Compositions and additives derived from Ogataea polymorpha fungi

Abstract

To provide a food composition that does not contain animal-derived substances and that can be used as a food additive or flavoring to impart spice, umami, or meat seasoning to a food composition by utilizing fungi, yeast, and extracts of Ogataea pholymorpha. SOLUTION: The Ogataea polymorphic fungus yeast, extract, and biomass extract of the Ogataea polymorphic fungus of the present invention contain 0.03 to 3.1% w / w heme. They also contain 0.1 to 25% w / w amino acids. They also contain 0.1 to 2% w / w histidine. They are dissolved or formed by hydrolysis. The Ogataea polymorphic fungus of the present invention further contains proteins, amino acids, carbohydrates, and vitamins. They also contain glutathione. The present invention also relates to the production of recombinant heme proteins in methylotrophic yeast such as Ogataea polymorphic fungus. The Ogataea polymorphic fungus extract contains 0.007% or more 5' ribonucleotides. The 5' ribonucleotides are extracted from 5' GMP.

Description

[Technical Field]

[0001] The present invention relates to the use of Ogataea pholymorpha, in particular as a flavoring agent for imparting spice, umami, flavor, or meat flavor (hereinafter collectively referred to as "aroma") to a food additive or flavoring or to a food composition. [Background technology]

[0002] The increasing demand for sustainable alternative foods (foods that do not contain animal-derived substances for their preparation) presents a challenge in providing appropriate flavors and flavorings for consumer acceptance. Flavoring animal protein analogs presents a challenge for the industry to expand alternative foods and food varieties.

[0003] Yeast extracts are widely used in the food industry to adjust desired flavor, mouthfeel, pH, color, baking characteristics, chewiness, etc. Yeast extracts are typically used as the primary raw material to reduce sugars, vitamins, and amino acids and combine with the Maillard reaction to obtain products with distinctive flavors. The yeasts commonly used for this purpose are Saccharomyces cerevisiae and, to a lesser extent, Cyberlindnera jadiini. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Publication No. 2005 / 003071 [Patent Document 2] U.S. Patent No. 7,070,827 [Patent Document 3] U.S. Patent No. 10,039,306 [Patent Document 4] U.S. Patent No. 10,863,761 [Non-patent literature]

[0005] [Non-Patent Document 1] Zhang, B. et al. Efficient secretory expression and purification of food-grade porcine myoglobin in Komagataella pHaffii, Journal of Agricultural and Food Chemistry, 69(35), 10235-10245(2021) [Non-patent document 2] Hopp, MT, et al., Heme Determination and Quantification Methods and their Suitability for Practical Applications and Everyday Use.Anal.Chem.92, 14, 9429?9440 (2020) [Non-patