In certain embodiments, peptide and / or amino acid are supplied to the microorganism or organism with the transport system for peptide and / or amino acid, and the microorganism or organism may be or may not be the same organism as the microorganism providing extracellular protease and / or proteolytic system. In certain embodiments, however, to the microorganism feeding protein hydrolysate and / or peptide composition and / or amino acid composition that can synthesize and secrete one or more extracellular proteases and / or have one or more proteolytic systems to save the consumption of other required cell energy of enzyme production, and improve the productive rate of cell growth and / or product thus. In addition, when the extracellular protease and / or proteolytic system of microorganism release essential amino acid from protein nutrition too slowly and can not support optimal growth and / or production, in certain embodiments, these essential amino acids are provided and / or with the form of protein hydrolysate and / or peptide composition and / or amino acid composition and / or free amino acid produced as described herein to supplement.

[0256] Some LAB lack proteolytic systems and require a culture medium containing basic structural units such as amino acids and ammonia as a nitrogen source. In certain embodiments, a co-culture is provided, the co-culture comprising a first microorganism capable of synthesizing and secreting extracellular proteases and / or having a proteolytic system, a second different microorganism, the second different microorganism lacking a proteolytic system and / or requiring a culture medium containing basic structural units such as amino acids and ammonia as a nitrogen source. In certain embodiments, the first microorganism is a Lactococcus microorganism, such as Lactococcus lactis, and / or the second microorganism is a LAB.

[0257] LAB does not usually have a functional tricarboxylic acid (TCA) cycle, which makes their energy generation pathways relatively inefficient. Homofermentative organisms such as Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus helveticus and Lactobacillus acidophilus produce energy through glycolysis (Embden-Meyerhoff-Parnas-EMP) pathway, wherein 2 moles of ATP are formed per 1 mole of hexose consumed. Leuconostoc species produce energy through heterofermentative pathways, and only 1 mole of ATP is formed per 1 mole of hexose consumed. In addition, ATP can be produced by a chemiosmotic energy process (e.g., lactate outflow). In addition, LAB generally does not produce any endogenous energy storage compounds, such as glycogen, polyphosphates and poly-β-hydroxybutyrate, except a small amount of phosphoenolpyruvic acid collection. An exception is Bifidobacterium bifidum, which forms storage compounds, such as glycogen and polyphosphates, in the stationary phase. Therefore, in order to support the anabolic process and cell growth of LAB cultures, energy and other nutrient sources must usually be provided to the culture medium.

[0258] In some embodiments herein, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are provided to LAB culture, which compensates for incomplete tricarboxylic acid (TCA) cycle. In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are provided to LAB culture, which preserves energy in culture, otherwise the energy will be consumed when synthesizing biochemical substances (such as organic compounds) by anabolic pathways driven by inefficient LAB metabolism, thereby preserving and / or more effectively utilizing carbon sources, such as lactose or glucose and / or ATP. In certain embodiments, the biochemical substances synthesized by anabolic pathways include but are not limited to one or more of the following: amino acids; peptides; lipids; carbohydrates; polysaccharides; nucleic acids; vitamins; and / or cofactors.

[0259] In certain embodiments, the lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein are used to supplement the phosphoenolpyruvate (PEP) pool and / or increase the concentration of PEP in the pool for use in microbial cultures, such as but not limited to LAB cultures.

[0260] In certain embodiments, the lysate and / or protein hydrolysate and / or extract produced as described herein provide one or more of the following to the culture (such as LAB culture): cell wall components, such as lipid, teichoic acid and / or peptidoglycan; and / or RNA and / or DNA precursors, such as purine and pyrimidine bases. In certain embodiments, these supplements are provided to the culture medium to preserve sugar and / or ATP, which are otherwise consumed by the culture when synthesizing biochemical substances.

[0261] Lactococci, the main component of dairy cultures used to produce many hard and semi-hard cheeses, fermented milks and creams, have lost their characteristic strong proteolytic activity and have acquired auxotrophies for most amino acids (Bringel, F., and Hubert, J.C. (2003) Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: Prevalence of CO2-dependent auxotrophs and characterization of deficient arg genes in L. plantarum. Applied and Environmental Microbiology. https: / / doi.org / 10.1128 / AEM.69.5.2674-2683.2003; Morishita, S., and Tarui, S. (1981. Lactic acidosis. Nihon Rinsho. Japanese Journal of Clinical Medicine, 39(11), 3459–3465), due to their long-term adaptation to milk, which is a fairly nutritious growth medium. Amino acid requirements and transport systems are known to be growth limiting factors in Lactococci (Poolman and Konings 1988). In certain embodiments, the protein hydrolysate and / or peptide composition and / or amino acid composition produced as described herein is provided to a culture comprising a microorganism that has lost its proteolytic activity and / or acquired amino acid auxotrophy due to evolution on a milk matrix. In certain embodiments, the microorganism that has lost its proteolytic activity and / or acquired amino acid auxotrophy is a Lactococcus strain.

[0262] Lactococcus lactis subsp. lactis is auxotrophic for at least seven amino acids: Gln, Met, Leu, Ile, Val, Arg, and His (Law et al. (1976)). Lactococcus lactis cream strains are reported to have higher requirements than Lactococcus lactis lactis strains and generally require additional Tyr, Asn, and Ala. In certain embodiments, amino acids including, but not limited to, one or more of the following: Gln; Met; Leu; Ile; Val; Arg; His; Tyr; Asn and / or Ala, produced as described herein, are provided to another microbial strain that is auxotrophic for one or more of those amino acids. In certain embodiments, the amino acid or protein supplement composition is provided to the auxotrophic strain in the form of one or more of the following: free amino acids; peptides; protein hydrolysates; proteins; lysates; and / or whole cell biomass. In certain embodiments, the auxotrophic strain is a dairy Lactococcus lactis subsp. lactis and / or Lactococcus lactis cream species. In certain embodiments, the growth medium supplemented with amino acids in one or more of the following forms produced as described herein: free amino acids; peptides; protein hydrolysates; proteins; lysates; and / or whole cell biomass causes the microbial strain as described herein (such as auxotrophic strain) to increase its growth rate compared to its growth rate on milk. In certain embodiments, the amino acid or protein supplement is provided to the culture medium at a level of up to about 4% (w / v) or up to about 2.5% (w / v). In some embodiments, the amount of the amino acid or protein supplement is about 0.5% (w / v) to about 2.5% (w / v). In certain embodiments, the amino acid supplement is provided at a level greater than about 4% (w / v). In certain embodiments, the amino acid or protein supplement includes, but is not limited to, one or more of the following amino acids: Leu; Phe; and / or Glu. In certain embodiments, the growth rate on the supplemented medium is at least about 10%, 20%, or 40% higher than the growth rate on milk.

[0263] Proline (Pro) has been found to stimulate the growth of Lactococcus lactis strains, regardless of their ability to synthesize this amino acid (Smid and Konings (1990)). Although milk is rich in Pro, this amino acid is reported to be not readily available because some strains lack a proline transport system. Proline can only be transported into cells by passive diffusion or by transporting proline-containing peptides (Smid and Konings, supra). In certain embodiments, Pro produced as described herein is provided to another culture (e.g., a culture of a microorganism different from the microorganism from which Pro is derived). In certain embodiments, Pro is provided as a free amino acid and / or is provided within a peptide and / or protein containing Pro. In certain embodiments, the culture to which the Pro supplement in the form described is provided contains one or more Lactococcus lactis strains. In certain embodiments, a microbial strain to which Pro produced as described herein is provided exhibits a higher growth rate than the same culture not provided with Pro. In some embodiments, the microbial strain is a Lactococcus lactis strain, such as one or more of the following: Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. cremoris strains.

[0264] Many chemically defined culture media have been developed for Lactococcus lactis. The standard synthetic culture media (MCD) developed by the people such as Otto (1983) and modified by Poolman and Konings (1988) the same contains 47 components, including 18 kinds of amino acids and 14 kinds of vitamins. In certain embodiments, one or more amino acids and / or vitamins contained in the chemically defined culture media for another microorganism (for example, a microorganism different from the microorganism from which amino acids and / or vitamins are derived) are produced as described herein. In certain such embodiments, amino acids and / or vitamins are produced by CO as a sole carbon source. In certain embodiments, the microorganism receiving amino acids and / or vitamins is a Lactococcus microorganism, such as Lactococcus lactis. In certain embodiments, one or more amino acids produced as described herein are provided to the culture media, wherein for a culture provided without the amino acidic culture media, the omission of amino acids will cause a growth rate reduction of at least about 75% and / or a biomass yield reduction of at least about 50%.

[0265] Several lactobacillus species are used as components of starter cultures for the production of yogurt (Lactobacillus delbrueckii bulgaricus species) and various types of cheese (Lactobacillus helveticus, Lactobacillus paracasei), and as probiotics in fermented milk and / or nutritional products (Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus reuteri). Nutritional and nitrogen requirements vary significantly from species to species, and even between strains of the same species / subspecies. Several studies have been conducted to elucidate the general nutritional requirements of Lactobacillus species. Elli et al. (2000) and Chervaux et al. (2000) described the nutritional requirements of 22 lactobacillus strains using a chemically defined culture medium containing 21 amino acids and other nutrients, comprising 60 components. In general, in order to obtain optimal growth and vigor, these lactobacilli need to be supplemented with a fermentation medium rich in carbon and nitrogen sources, vitamins, micronutrients and macronutrients, and nucleotide bases. In certain embodiments, one or more of the following: biomass produced as described herein; protein; lysate; protein hydrolysate; peptide composition; amino acid composition; and / or other extracts are used to provide one or more of the following: a carbon source; a nitrogen source; vitamins; macronutrients; micronutrients; and / or nucleotide bases to a culture comprising one or more Lactobacillus strains.

[0266] Lactobacillus helveticus is reported to have a greater amino acid requirement than most other lactobacillus or lactococcal strains. Morishita et al. (1981), supra, showed that strain ATCC15009 was auxotrophic for 14 amino acids, four vitamins and uracil, while strain CRL 1062 required 13 amino acids (Hebert et al. 2000). In certain embodiments, one or more of the following: amino acids produced as described herein; vitamins; and / or RNA and / or DNA bases such as uracil are provided to a culture containing auxotrophic for one or more amino acids; vitamins; and / or nucleobases such as, but not limited to, uracil. In certain embodiments, the auxotrophic is a strain of Lactobacillus helveticus.

[0267] The probiotic Lactobacillus acidophilus (LA) is reported to require the presence of Pro, Arg, Glu (Morishita, et al. (1981), supra), aromatic amino acids, and His (Hebert et al. (2000), supra) for growth. The requirement for aromatic amino acids and His is reported to be associated with LA that does not have a fully functional pentose phosphate pathway. However, it is reported that almost all 18 amino acid types greatly stimulate LA. In certain embodiments, amino acids produced as described herein, including but not limited to one or more of the following: Pro, Arg, Glu, aromatic amino acids, and / or His, are provided to one or more other strains (e.g., a microbial strain different from the microorganism from which the amino acids are derived). In certain such embodiments, the strain requires one or more of the following: Pro, Arg, Glu, aromatic amino acids, and / or His for growth. In certain such embodiments, those amino acids produced according to the present disclosure (i.e., Pro, Arg, Glu, aromatic amino acids, and / or His) are provided to one or more microorganisms that do not have a fully functional pentose phosphate pathway. In certain such embodiments, the other strains include LA strains.

[0268] Although it is not considered necessary, it is reported that Arg can stimulate the growth of Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus reuteri, Pediococcus pentosaceus and Streptococcus thermophilus strains. In certain non-limiting embodiments, Arg is provided to one or more other strains, such as microbial strains different from the microorganisms that produce Arg as described herein, and its growth is stimulated by Arg. In certain embodiments, Arg is provided to a culture comprising one or more of the following microorganisms: Lactobacillus bulgaricus; Lactobacillus acidophilus; Lactobacillus reuteri; Pediococcus pentosaceus; and / or Streptococcus thermophilus strains. In certain embodiments, all 20 protein amino acids directly encoded by triplet codons are produced as described herein and provided to one or more other strains (e.g., one or more microbial strains different from the microorganisms from which the amino acids are derived). In certain embodiments, the growth of one or more other strains is stimulated by providing one or more of the 20 amino acids produced according to the present disclosure and provided as described herein. In certain embodiments, other strains include LA strains and / or Lactobacillus reuteri strains.

[0269] As is well known, Lactobacillus reuteri is a particularly demanding organism that requires the amino acids Met, Glu, Tyr, Trp, His, Leu, Val and Ala for growth, and when other amino acids are not provided to the organism, its growth rate is reduced. In certain embodiments, the amino acids produced as described herein, including but not limited to one or more of the following: Met, Glu, Tyr, Trp, His, Leu, Val and / or Ala are provided to one or more other strains, such as microbial strains different from the microorganisms that produce amino acids as described herein. In certain such embodiments, the other strains require one or more of the following: Met, Glu, Tyr, Trp, His, Leu, Val and / or Ala for growth. In certain embodiments, the other strains include Lactobacillus reuteri. In certain embodiments, additional amino acids other than Met, Glu, Tyr, Trp, His, Leu, Val and Ala produced as described herein are also provided to Lactobacillus reuteri.

[0270] In certain embodiments, all twenty kinds of protein amino acids directly encoded by triplet codons in the genetic code and referred to as "standard" amino acids are produced as described herein, and provided to one or more other strains and / or organisms, for example, microbial strains and / or organisms different from the microorganisms producing amino acids as described herein. In certain embodiments, all 22 kinds of protein ("protein building") amino acids are produced as described herein and provided to one or more other strains and / or organisms, for example, microbial strains and / or organisms different from the microorganisms producing amino acids as described herein. In certain embodiments, one or more of approximately 500 kinds of known naturally occurring amino acids are produced as described herein and provided to one or more other strains and / or organisms, for example, microbial strains and / or organisms different from the microorganisms producing amino acids as described herein.

[0271] Streptococcus thermophilus (ST) strain is an important component of yogurt culture and some cheese cultures. Except lactic acid, some strains also produce exopolysaccharide (EPS), which contributes to the texture of fermented milk, and can improve the output of certain types of cheese (Petersen et al. (2000)). The growth of Streptococcus thermophilus requires nitrogen source in culture medium. Milk contains nitrogen suitable for the growth of Streptococcus thermophilus. However, the natural supply of amino acids and non-protein nitrogen present in milk is not enough to support the growth of Streptococcus thermophilus to high cell number. In certain embodiments herein, nutrients such as whole cell biomass products and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions and / or other nutrients, cofactors or components are provided to produce polysaccharides, such as but not limited to EPS and / or lactic acid cultures. In certain embodiments, when provided to Streptococcus thermophilus, nutrients cause the output of EPS to increase and / or larger capsule. In certain embodiments, nutrients improve the texture of fermented milk and / or improve the output of cheese. In certain embodiments, the nutrient serves as a nitrogen source of thermophilus streptococcus or a supplementary nitrogen source of thermophilus streptococcus. In certain embodiments, compared with only growing on milk, supplementing milk with one or more nutrients or replacing milk with one or more nutrients makes the microbial strain grow to higher cell quantity. In certain embodiments, the bacterial strain that grows to higher cell quantity is thermophilus streptococcus.

[0272] Streptococcus thermophilus relies on cell wall-associated proteases to digest whole proteins, or grows on protein hydrolysates as a source of amino acids, peptides and oligopeptides. In certain embodiments, nutrients such as whole cell biomass and / or lysates and / or whole proteins produced as described herein are provided to Streptococcus thermophilus, and rely on its natural proteases to convert these nutrients into amino acids, peptides and / or oligopeptides required for growth and / or the production of EPS and / or lactic acid.

[0273] For thermophilic streptococcus, Gln and Glu and sulfur-containing amino acids are considered to be essential amino acids. In addition, the branched-chain amino acid (BCAA) biosynthetic pathway is functional, but not sufficient to allow optimal growth of thermophilic streptococcus (Garault et al. (2000)) without supplementing BCAA. Therefore, BCAA is usually supplemented. In certain embodiments, one or more of the following will be provided, including but not limited to: Gln; Glu; sulfur-containing amino acids; and / or BCAA's produced as described herein in peptides and / or proteins free or bound amino acids are provided to one or more other strains or organisms, for example, microbial strains and / or organisms different from the microorganisms producing amino acids as described herein. In some such embodiments, the strain is thermophilic streptococcus.

[0274] According to reports, the combination of protein hydrolysate and yeast extract can provide suitable nitrogen source for the optimal production of thermophilic streptococcus.In certain embodiments, the protein hydrolysate produced as described herein and yeast extract are combined in the growth medium provided to microorganism (such as but not limited to thermophilic streptococcus).In certain embodiments, yeast extract is substituted with lysate, hydrolysate and / or other extracts produced as described herein in the culture medium provided to microorganism (such as but not limited to thermophilic streptococcus).In certain embodiments, one or more components of Elliker broth (also referred to as lactobacillus broth) are substituted with whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients and / or cofactors produced as described herein, thereby produce improved Elliker broth.In certain embodiments, the components substituted in improved Elliker broth include but are not limited to casein hydrolysate and / or yeast extract. In certain embodiments, Elliker broth is supplemented with whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients and / or cofactors produced as described herein, thereby producing supplemented Elliker broth. In certain embodiments, the modified and / or supplemented Elliker broth is used to culture Streptococcus and / or Lactobacillus.

[0275] In certain embodiments, the protein hydrolysate produced as described herein is provided to the culture medium with a concentration of about 5g / L to about 20g / L or about 5g / L to about 25g / L or about 20g / L to about 25g / L. It is reported that the hydrolysate of adding casein and whey in milk can improve the growth and acidification rate of thermophilic streptococcus ST-7, thereby shortening the fermentation time of yogurt (Lucas et al.). In certain embodiments, the hydrolysate produced as described herein improves the growth and / or acidification rate of another culture. In certain embodiments, the culture comprises thermophilic streptococcus ST-7 or is composed of it. It is reported that when tested in frozen dairy desserts, adding 2% acid casein hydrolysate and cysteine ​​to milk can improve the vigor of thermophilic streptococcus WJ7 within 12 weeks (Ravula and Shah, 1998). In certain embodiments, the protein hydrolysate produced as described herein is added to the culture medium with a concentration of about 2% (w / v). In certain embodiments, protein hydrolysate and / or amino acid composition are used to replace casein hydrolysate and / or cysteine.In certain embodiments, protein hydrolysate and / or amino acid composition produced as described herein improves the vigor of microorganism (e.g., Streptococcus thermophilus culture).

[0276] Bifidobacterium species are used as components of starter cultures for fermented milk and as freeze-dried materials for encapsulation. All bifidobacteria can utilize lactose, which enables them to grow in milk, although growth is generally weak due to low proteolytic activity (Klaver et al., 1993; Collins and Hall, 1984). However, it is reported that species such as Bifidobacterium bifidum and Bifidobacterium adolescentis do produce intracellular and extracellular proteases. Most strains contain leucine aminopeptidase, while a few strains contain valine aminopeptidase (Desjardins et al. 1990). Most bifidobacteria can use ammonium salts as their only nitrogen source (Azaola et al., 1999), but supplementing peptides and amino acids is considered to be a requirement for the economic production of these strains. The specific nitrogen demand depends on the strain, but typical nitrogen sources are peptides / amino acids, cysteine ​​and ammonium salts. It is reported that bifidobacteria have a relatively high demand for growth factors and vitamins (including biotin and calcium pantothenate) (Kurmann and Rasic, 1991). Several proteinaceous growth promoters are formed in milk, such as peptides containing disulfide / thiol groups, lactoferrin with bound metals (Fe, Cu, Zn), α-lactalbumin and β-lactoglobulin (Petschow and Talbott, 1991). In certain embodiments, nutrients such as whole cell biomass and / or lysate and / or protein hydrolysate and / or protein hydrolyzate and / or protein hydrolyzate and / or peptide composition and / or amino acid composition and / or other nutrients and / or cofactors produced as described herein are combined with milk and / or a lactose-containing culture medium and then supplied to another culture, such as a microbial culture of a microorganism different from the microorganism from which the whole cell biomass and / or lysate and / or protein hydrolyzate and / or protein hydrolyzate and / or protein hydrolyzate and / or peptide composition and / or amino acid composition and / or other nutrients and / or cofactors are derived. In certain embodiments, whole cell biomass and / or lysate and / or protein hydrolysate and / or proteolytic digest and / or peptide composition and / or amino acid composition and / or other nutrients and / or cofactors, which may be combined with other media components such as lactose, are used to replace milk. In certain embodiments, the culture comprises one or more strains of Bifidobacterium.

[0277] In certain embodiments, protein hydrolysate and / or peptide composition and / or amino acid composition are provided to microbial culture, and the microbial culture shows slow growth on a substrate such as milk, for example due to low proteolytic activity. In certain embodiments, the culture comprises one or more bifidobacterium strains. In certain embodiments, nutrients, supplements or culture medium preparations (e.g., containing protein hydrolysate and / or peptide composition and / or amino acid composition produced as described herein) are provided to a culture comprising one or more bifidobacterium strains, and faster growth of the same culture grown only on milk is observed.

[0278] In certain embodiments, a culture containing a microorganism producing intracellular and / or extracellular proteases is provided with nutrients produced as described herein (e.g., including whole cell biomass and / or lysate and / or protein concentrate and / or protein isolate and / or whole protein), and the microorganism producing the protease is capable of digesting the nutrients into peptides and / or free amino acids, which can be used by the microorganism and other microorganisms for growth and production of biomass and / or bioproducts. In certain embodiments, the microorganism producing the protease can be, but is not limited to, Bifidobacterium bifidum and / or Bifidobacterium adolescentis.

[0279] In certain embodiments, the culture is supplemented with peptides and / or amino acids produced as described herein, for economic production of culture and / or culture products. In certain embodiments, the culture comprises one or more strains of the genus Bifidobacterium. In certain embodiments, a nitrogen source is prepared, the nitrogen source comprising peptides and / or amino acids produced as described herein, including but not limited to cysteine. In certain embodiments, the nitrogen source also includes inorganic forms of nitrogen, including but not limited to ammonium salts. In certain embodiments, the nitrogen source is provided to another culture, such as a microbial culture of a microorganism different from the microorganism from which the nitrogen source is derived, such as but not limited to a culture comprising one or more strains of Bifidobacterium.

[0280] It has been reported that for some strains of Bifidobacterium, small peptides are a better source of amino acids than free amino acids (Proulx et al., 1994). In certain embodiments, the hydrolysis of proteins produced as described herein is designed to maximize small peptides and minimize free amino acids. In certain embodiments, enzymatic hydrolysis is used to prevent the presence of high concentrations of free amino acids. In certain embodiments, peptides with low or no or substantially no free amino acid content are provided to another culture, such as a microbial culture of a microorganism different from the microorganism from which the peptides are derived, such as but not limited to a culture comprising one or more strains of Bifidobacterium. It has been reported that the choice of protease affects the value of protein hydrolysates as growth promoters. It has been reported that with Peptides from tryptic digestion of casein have a better growth promoting effect on Bifidobacterium longum and Bifidobacterium infantis than enzymatic digests of casein or chymotrypsin (Proulx et al., 1994, supra). In certain embodiments, a protein hydrolysate is enzymatically produced (e.g., chemoautotrophically, e.g., produced from C1 compounds such as CO2, CO and / or CH4) from a protein produced as described herein and provided to another culture, such as, but not limited to, a microbial culture comprising one or more of Bifidobacterium longum and / or Bifidobacterium infantis.

[0281] It has been reported that for Bifidobacterium longum, an E. coli extract used in a complex medium produces a significant growth promoting effect (Ibrahim and Bezkorovany, 1994). In certain embodiments, an extract from a chemolithoautotrophic microorganism grown, for example, on a C1 substrate, such as, but not limited to, CO2, CO and / or CH4, is provided to another organism. In certain embodiments, the addition of the extract to the growth medium produces a growth promoting effect in another organism, such as, but not limited to, a bifidobacterial microorganism, e.g., Bifidobacterium longum.

[0282] In certain embodiments, the nutrients produced as described herein include growth factors and vitamins, such as, but not limited to, biotin and / or calcium pantothenate. In certain embodiments, the growth factors and / or vitamins are provided to another culture, such as a culture of an organism and / or microorganism different from the microorganism from which the growth factors and / or vitamins are derived. In certain embodiments, the vitamins include biotin and / or calcium pantothenate, and the culture to which these nutrients are provided comprises one or more strains of Bifidobacterium.

[0283] In certain embodiments, nutrients produced as described herein include proteinaceous growth promoters, such as, but not limited to, disulfide / thiol-containing peptides. In certain embodiments, proteinaceous growth promoters produced as described herein are combined with one or more growth promoters, such as lactoferrin, α-lactalbumin, and / or β-lactoglobulin with metal binding (e.g., Fe, Cu, Zn) from another matrix such as milk. In certain embodiments, one or more of the above proteinaceous growth promoters are provided to another culture, such as a culture of an organism and / or microorganism different from the microorganism from which the proteinaceous growth promoter is derived, such as, but not limited to, a culture comprising one or more strains of Bifidobacterium.

[0284] Bifidobacterium species require D-glucosamine, which is a structural unit of the peptidoglycan units N-acetylglucosamine and muramic acid and is therefore an essential component of the cell wall. (Poupard et al. (1973), supra). The composition and strength of the cell wall affect the survival rate of bifidobacteria during downstream processing. Milk contains N-acetylglucosamine in the form of oligosaccharides, and it is most commonly used as a source of glucosamine (Exterkate and Veerkamp, ​​1969). In certain embodiments, the whole cell biomass product and / or lysate and / or hydrolyzate and / or extract produced as described herein include peptidoglycan and / or peptidoglycan units, such as one or more of the following: D-glucosamine; N-acetylglucosamine; and / or muramic acid. In certain embodiments, the peptidoglycan and / or peptidoglycan units are provided to another culture, such as a culture of an organism and / or microorganism different from the microorganism from which the peptidoglycan and / or peptidoglycan units are derived. In certain embodiments, peptidoglycan and / or peptidoglycan unit are used to substitute or supplement from another source, such as plant animal source, such as peptidoglycan and / or peptidoglycan unit of milk, such as N-acetyl-glucosamine.In certain embodiments, culture needs peptidoglycan and / or peptidoglycan unit to be used for the cell wall generation and / or growth of organism (for example microorganism).In certain embodiments, peptidoglycan and / or peptidoglycan unit are provided as culture medium components and / or supplement to improve composition and the cell wall strength of microorganism in culture, and / or improve the survival rate of the microorganism in culture in downstream processing.In certain embodiments, culture comprises one or more bifidobacterium strains.

[0285] In addition to the complex nutritional requirements, the cultivation of bifidobacteria also needs to address the extreme sensitivity of these strains to oxygen. This problem is usually solved by adding substances that can maintain a low redox potential. Cysteine, ascorbic acid or sodium sulfite are usually used for this purpose. In certain embodiments, one or more nutrients and / or biochemical substances (e.g., organic compounds) produced as described herein help to maintain a low redox potential in the culture medium and / or culture environment to which they are added. In certain embodiments, the redox potential reducing components produced as described herein include but are not limited to cysteine ​​and / or ascorbic acid. In certain embodiments, the redox potential reducing components are provided to a culture comprising one or more bifidobacterium strains.

[0286] In certain embodiments, cultures grown on one or more nutrients and / or culture media described herein are used as fermentation products, such as fermented food products, e.g., starter cultures for fermented milk, and / or are further processed into encapsulated freeze-dried materials.

[0287] Pediococcus is used as a component of the starter culture for traditional fermented sausages. Unlike milk, meat is not pasteurized before inoculation of the starter culture and still contains a large amount of indigenous microflora. For the production of Pediococcus species, glucose or sucrose is used as an energy source and carbon source. Although not required, it is reported that the addition of acetate can reduce the lag phase and stimulate the growth of the organism. In certain embodiments, nutrients such as whole cell biomass and / or lysates and / or protein hydrolysates and / or protein hydrolyzed digests and / or peptide compositions and / or amino acid compositions and / or other nutrients and / or cofactors produced as described herein are combined with glucose and / or sucrose in the culture medium and then supplied to another culture, such as a culture of organisms and / or microorganisms different from the microorganisms from which nutrients such as whole cell biomass and / or lysates and / or protein hydrolysates and / or protein hydrolyzed digests and / or peptide compositions and / or amino acid compositions and / or other nutrients and / or cofactors are derived. In certain embodiments, the culture comprises one or more Pediococcus strains. In certain embodiments, the culture medium additionally comprises acetate. In certain embodiments, the acetate is, for example, produced by a C1 substrate, such as, but not limited to, CO2, CO and / or CH4 chemoautotrophic. In certain embodiments, one or more Pediococcus strains are fermented on a protein substrate other than meat. In certain embodiments, the protein substrate comprises whole cell biomass and / or lysate and / or protein hydrolysate and / or protein hydrolyzate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein. In certain such embodiments, the substrate is not pasteurized prior to inoculation of the starter culture (such as, but not limited to, one or more Pediococcus strains) and therefore may contain indigenous microflora.

[0288] Pediococcus pentosaceus is reported to have the following amino acid requirements: Val, Ala, Met, Pro, Arg, Glu, Cys, Tyr and His, while other amino acids are reported to have stimulatory effects. Cys-hydrochloride is reported to be irritating, but some of its stimulatory effects may be related to its function as an oxygen scavenger. In certain embodiments, amino acids produced as described herein, including but not limited to one or more of the following: Val, Ala, Met, Pro, Arg, Glu, Cys, Tyr and His are provided as free amino acids and / or amino acids bound in peptides and / or proteins to one or more other strains, such as organisms and / or microorganisms different from the microorganisms from which the amino acids are derived. In certain such embodiments, the strain requires one or more of the following: Val, Ala, Met, Pro, Arg, Glu, Cys, Tyr and / or His for growth. In certain embodiments, the strain includes one or more Pediococcus microorganisms, such as but not limited to Pediococcus pentosaceus.

[0289] It is reported that Pediococcus acidilactici is capable of hydrolyzing meat proteins. In certain embodiments, whole cell biomass, lysate and / or whole protein produced as described herein is provided to another organism, such as an organism and / or microorganism that is different from the microorganism from which the whole cell biomass, lysate and / or whole protein is derived and is capable of hydrolyzing them. In certain embodiments, the resulting peptides and / or amino acids produced by the hydrolysis are then used for nutrition by the organism itself, or by other organisms. In certain embodiments, the resulting peptides and / or amino acids produced by the hydrolysis are included in the final food or feed product. In certain embodiments, the one or more organisms performing protein hydrolysis include one or more Pediococcus strains, such as but not limited to Pediococcus acidilactici.

[0290] Even when a microbial strain possesses sufficient proteolytic systems for a given protein matrix, growth on a particular protein (e.g., casein) can be limited due to the low levels of certain amino acids (e.g., His, Leu, Gln, Val, and Met) in the case of casein (Kunji et al., 1995). In certain embodiments, a given protein matrix is ​​supplemented with one or more of the following as a source of amino acids: free amino acids produced as described herein; peptides; protein hydrolysates; proteins; lysates; and / or whole cell biomass, which provide amino acids lacking in the protein matrix. In certain embodiments, providing the lacking amino acids removes limitations on culture growth. In some embodiments, the supplemented protein matrix is ​​a milk protein, such as casein. In some embodiments, the amino acids supplemented by providing an amino acid source produced as described herein include, but are not limited to, one or more of the following: His; Leu; Gln; Val; and / or Met.

[0291] Several Lactobacillus species (Lactobacillus johnsonii, Lactobacillus gallinaceus, Lactobacillus gasseri, Lactobacillus helveticus) cannot synthesize purines and pyrimidines de novo (Elli et al., 2000). Milk and milk-derived hydrolysates do not contain purine and pyrimidine precursors. In certain embodiments, the purines and / or pyrimidines produced as described herein are provided to one or more microbial strains that cannot synthesize purines and / or pyrimidines de novo. In certain embodiments, the strains include Lactobacillus species, including but not limited to one or more of the following: Lactobacillus johnsonii, Lactobacillus gallinaceus, Lactobacillus gasseri and / or Lactobacillus helveticus. In certain embodiments, the purines and / or pyrimidines produced as described herein are provided to cultures grown on milk or milk-derived hydrolysates. In certain embodiments, the compositions comprising purines and / or pyrimidines produced as described herein are used to replace another typical purine and / or pyrimidine source used in a culture medium, such as, but not limited to, yeast extract.

[0292] In certain embodiments, the lysate as described herein and / or hydrolysate and / or peptide composition and / or amino acid composition supplemented in the nutrient medium enhances the cell proliferation and / or biological yield and / or cell density of the cell grown in the nutrient medium. In certain embodiments, the cell is an animal cell. In certain embodiments, compared with the same cells grown in the same medium not comprising lysate and / or hydrolysate and / or peptide composition and / or amino acid composition, lysate as described herein and / or hydrolysate and / or peptide composition and / or amino acid composition improves viable cell density and / or biomass amplification and / or product yield. In certain embodiments, when included in the nutrient medium, lysate as described herein and / or hydrolysate and / or peptide composition and / or amino acid composition increase cell density. In other embodiments, lysate as described herein and / or hydrolysate and / or peptide composition and / or amino acid composition improve cell density and product yield, and in other embodiments, inhibit cell growth but improve the yield of one or more bioproducts. In certain embodiments, lysate and / or hydrolyzate and / or peptide composition as described herein contain oligopeptides that serve as external molecular signals affecting cell growth and death. In certain embodiments, oligopeptides produced as described herein stimulate cell growth, thereby produce higher biomass production, and / or stimulate the production of secreted proteins and / or enhance the viable cell density of culture. In certain embodiments, oligopeptides produced as described herein serve as agents for delaying apoptosis death in cell culture. In certain embodiments, peptides produced as described herein cause lasting transformation of cell culture metabolism and / or changes in gene expression and / or cell proliferation. In certain embodiments, transformation and / or change last for several days. In certain embodiments, the distribution of cell cycle phases in the culture medium comprising peptides produced as described herein is changed. In certain embodiments, peptides produced as described herein regulate proliferation and / or signal transduction cascades and / or activation or inhibition of cultured animal cells. In certain embodiments, peptides produced as described herein are provided to cell culture with a concentration greater than or equal to about 1mM.

[0293] In certain embodiments, the chromatographic fractions of lysate and / or hydrolysate and / or peptide composition and / or amino acid composition produce different activities in the cell culture that provides them.In certain embodiments, lysate and / or hydrolysate and / or peptide composition and / or amino acid composition as described herein are not only used as the source of available amino acid, but also as the source of the peptide that cell growth and / or productivity play a specific role.In certain embodiments, one or more of the following culture parameters are improved by using lysate and / or hydrolysate and / or peptide composition and / or amino acid composition: viable cell density; Long-term vigor; And / or the productive rate of one or more biological products.In certain embodiments, the concentrated mixture of amino acid and / or other nutrients produced as described herein increases the productive rate of one or more biological products in the culture that provides the amino acid and / or other nutrients.

[0294] In certain embodiments, the protein hydrolysate composition is added to the culture medium at a concentration of about 0.001% w / v or higher, for example, about 0.01% w / v or higher, about 0.05% w / v or higher, about 0.1% w / v or more, including about 1% w / v or higher (as measured by the dry weight of the protein hydrolysate composition). In some embodiments, the protein hydrolysate composition is added to the culture medium at a range of about 0.001% w / v to about 5% w / v, for example, about 0.01% w / v to about 2% w / v, about 0.05% w / v to about 1% w / v, including about 0.1% w / v to about 1% w / v (as measured by the dry weight of the protein hydrolysate composition). The amount of the protein hydrolysate composition added to the culture medium can vary according to one or more of a variety of considerations, such as cell type, growth, reproduction, productivity, differentiation, etc. In certain embodiments, the culture medium is provided to the culture of animal cells.

[0295] In certain embodiments, the peptide is added to the culture medium at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 1 mM to about 7 mM, about 7 mM to about 10 mM, or greater than about 10 mM. In certain embodiments, the oligopeptides produced as described herein are added to the culture medium at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 1 mM to about 7 mM, about 7 mM to about 10 mM, or greater than about 10 mM. In certain embodiments, the culture medium is provided to the culture of the animal cell.

[0296] In some embodiments, the protein hydrolysate composition added to the culture medium comprises a biostimulant polyester. The biostimulant polyester can be a PHA polymer, such as PHB and / or PHV. The biostimulant can be a monomer, such as hydroxybutyrate (HB), or an oligomer. When added to the culture medium, the protein hydrolysate composition provides an effective amount of a biostimulant polyester, such as a PHA, such as PHB and / or PHV, to the culture medium to promote the growth and / or development of the cultured cells.

[0297] In some embodiments, the protein hydrolysate composition added to the culture medium contains vitamins, such as vitamin B1, vitamin B2 and / or vitamin B 12 When added to a culture medium, the protein hydrolysate composition provides an effective amount of vitamins to the culture medium to promote the growth and / or development of the cultured cells. In some embodiments, the protein hydrolysate composition comprises vitamin B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18, B19, B19, B21, B22, B23, B24, B25, B26, B27, B28, B29, B29, B2A, B2A, B2B, B2B, B2B, B2B, B2B, B2C, B2C, B2A, B2B, B2B, B2B, B2C, B2B, B2B, B2 12 and / or one or more of their vitamin equivalents (e.g., cyanocobalamin, hydroxocobalamin, adenosylcobalamin and / or methylcobalamin).

[0298] In some embodiments, the protein hydrolysate is produced from biomass that has been produced from a C1 substrate such as CO2, CO and / or CH4 and contains vitamin B12 at a concentration of about 2 μg / 100 g dry biomass, up to about 6.5 μg / 100 g dry biomass, relative to the dry weight of the biomass. 12 In some embodiments, the culture medium produced by the C1 matrix contains vitamin B 12 , such as a concentration of about 6.5 μg / 100 g dry biomass, up to about 13 μg / 100 g dry biomass, or greater than about 13 μg / 100 g dry biomass relative to the dry weight of the biomass.

[0299] In some embodiments, the method includes culturing cells in a culture medium, and the culture medium includes one or more protein hydrolysates derived from plants or yeast, in addition to a protein hydrolysate composition derived from a microorganism (e.g., chemoautotrophic microorganism) as described herein. Suitable plant-based protein hydrolysates include those derived from, but not limited to, soybeans, rice, potatoes, or corn. In some embodiments, the method includes culturing cells in a culture medium, and the culture medium includes a protein hydrolysate based on plants and a protein hydrolysate composition derived from a microorganism (e.g., chemoautotrophic microorganism) as described herein. In some embodiments, the method includes culturing cells in a culture medium, and the culture medium includes a protein hydrolysate based on yeast and a protein hydrolysate composition derived from a microorganism (e.g., chemoautotrophic microorganism) as described herein. In some embodiments, the method includes culturing cells in a culture medium, and the culture medium includes a protein hydrolysate based on yeast, a protein hydrolysate based on plants, and a protein hydrolysate composition derived from a microorganism (e.g., chemoautotrophic microorganism) as described herein. Suitable plant and / or yeast based protein hydrolysates are described, for example, in US Pat. No. 8,093,045 and PCT Publication No. WO1999057246, which are hereby incorporated by reference in their entirety.

[0300] The protein hydrolysate based on plant or yeast can be added to the culture medium in any suitable amount. In certain embodiments, the protein hydrolysate based on plant or yeast is added to the culture medium at a concentration of about 0.001% w / v or higher, for example, about 0.01% w / v or higher, about 0.05% w / v or higher, about 0.1% w / v or more, including about 1% w / v or higher. In some embodiments, the protein hydrolysate based on plant or yeast is added to the culture medium at a concentration of about 0.001% w / v to about 5% w / v, for example, about 0.01% w / v to about 2% w / v, about 0.05% w / v to about 1% w / v, including about 0.1% w / v to about 1% w / v.

[0301] Cell can cultivate the time of any suitable amount in the substratum of the protein hydrolysate composition with microorganism (for example chemoautotrophic microorganism) source as described herein.In some embodiments, in the substratum, there is protein hydrolysate, cell is continuously cultured in whole growth and reproduction process.In some embodiments, cell is cultivated about 1 hour or longer in the substratum containing the protein hydrolysate in chemoautotrophic microorganism source, for example, about 5 hours or longer, about 12 hours or longer, about 24 hours or longer, about 5 days or longer, about 2 weeks or longer, about 6 weeks or longer, about 3 months or longer, about 6 months or longer, including about one year or longer.In some embodiments, cell is cultivated about 1 hour to about 3 years in the substratum containing the protein hydrolysate in chemoautotrophic microorganism source, for example, about 5 hours to about 1 year, about 12 hours to about 6 months, about 24 hours to about 3 months, including about 5 days to about 6 weeks time period.

[0302] In some embodiments, the protein hydrolysate composition derived from a microorganism (e.g., a chemoautotrophic microorganism) is temporarily present in the culture medium during cell culture. When cells grow in the absence of a microbial-derived protein hydrolysate in the culture medium, other culture medium supplements, such as a plant or yeast-derived protein hydrolysate, may be present in the culture medium.

[0303] Any suitable cell can be cultured using the method of the present invention. The cell can be derived from mammals, birds, fish, insects or other animal sources. The cell can be a stem cell. The cell can be a fungal cell, a plant cell, a eukaryotic cell or a prokaryotic cell. The cell can be a probiotic. The cultured cell can be a primary cell, an immortalized cell line, a hybridoma, an established cell line, a stem cell-derived cell or a genetically engineered cell, such as a recombinant cell expressing a heterologous polypeptide or protein. The cell can be a single cell, a tissue, an organ. Suitable non-mammalian cells include insect cells, bird cells (including chicken cells) and fish cells. Suitable cells include mammalian cells of human or non-human origin. Suitable mammalian cells include, but are not limited to, cattle, pigs, sheep, rabbits or horse cells. The cultured cell can be a monkey kidney cell, a cattle kidney cell, a dog kidney cell, a pig kidney cell, a rabbit kidney cell, a mouse kidney cell, a rat kidney cell, a sheep kidney cell, a hamster kidney cell, a Chinese hamster ovary cell or an animal cell derived from any tissue. Suitable mammalian cells include, but are not limited to, CHO cells, COS cells, VERO cells, HeLa cells, 293 cells, HEK-293 cells, HEK cells, PER.C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS0 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, BHK cells, stem cells and their derivatives. Suitable non-mammalian cells include, but are not limited to, AGE1.CR cells, EB66 cells, Sf9 cells, stem cells and their derivatives. In certain embodiments, the culture cells grown on the culture medium components produced as described herein have been transfected with exogenous nucleic acids.

[0304] In some embodiments, the method includes culturing muscle cells in a culture medium containing a lysate and / or protein hydrolysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism as described herein (e.g., chemoautotrophic microorganism). In some embodiments, the method includes culturing cells in a culture medium to produce and maintain muscle cells. Suitable cells for producing muscle cells include, but are not limited to, embryonic stem cells, satellite cells, and myoblasts. In some embodiments, cells include adipocytes. In some embodiments, cells include fibroblasts. In some embodiments, any two or more of muscle cells, adipocytes, and fibroblasts are cultured together in a culture medium containing a protein hydrolysate composition derived from a microorganism as described herein (e.g., chemoautotrophic microorganism).

[0305] Any suitable method can be used to cultivate cells. Cells can be grown in suspensions, roller bottles, flasks, etc. Large-scale methods are also included, such as bioreactors, including adherent cells grown on microcarriers attached to stirred fermenters. In some embodiments, cells are grown in suspension. If cells grow on microcarriers, microcarriers can be selected from the group of microcarriers based on dextran, collagen, plastics, gelatin and cellulose, etc. In certain embodiments, microcarriers can include PHA (such as PHB and / or PHV) produced by microorganisms as described herein. In certain embodiments, microcarrier support systems can be used for pseudo-suspension cultures in stirred tank bioreactors.

[0306] In some embodiments, the method includes culturing cells on a three-dimensional support or scaffold. The scaffold can provide a microenvironment suitable for supporting the proliferation, maintenance, development and / or differentiation of cells in the presence of a protein hydrolysate composition derived from a microorganism (e.g., a chemoautotrophic microorganism) in a culture medium. In some embodiments, the three-dimensional support or scaffold is porous. In certain embodiments, the three-dimensional support or scaffold includes PHA (e.g., PHB and / or PHV) produced by a microorganism as described herein.

[0307] The three-dimensional support or scaffold can be made of any material suitable for the cultured cells to grow thereon. In some embodiments, the scaffold is biodegradable. In some embodiments, the scaffold is made of biodegradable materials, such as biodegradable polyesters or hydrogels. In some embodiments, the scaffold is made of PHA polymers, such as but not limited to PHB. In some embodiments, biodegradable polyesters (such as PHA or PHB) are produced by microorganisms (such as chemoautotrophic microorganisms). In certain embodiments, the scaffold is consumable, for example, suitable for human consumption. Edible supports or scaffolds can be made of but not limited to gellan gum, alginate, pectin or cellulose. In certain embodiments, the scaffold is 3-D printed.

[0308] Cells can be cultured at appropriate temperature and pH. Mammalian cells are typically cultured in a cell incubator at about 37°C, with the optimum pH of the culture medium being between about 6.8 and 7.6, including within the range of between 7.0 and 7.3. In some embodiments, cells in batch culture can be replaced with complete culture medium approximately every 2 to 3 days, or more or less frequently as needed. Cells in perfusion culture (e.g., in a bioreactor or fermenter) can be replaced with fresh culture medium on a continuous circulation basis.

[0309] In certain embodiments, the method includes culturing animal cells to produce products suitable for human consumption, such as meat products. Therefore, provided herein is a method for culturing muscle cells to cultivate meat using a culture medium containing a lysate and / or protein hydrolysate and / or a peptide composition and / or an amino acid composition derived from a microorganism as described herein, with or without other cells (such as adipocytes or fibroblasts). When the culture medium is a serum-free or animal component-free culture medium, the method of the present invention provides a humane method for producing meat products. In certain embodiments, serum of animal origin is used in the culture medium; however, some amino acids or protein hydrolysate fractions are derived from microorganisms as described herein.

[0310] In some embodiments, the microbial-derived protein hydrolysate composition comprises a component that improves the flavor of a cultured food product, such as cultured meat. In certain embodiments, the protein hydrolysate composition stimulates the development of flavor enhancement elements in a cultured food product, such as cultured meat. As used herein, enhancing the flavor of a food product includes making the product more palatable, or imparting one or more flavor components found in the naturally occurring counterpart of the cultured product.

[0311] In some embodiments, the method includes culturing muscle cells in a serum-free medium containing whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism as described herein (e.g., chemoautotrophic microorganism). In some embodiments, the method includes culturing muscle cell precursor cells in a serum-free medium containing whole cell biomass and / or extract and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism as described herein (e.g., chemoautotrophic microorganism). In some embodiments, the method includes culturing muscle cells in a medium containing serum of animal origin, but also containing protein whole cell biomass and / or extract and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism as described herein (e.g., chemoautotrophic microorganism). In some embodiments, the method includes culturing muscle cell precursor cells in a culture medium comprising serum of animal origin, but also containing whole cell biomass and / or extracts and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions derived from microorganisms as described herein (e.g., chemoautotrophic microorganisms). Muscle cell precursor cells can be cultured under conditions sufficient to promote the proliferation of precursor cells in the culture medium. In some embodiments, muscle cell precursor cells can be cultured under conditions sufficient to induce the differentiation of precursor cells into muscle cells. Precursor cells can be any suitable cells that can be induced to produce muscle cells. Suitable precursor cells include, but are not limited to, satellite cells, embryonic stem cells, and myoblasts. Therefore, the method of the present invention includes adding an effective amount of a protein hydrolysate derived from a chemoautotrophic microorganism to the culture medium to promote the proliferation, maintenance, development, and / or differentiation of muscle cells.

[0312] In certain embodiments, myocyte is cultivated together with one or more other cell types. Suitable cells for co-cultivation with myocyte include but are not limited to adipocyte and fibroblast or their precursor. Therefore, the method of the present invention may include cultured cells in the culture medium containing whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition in serum-free medium or containing animal-derived serum containing microorganism (such as chemoautotrophic microorganism) source as described herein, to produce the collection of muscle cell, adipocyte and connective tissue. Cells can be cultivated under the conditions that are enough to induce myogenesis or myofiber formation. In some embodiments, the method includes adding whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition in the microorganism (such as chemoautotrophic microorganism) source of effective dose to promote adipocyte and / or fibroblast proliferation, maintenance, growth and / or differentiation in culture medium. In some embodiments, the method comprises adding an effective amount of whole-cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism (e.g., a chemoautotrophic microorganism) to the culture medium to induce, maintain and / or promote myogenesis.

[0313] In some embodiments, myocytes (with or without adipocytes and / or fibroblasts) are cultured on a three-dimensional support or scaffold. In some cases, the scaffold may be porous. The scaffold may be made of any suitable material for supporting cell growth and / or myogenesis. The scaffold may be a biodegradable and / or consumable material. In some embodiments, the scaffold comprises a biodegradable polyester or a biodegradable hydrogel. In certain embodiments, the scaffold comprises a PHA polymer, such as PHB and / or PHV. In some embodiments, the scaffold is made of a material derived from a microorganism (e.g., a chemoautotrophic microorganism). In some embodiments, the scaffold comprises a biodegradable polyester, such as a PHA (e.g., PHB and / or PHV), that is sustainably produced by the growth of a microorganism (e.g., a chemoautotrophic growth).

[0314] In some embodiments, an effective amount of whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition derived from a microorganism as described herein (e.g., a chemoautotrophic microorganism) is added to the culture medium to provide a source of vitamins to the growing cells. Whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition can be, for example, vitamins for muscle cells and / or other cells (such as, but not limited to, vitamin B1, vitamin B2 and / or vitamin B 12Whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition can be a source of one or more vitamins for muscle cells and / or other cells, including but not limited to: vitamin A, β-carotene, lutein, zeaxanthin, thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), vitamin B6, folic acid (B9), vitamin B 12 , choline, vitamin C, vitamin D, vitamin E, vitamin K. Whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition can be a source of one or more minerals for muscle cells and / or other cells, including but not limited to: calcium, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc.

[0315] In certain embodiments, whole-cell biomass and / or lysates and / or extracts and / or protein hydrolysates produced as described herein serve as a source of one or more of: amino acids; oligopeptides; lipids; and / or iron for cell culture of cells different from the microbial cells from which the amino acids, oligopeptides, lipids and / or iron are derived.

[0316] In some embodiments, the food products (e.g., but not limited to, meat products or meat-like products) produced by the methods described herein can be vitamins (e.g., vitamin B1, vitamin B2 and / or vitamin B 12 In certain embodiments, the food product contains vitamins such as vitamin B1, vitamin B2 and / or vitamin B 12 In certain of the embodiments, vitamin B1, vitamin B2 and / or vitamin B 12 Provided by whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition produced as described herein added to the culture medium of at least a portion of the cultured cells used to produce the food product. In certain embodiments, the food product contains one or more vitamins, including but not limited to: vitamin A, beta-carotene, lutein, zeaxanthin, thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), vitamin B6, folic acid (B9), vitamin B 12, choline, vitamin C, vitamin D, vitamin E and / or vitamin K, some or all of which may ultimately be derived from the microorganism from which the whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or extract is derived. In certain embodiments, the vitamins are provided by whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition added to the culture medium of at least a portion of the cultured cells used to produce the food product.

[0317] In some embodiments, the food products (e.g., meat or meat-like products) produced by the methods described herein can be a nutritional source of minerals, including but not limited to one or more of the following: calcium, iron, magnesium, manganese, phosphorus, potassium, sodium and / or zinc. In certain embodiments, the minerals are provided by whole cell biomass and / or lysates and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions produced as described herein added to the culture medium of the cultured cells for producing the food products. In certain embodiments, the iron content of whole cell biomass and / or lysates and / or extracts and / or protein hydrolysates and / or peptide compositions and / or amino acid compositions includes iron in the form of heme.

[0318] In certain embodiments, a preparation is provided, comprising a combination of whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein and other ingredients, including but not limited to biomass and / or whole cell and / or protein concentrate and / or protein isolate and / or protein hydrolysate and / or extract from one or more of the following sources: meat; dairy products; eggs; soybeans; wheat; rice; peas; other plant proteins; yeast; probiotics; LAB; and / or other GRAS microorganisms or large organisms. In certain embodiments, the yogurt mixture is supplemented with one or more of the following: whole cell biomass and / or lysate and / or protein hydrolysate and / or peptide composition and / or amino acid composition and / or other nutrients or cofactors produced as described herein. In certain embodiments, the preparation does not include protein or other biomass components from meat and / or soybean sources. In certain embodiments, the preparation does not include protein, protein hydrolysate and / or other biomass components from dairy products and / or non-soybean plant sources. In some cases, the formulation does not include genetically engineered microorganisms (i.e., does not contain GMOs). In some cases, the formulation does not contain meat and / or dairy products. In certain embodiments, the formulation is used in food, feed, or beverage products. In certain embodiments, the food, feed, or beverage products are vegetarian or vegan. In certain embodiments, the formulation is considered to be GRAS for human consumption.

[0319] In certain embodiments, the cell culture grown on whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition as described herein secretes protein product. In certain embodiments, the protein purification process is used to recycle and / or purify protein product. As well known in the art, any suitable protein purification method can be used to recycle and / or purify protein product.

[0320] In certain embodiments, after using whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition as described herein to produce products as components of culture medium, whole cell biomass and / or lysate and / or extract and / or protein hydrolysate and / or peptide composition and / or amino acid composition materials are removed from culture medium or fermentation broth and / or product. In some cases, these removed nutrients are recycled back to one or more upstream steps to utilize more completely, and / or are used as byproducts.

[0321] The following examples are intended to illustrate but not to limit the present invention.

[0322] Example

[0323] Example 1: Protein hydrolysate produced from a culture of Cupribothrium necrotum

[0324] The Cupriavidus necator strain was cultivated chemoautotrophically in a mineral salt growth medium with CO2 as a carbon source and H2 as an electron donor. After growth, the whole cell biomass was separated from the growth medium by centrifugation and dried by lyophilization. The dried biomass was processed as follows.

[0325] Delipidation of whole cell biomass: Whole cell biomass was delipidated (lipids were extracted) with ammonium hydroxide and methanol (1:1:0.4, WCB:NH4OH:MeOH) by stirring the mixture in a fume hood in a tightly covered container for 1 hour. The mixture was vacuum filtered with Whatman 4 filter paper. The filtrate contained the extracted lipids. The retentate on the filter was the defatted biomass, which was dried overnight in an incubator at 40°C.

[0326] Protein hydrolysis with NH4OH and neutralization with CO2: Prepare 2% defatted dry matter solid load in the hydrolysis step by rehydrating with the required amount of deionized (DI) water. The slurry is fully mixed for 1 minute with Turret Stick at 15,000rpm. NH4OH 28%-30% (pre-made) solution is used in a fume hood to increase the pH of the reaction mixture to 10.85. The mixture is transferred to a pressure tube (size: 120mL) with a 50mL working volume. The mixture is then autoclaved at 110°C for 10 minutes, slowly vented. The pH of the solution after autoclaving is 10.82. Then by bubbling CO2 through the cannula / 18G needle inserted in the solution for 10-20 minutes to reduce the pH to pH 9. Then at 55°C, with 110rpm, enzyme digestion is carried out overnight with bacterial alkaline protease at pH 9. By centrifugation at 20000xg, 20 minutes, 5°C, the supernatant containing soluble hydrolyzed protein is separated from the coarse mass rich in PHB. The supernatant protein hydrolyzate is then freeze-dried. The ash content measured for this protein hydrolyzate (PH) is 5%. The ash content is measured by placing a minimum of 300 mg of protein hydrolyzate powder in a tared crucible and running an ash cycle in a muffle furnace. It is also independently measured using AOAC method 942.05 by external laboratory analysis (SGS, North America). The total amino acid content and amino acid spectrum of PH are also measured by SGS, North America using AOAC method AOAC 994.12. The results are shown in Table 1.

[0327] Table 1. NH4OH / CO2 low ash pH

[0328]

[0329]

[0330] Example 2: Protein hydrolysate produced from a culture of Cupribothrium necrotum

[0331] The Cuprix necator PHB-negative mutant strain (DSM 541) was cultivated chemoautotrophically in an inorganic mineral growth medium with CO2 as a carbon source and H2 as an electron donor. After growth, the whole cell biomass was separated from the growth medium by centrifugation and dried by lyophilization. The dried biomass was processed as follows.

[0332] Degreasing of whole cell biomass: Dry whole cell biomass was degreased by treatment with ammonium hydroxide and ethanol (1:4:0.5 w / v). The biomass and solvent slurry was stirred in a tightly capped glass bottle for 30 minutes and then vacuum filtered through a Whatman 4 filter paper in a fume hood. The filtrate contained the lipid fraction. The defatted retentate recovered from the filter was air dried overnight and then dried in an incubator at 40°C for 4-6 hours.

[0333] The defatted biomass was hydrolyzed with Ca(OH)2 and neutralized with H3PO4: The dried defatted biomass was resuspended in DI water to a final concentration of 2%. The biomass solution was mixed with IKA Ultra-Turax at 15000rpm until completely and evenly resuspended. The biomass solution was brought to pH 11 by adding Ca(OH)2. The solution was transferred to a glass medium bottle, autoclaved at 110°C for 10 minutes, and then cooled to room temperature. The solution was neutralized to pH 9 using H3PO4. Bacterial alkaline protease (Sigma P8038) was added to the solution at a concentration of 2.6 active substance units / g biomass. The biomass solution was placed in a 55°C shaking water bath overnight. After digestion for 16-24 hours, the enzyme was inactivated by incubating the slurry in a 95°C water bath for 10 minutes. The biomass slurry was then cooled to room temperature and the protein hydrolysate (supernatant fraction) was separated by centrifugation at 7°C for 30 minutes at 26000xg. The resulting protein hydrolysate solution was frozen in a -80°C freezer and then freeze-dried. The moisture, ash and N content of the dried powder were determined, and the protein profile was analyzed by SDS-PAGE analysis. There was no protein above 2000 Daltons in the protein hydrolysate, and the resulting ash content was 10.5%. The total amino acid content and amino acid profile of PH were also determined by SGS, North America using AOAC method AOAC 994.12. The mineral profile was determined using AOAC method AOAC968.08. The results are shown in Table 2.

[0334] Table 2

[0335]

[0336]

[0337] Example 3: Protein hydrolysate design.

[0338] The resulting protein hydrolysates produced from chemoautotrophic biomass were analyzed for various digestion processes (acidic vs. alkaline vs. enzymatic hydrolysis vs. autolysis), selection of enzymes with different enzyme specificities (purified or blends of exogenous animal / plant / microbial enzymes and / or endogenous enzymes) and process parameters (pH, temperature and incubation / processing time). The release of peptides represented by peptide spectra and the size of the peptides were performed. The average molecular weight (MW) of the peptides (Da) was determined. The ratio of amino nitrogen to total nitrogen (AN / TN ratio) and the degree of hydrolysis (DH%), as well as the level of free amino acids were measured. The effects of various hydrolyzing agents were compared based on DH% and peptide and amino acid distribution spectra. Comparisons of protein hydrolysates produced as described herein were also performed for common and / or commercial sources of protein hydrolysates, including but not limited to dairy, meat and / or soy protein hydrolysates and / or yeast extracts produced by various hydrolysis methods (including enzymatic, acidic, alkaline and / or autolysis). This comparison was based on the total amino acid profile, AN / TN ratio and all other parameters and characteristics described above.

[0339] When providing a given protein hydrolysate, also compare according to the growth performance of the culture.This test culture may include one or more LAB.LAB may include Lactobacillus helveticus strains.Protein hydrolysates for comparison may include casein (casein) casein-pancreatic digest - peptone made by casein pancreatic digestion.Measurement of culture growth performance includes cell number and specific growth rate measured over time.A negative control for testing may be a basal medium without any protein hydrolysate supplement.Another negative control may be a culture medium without any protein hydrolysate supplement or any amino acid culture medium component.Another experimental test and comparison is in a culture medium (that is, other amino acid components are not included in the culture medium) in which protein hydrolysate constitutes a sole amino acid source, including between different protein hydrolysates prepared as described herein.Except for comparisons for other protein hydrolysates, comparisons are also made for culture mediums without protein hydrolysate supplements but with free amino acid compositions (e.g. casamino acids) or with full protein (e.g. casein).

[0340] Example 4: Testing the effects of whole cell biomass, lysates and hydrolysates on the beneficial fungus Trichoderma aureum.

[0341] Trichoderma aureum is a saprophytic fungus that lives in the rhizosphere and soil under a wide range of temperature and soil pH conditions. It is beneficial to many different crops because it can resist plant pathogenic fungi, including Rhizoctonia solani and Botrytis cinerea, which cause diseases in hundreds of plant crops including tomatoes, beans, cucumbers, strawberries, cotton and grapes. It also increases the plant's uptake of micronutrients and is associated with root growth stimulation. Trichoderma aureum is mainly used as a microbial inoculant in agricultural cropping systems.

[0342] The purpose of this in vitro experiment was to evaluate the stimulatory activity of seven different lysates and protein hydrolysates and whole cell biomass (WCB) produced from C. necator biomass grown autotrophically on CO as a carbon source using H2 as an electron donor as described herein. These seven biomass products were compared with two commercial biostimulants; one containing a plant-derived PH and the other containing an animal-derived PH.

[0343] The ability of different WCBs, lysates and PH samples to promote the growth of Trichoderma aureum AT10 in sterile media was evaluated. Pure cultures of Trichoderma aureum AT10 were grown on potato dextrose agar (PDA) medium as a control and on PDA medium supplemented with 3 ml / L of 9 WCBs, lysates and PH samples at 25°C in the dark for 24 hours.

[0344] PDA medium rich in lysate and PH products was prepared by dissolving the required amount of each organic product in deionized water. The resulting solution was filtered into a sterile bottle containing PDA using a 0.25 μm filter at 45°C. The matrix was gently stirred and then poured into a separate 9-cm petri dish. When the matrix in the plate cooled and solidified, a 5 mm mycelial disk of dark green Trichoderma AT10 taken from the edge of the pure culture after 4 days of growth was placed in the center of each petri plate and incubated at 25°C. The radial growth of the mycelium was measured after 24 hours of incubation. Twelve petri dishes were used for each treatment. SPSS v.21 (IBM Corp., Armonk, NY, USA) was used for statistical analysis of all data. Duncan's multiple range test was performed on each significant variable measured at P = 0.05. In Table 3, the standard error is followed by the mean value. The different letters in each column represent significant differences according to Duncan's multiple range test P = 0.05.

[0345] Table 3

[0346]

[0347] It was found that the PDA matrix enriched with organic products can significantly affect the radial growth of Trichoderma mycelium after 24 hours of incubation. The highest Trichoderma growth was recorded for the pH produced by Uncaria nebulosa by alkali treatment followed by protease treatment. Commercial biostimulants based on plant-derived pH had lower average growth, but there were no significant differences. The pH produced by Uncaria nebulosa by alkali treatment followed by protease treatment produced significantly greater growth compared to the control and commercial biostimulants based on animal-derived pH. Significantly more intensive sporulation was observed after treatment with Uncaria nebulosa pH compared to the control ( Figure 5 ), where spores are a means for Trichoderma to replicate and reproduce.

[0348] The results of this preliminary trial indicate that the pH generated by C. necator via alkaline treatment followed by protease treatment has the potential to enhance the growth of Trichoderma in vitro. The pH from C. necator can be applied to the root zone of crops to stimulate root growth and nutrient uptake and increase soil populations of naturally occurring or artificially inoculated Trichoderma species.

[0349] Example 5: Testing the addition of protein hookworm and copper-grazing bacteria biomass to Indonesian fermented black bean fermentation substrate

[0350] The aim was to test whether high-protein biomass from Cupriavidus necator grown on CO2 could be used to supplement plant substrates used in the production of tempeh.

[0351] A commercial starter culture of Rhizopus oryzae was obtained.

[0352] Soak the chickpeas overnight in sterile water (boiled) and then cook them in a pressure cooker on normal pressure mode for one minute. The chickpeas are cooked just enough to be tender and soft to bite. The chickpeas are then quickly drained while still hot. The chickpeas are then spread out on a paper towel in a thin layer to dry. Shell the beans.

[0353] The chickpeas were then divided into two flat wide-mouth plastic containers. Container #1 served as a control with chickpeas alone added. In container #2, chickpeas were added with dry whole-cell Cupriavidus necrotus biomass at a ratio of 10% of the starting chickpea weight.

[0354] The vinegar was gradually added to both containers and mixed well to give a starting pH of 3.5.

[0355] Sprinkle 1 teaspoon of Rhizopus oryzae starter culture per pound of cooked dry chickpeas on the substrate. Then use a spatula to mix the chickpeas + culture thoroughly so that the culture sticks evenly to all the substrate.

[0356] Both wide-mouth plastic containers (i.e., chickpea control and chickpea + C. necator experiment) had lids with punched holes spaced 0.3-0.5" apart, and the lids were loosely held on top. The containers were transferred to an incubator at 30.5°C and incubated for 48-72 hours.

[0357] The white mycelium of R. oryzae started to grow after 20-24 hours of incubation, after which the fermentation substrate was transferred from the incubator to room temperature. Full growth of the mycelium of R. oryzae was observed after 48-72 hours.

[0358] It was observed that R. oryzae grew equally well on chickpeas with or without the addition of C. necator biomass, and even had white mycelium growth. Therefore, tempeh can supplement the protein and other nutrients (such as B vitamins) provided by C. necator.

[0359] Further analyses were conducted to investigate any compositional or nutritional differences between supplemented and unsupplemented tempeh.

[0360] Similar experiments can be conducted to test other edible fungal strains used in the production of tempeh or other fermented products such as miso or soy sauce, such as Aspergillus oryzae (NRRL 3485), Rhizopus oligosporus (NRRL 2710), and Rhizopus oryzae (NRRL 3613). Additional fermentation substrates such as white rice, barley, mung bean, okara flour, and soybeans can also be tested.

[0361] Example 6: Testing the growth of lactic acid bacteria on protein hydrolysates and protein isolates

[0362] GRAS lactic acid bacteria (LAB) were tested on nutrients derived from C. necator protein biomass produced by CO2. The protein sources of nutrients to be tested included: alkaline protein hydrolysate (PH) produced by alkaline treatment with NH4OH, followed by neutralization with CO2 and enzymatic hydrolysis using bacterial alkaline protease (BAP); acidic protein hydrolysate produced by acid treatment with H3PO4, followed by neutralization with Ca(OH)2, and enzymatic hydrolysis with BAP; and protein isolate (PI) formed by sonicating the biomass, followed by centrifugation, discarding the pellet, and recovering and drying the protein-rich supernatant.

[0363] The products of these and other protein sources were tested on LAB strains (including but not limited to Streptococcus thermophilus, Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus acidophilus and Bifidobacterium lactis). The LAB strains will be tested individually, as well as in co-culture, mixed culture and consortium. The growth medium used in the control is ordinary milk in one control and Elliker broth (Sigma #17123) in another control. The composition of Elliker broth is: 0.5g / L ascorbic acid; 20g / L casein hydrolysate; 5g / L glucose; 2.5g / L gelatin; 5g / L lactose; 5g / L sucrose; 1.5g / L sodium acetate; 4g / L sodium chloride; 5g / L yeast extract.

[0364] A control case of yogurt from milk is produced as follows: 1-2 quarts of pasteurized whole milk is heated to 180°F and then cooled to 115°F. The milk is poured into a glass container and a package of commercial yogurt starter culture containing Streptococcus thermophilus, Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus acidophilus and Bifidobacterium lactis is added and mixed thoroughly. The milk culture is then covered and incubated in a pressure cooker in yogurt mode at 105°F-112°F for approximately 8 hours. The culture is checked by gently tilting the jar. When the yogurt moves away from the side of the jar in a mass instead of running to the side, it has completed the culture. Once the yogurt is set, the lid is covered and cooled at room temperature for 2 hours and then refrigerated.

[0365] A control case for LAB growth on Elliker broth is as follows: 48.5 grams of Elliker broth powder is suspended in 1 liter of distilled water. Mix and boil to completely dissolve the medium, then sterilize by autoclaving at 121°C for 15 minutes. The medium solution is light to medium amber transparent and has a pH between 6.6 and 7. The solution is cooled to 115°F and inoculated with one package of commercial yogurt starter culture containing Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, and Bifidobacterium lactis per 1-2 quarts of medium. The medium is then overlaid and incubated at 105-112°F for approximately 24 to 48 hours. The optical density (OD) at 600 nm of the LAB culture in the Elliker broth will be monitored over time. 600 ).

[0366] One experiment tested against a milk control involved mixing C. necator-derived PH or PI into milk at a 1:1 C. necator nitrogen (N) to milk nitrogen ratio, followed by the same yogurt fermentation steps as described above.

[0367] Another experiment tested against an Elliker broth control involved replacing casein enzymatic hydrolysate in Elliker broth with an amount of test substrate (PH / PI) to obtain the same N content in the broth as 20 g / L casein enzymatic hydrolysate. The rest of the medium composition remained unchanged. LAB growth performance on the experimental broth was compared to the Elliker broth control in terms of growth rate and final titer. Similar experiments involved replacing casein enzymatic hydrolysate and gelatin in Elliker broth with experimental pH and / or PI on an isonitrogenous basis; and replacing casein enzymatic hydrolysate, gelatin, and yeast extract in Elliker broth, wherein casein enzymatic hydrolysate and gelatin were replaced on an isonitrogenous basis, and yeast extract was replaced on an equivalent B vitamin basis or nucleotide basis.

[0368] Example 7: Testing the effects of protein hydrolysates on cell viability and other performance indicators.

[0369] The effects of lysates and / or protein hydrolysates prepared as described herein by CO2 as the sole carbon source entering the production process on cell cultures provided with the lysates and / or protein hydrolysates were investigated. The effects of adding 2% (w / v) of an acid hydrolysate of a protein produced as described herein to a milk matrix on the viability of the LA strain were determined. The viability of the LA strain after 12 weeks in terms of colony forming units (CFU) / gram was determined for both the experimental and control. The negative control may be a milk matrix alone. The positive control may include the addition of 2% acid casein hydrolysate (ACH) and cysteine. Other controls (positive and negative) may include the addition of cysteine ​​alone, ACH alone, whey powder (WP), whey protein concentrate (WPC), tryptone (tryptic digest of casein), soy protein hydrolysate, including but not limited to commercial soy PH such as NZ Soy-BL, Hy-soy and Amisoy, or whole soy protein. The viability of other strains including thermophilic Streptococcus and Bifidobacterium species and other performance indicators were tested. In addition to or in lieu of cell viability, other culture parameters measured, tested, and compared included specific growth rate, fermentation time required to reach pH 4.50, and lactate titer (g / L).

[0370] Example 8: Evaluation of growth and acid production of probiotic strains grown using protein hydrolysates.

[0371] The growth and acid production of Bifidobacterium lactis strains were evaluated using a culture medium supplemented with one or more protein hydrolysates produced as described herein. Positive controls for comparison may include sheep and goat milk as culture medium, supplemented with milk protein hydrolysate (MPH) prepared by using commercial proteases (e.g., Aspergillus species protease 2A; Amano Pharmaceutical, Nagoya, Japan) (Gomes and Malcata 1998). Free amino acids from various sources, including free amino acids produced as described herein (e.g., by chemoautotrophic conversion of CO2), can also be used as nitrogen enrichment supplements in experimental and control cases. Free amino acid concentrates are added to the culture medium at a ratio of 25–50 mL / L. The viable counts of Bifidobacterium lactis in the experimental and control samples are measured and compared.

[0372] Example 9: Evaluation of starter culture production using protein hydrolysate and / or protein.

[0373] Pediococcus pentosaceus is grown on a substrate. A positive control is compared with proteins and / or protein hydrolysates produced as described herein as a nitrogen source for producing Pediococcus pentosaceus, wherein the proteins and / or protein hydrolysates produced as described herein include: casein peptone (hydrolyzed casein); primatone (hydrolyzed meat protein); and / or yeast paste. Performance indicators are measured, including fermentation time and / or biomass yield. The optimal nitrogen / carbon (N / C) ratio is determined.

[0374] The effects of various whole cell biomasses, lysates, and whole proteins produced as described herein were compared to various dried and frozen meat stocks (1%-5%) as growth promoters for Pediococcus acidilactici.

[0375] Example 10: Testing the growth performance of different cell lines on libraries of lysates, hydrolysates, peptide compositions and / or amino acid compositions.

[0376] Libraries of various lysates, hydrolysates, peptide compositions, and / or amino acid compositions generated using different starting materials and methods as described herein are evaluated by compositional analysis and based on the growth performance of various model cell types on media containing the library members.

[0377] Compositional analysis and characterization of lysates, hydrolysates, peptide compositions and / or amino acid compositions include: α-amino nitrogen % (AN); total nitrogen (TN) %; ratio (AN / TN); free amino acids (% of total amino acids); peptide size range, e.g., 100-200 Da (%); 200-500 Da (%); 500-1000 Da (%); >1000 Da (%); average MW; ash (%); moisture (%); pH; sodium (%); potassium (%); calcium (mg / g); magnesium (mg / g); chloride (mg / g); sulfate (mg / g); phosphate (mg / g); / g; amino acid content, namely alanine (mg / g), arginine (mg / g), aspartic acid (mg / g), cysteine ​​(mg / g), glutamic acid (mg / g), glycine (mg / g), histidine (mg / g), isoleucine (mg / g), leucine (mg / g), lysine (mg / g), methionine (mg / g), phenylalanine (mg / g), proline (mg / g), serine (mg / g), threonine (mg / g), tryptophan (mg / g), tyrosine (mg / g), valine (mg / g) and total amino acids (mg / g).

[0378] The proliferation of the cell type is tested on culture medium supplemented with one or more lysates, hydrolysates, peptide compositions and / or amino acid compositions produced as described herein.

[0379] Three model cell types that can be used for this growth test are: High Five derived from cabbage louper TM Cells, and Sf9 and Sf21 cells derived from armyworm (Spodoptera frugiperda). Prior to quantitative performance evaluation, each cell line was cultured in serum-free medium supplemented with test lysates, hydrolysates, peptide compositions and / or amino acid compositions (e.g., at a concentration of 0.6% w / v). For each corresponding cell type, growth on supplemented medium was compared to growth on standard medium.

[0380] Example 11: Preparation of a cell culture medium free of animal-derived proteins.

[0381] The lysates, hydrolysates, peptide compositions and / or amino acid compositions produced as described herein are examined as supplements to the basal medium. The compositions are titrated into the basal medium. The cell growth of each supplemented medium is compared with the corresponding unsupplemented medium (negative control) or the medium supplemented with FBS (positive control). Growth is measured based on the growth rate per 25 cm 2The average cell count of three successive cultures in the flask is evaluated. The cell line grown on the experimental and control culture medium can be Vero (African green monkey kidney) cell. Vero cell growth is checked in the prototype serum-free preparation supplemented with various hydrolyzate sources, and after three adaptive passages, growth is compared with respect to the E-MEM reference culture medium supplemented with 5% (v / v) FBS. Every kind of lysate, hydrolyzate, peptide composition and amino acid composition are supplemented in the culture medium with a concentration of 200mg / L. Relatively, it is carried out according to the average cell count of the reference material for supplementing FBS.

[0382] The comparative yields of target model viruses obtained from Vero cell cultures maintained and infected in serum-free protein hydrolysates or serum-supplemented controls produced as described herein were also examined. The titers of Sindbis virus, poliovirus 1, pseudorabies virus, and reovirus produced by VERO cells grown in serum-free medium supplemented with protein hydrolysates produced as described herein were compared with the virus yields of VERO cells grown in EMEM with 2% serum as a control. The comparison was made based on plaque forming units (PFU).

[0383] Growth and production of canine adenovirus or infectious bovine rhinotracheitis (IBR) virus in MDCK (Madin-Darby canine kidney) cells in serum-free medium supplemented with protein hydrolysates produced as described herein were compared to growth and production on E-MEM plus 5% FBS. Growth and production of pseudorabies virus in PK-15 (porcine kidney) cells in serum-free medium supplemented with protein hydrolysates produced as described herein were compared to growth and production in E-MEM plus 5% FBS test medium. Comparisons were based on cell counts and 50% tissue culture infectious dose (TCID50).

[0384] Example 12: Recombinant protein production by CHO cells.

[0385] Comparative growth experiments of recombinant CHO cells were performed in serum-free medium supplemented with protein hydrolysates produced as described herein, and growth and production were compared with those in serum controls. Growth was compared in terms of peak cell density and sustained cell viability. Specific protein production rates and volumetric productivity were compared.

[0386] Example 13: Peptide supplements tested on model cells.

[0387] The activity of the peptide supplements produced as described herein was tested on the model mouse hybridoma ME-750 producing IgG2a antibodies. The protein-free medium was DMEM / F12 / RPMI 1640 (3:1:1) supplemented with 0.4 mM HEPES and 2.0 g / L sodium bicarbonate (Franek et al., 1992). The control was further supplemented with basal medium Eagle (BME) amino acids, 2.0 mM glutamine, survival-promoting amino acids (Franek and 1996a) and an iron-enriched growth promoting mixture containing 0.4 mM ferric citrate (Franek et al., 1992, supra). Experiments for comparison against controls used lysates, protein hydrolysates, peptide compositions and / or amino acid compositions produced as described herein to replace one or more of the following supplements: basal medium Eagle (BME) amino acids, 2.0 mM glutamine, survival-promoting amino acids (Franek and 1996a, supra) and / or an iron-enriched growth promoting mixture containing 0.4 mM ferric citrate (Franek, et al., 1992, supra). Another experiment for comparison with the control was to use a complete medium including the supplements provided to the control as listed above, and then further supplement this medium with the lysate, protein hydrolysate, peptide composition and / or amino acid composition produced as described herein. The concentration of the lysate, protein hydrolysate, peptide composition and / or amino acid composition at the time of inoculation was 0.1%, 0.2% or 0.3% (w / v).

[0388] Another set of experiments tested fed-batch cultures. In the control, a feed mixture of 0.25 mL volume was added every day, which contained DMEM fortified with 10x BME amino acids, 10x BME vitamins, and 20 mM glutamine. The experiments compared with the control replaced one or more of the following supplements with lysates, protein hydrolysates, peptide compositions, and / or amino acid compositions produced as described herein: 10x BME amino acids; 10x BME vitamins; and / or 20 mM glutamine.

[0389] Both batch and fed-batch cultures were maintained at 25 cm 2 The cells were grown in T flasks at 37°C in a humidified atmosphere with 5% CO2. The culture volume was 6.0 mL.

[0390] The concentration of monoclonal antibodies was determined by immunoturbidimetry using a calibration curve. The number of apoptotic cells in the culture was determined by microscopic counting of cells showing apoptotic morphology - contracted cells with ruffled membranes. The proportion of cell cycle phases was determined by permeabilization and staining with propidium iodide.

[0391] Batch and fed-batch experiments were compared to the respective controls based on: viable cells / mL; culture viability (%); concentration of monoclonal antibody (mg / L). Comparisons were made to the values ​​of these parameters six days after inoculation.

[0392] Example 14: Production of omega-7 oil from CO2 via a protein hydrolysate intermediate in a two-step process

[0393] In the first stage of the process, proteins and / or polyhydroxybutyrate (PHB) are produced on CO2 and H2 using Cupriavidus necator. In the second stage, the ω-7 producing microorganism Rhodococcus opacus is grown on the hydrolyzed protein and / or PHB produced in the first stage, and oil is extracted from the Rhodococcus...

Claims

1. A method for culturing a microorganism, the method comprising: culturing a first hydrogen-oxygen mixture microorganism under chemoautotrophic conditions to produce biomass; processing the biomass to produce a composition comprising a protein hydrolysate; as well as The composition is provided to a culture medium free of animal components, wherein the composition serves as a nutrient source for a culture comprising a second microorganism in the culture medium.

2. The method of claim 1, wherein the processing comprises treating the biomass with one or more of high temperature, high pressure, or proteases.

3. The method of claim 1, wherein the processing comprises adjusting the pH of a suspension comprising the biomass.

4. The method of claim 1, wherein the first hydrogen-oxygen mixed gas microorganism comprises a Cupriavidus species or a Xanthomonas species.

5. The method of claim 1, wherein the composition comprises the protein hydrolysate, and wherein the composition comprises polyhydroxyalkanoate (PHA).

6. The method of claim 5, wherein the PHA comprises polyhydroxybutyrate (PHB).

7. The method of claim 1, wherein the composition comprises vitamins.

8. The method of claim 7, wherein the vitamins include vitamin B1, vitamin B2 and vitamin B 12 One or more of .

9. The method of claim 1, wherein the total organic content of the composition is enriched in proteins, peptides and / or amino acids.

10. The method of claim 1, wherein the culture comprising the second microorganism comprises animal cells. The method of claim 10 , wherein the composition serves as a supplement for culturing recombinant animal cells.

12. The method of claim 10, wherein the composition serves as a supplement for cultured meat.

13. The method of claim 1, wherein the chemoautotrophic conditions include providing a gaseous substrate to grow the chemoautotrophic microorganisms.

14. The method of claim 13, wherein the gaseous substrate comprises one or more of CO2, CO or CH4 as a carbon source.

15. The method of claim 13 or 14, wherein the gaseous substrate comprises one or more of H2, CO or CH4 as an electron donor.

16. The method of claim 15, wherein the gaseous substrate comprises pyrolysis gas, producer gas, synthesis gas, natural gas or biogas.

17. The method of claim 1, wherein the chemoautotrophic microorganism is selected from one or more of the following: lysogens; Cupricobacter; Corynebacterium; Gordonia; Nocardia; Rhodopseudomonas; Rhodobacter; Rhodospirillum; Rhodococcus; Rhizobium; Thiothiopurpurogenum; Pseudomonas; Hydrogenomonas; Hydrogenobacter; Hydrogenophilus; Hydrogenovibrio; Thermus; Helicobacter; Xanthomonas; Hydrogenophage; Bradyrhizobium; Rhodococcus; Stoneella species; Alcaligenes species; Amycolatopsis species; Aquaspirillum species; Arthrobacter species; Azospirillum species; Acidovorax species; Bacillus species; Thermobacterium species; Dexeria species; Flavobacterium species; Microcyclotium species; Mycobacterium species; Paracoccus species; Perlecan species; Renobacter species; Cerebellum species; Streptomyces species; Thermotrix species; Wattella species; Anabaena species; Arthrospira species; Scenedesmus species; Chlamydomonas species; Cellulose species; or Pinellia species.

18. The method of claim 1, wherein the first oxyhydroxide microorganism is genetically modified.

19. The method of claim 10, wherein the composition acts as a supplement for the growth and / or differentiation of the animal cells.

20. The method of claim 1, further comprising promoting adhesion fibronectin-like activity by the composition for promoting attachment and spreading of the cells.

21. The method of claim 1, further comprising providing an osmoprotectant and / or an anti-apoptotic effect to the culture comprising the second microorganism via the composition.

22. The method of claim 1, wherein the composition comprises at least about 60% total amino acid content, wherein the total amino acid content comprises about 20% to about 50% free amino acids, about 5% to about 20% dipeptides and tripeptides, about 20% to about 60% oligopeptides having a MW of about 2,000 Da to about 3,000 Da, or a combination thereof.

23. The method of claim 1, wherein the organic content of the composition comprises amino acids in an amount ranging from about 10% to about 98% on a weight / weight basis of the organic content.

24. The method of claim 1, wherein the second microorganism comprises an archaeal cell, a bacterial cell, a fungal cell, an insect cell, a mammalian cell, a plant cell, or a yeast cell.

25. The method of claim 1, wherein the culture comprising the second microorganism produces a food product or a beverage.

26. The method of claim 1, wherein the composition has an amino nitrogen (AN) / total nitrogen (TN) ratio of about 20% to about 50% or about 50% to about 80%.

27. The method of claim 1, wherein the composition comprises the protein hydrolysate, and the method further comprises adding the composition to the culture medium at a concentration ranging from about 0.001% w / v to about 5% w / v.

28. The method of claim 1, wherein the culturing the first hydrogen-oxygen mixed gas microorganism comprises providing a gaseous substrate to grow the first hydrogen-oxygen mixed gas microorganism, wherein the gaseous substrate comprises one or more of CO2, CO or CH4 as a carbon source, the gaseous substrate comprises one or more of H2, CO or CH4 as an electron donor, the gaseous substrate comprises pyrolysis gas, producer gas, synthesis gas, natural gas or biogas, or a combination thereof.

29. The method of claim 10, wherein the animal cell is selected from the group consisting of recombinant cells, stem cells, myoblasts, muscle cells, adipocytes, and fibroblasts.

30. The method of claim 24, wherein the fungal cell is selected from one or more of the following: Pleurotus, Rhizomucor, Rhizopus, or Neurospora.

31. The method of claim 24, wherein the fungal cell is selected from one or more of the following: Aspergillus oryzae, Aspergillus sojae, Fusarium venenatum, Neurospora intermedia, Rhizopus oligosporus, Rhizopus oryzae, Trichoderma reesei, or Mucor miehei.

32. The method of claim 3, wherein said adjusting the pH of said suspension comprising said biomass comprises raising said pH to a pH in the range of about 9 to about 14.

33. The method of claim 32, wherein said processing said biomass further comprises heating said biomass to a temperature of about 100°C to about 130°C.

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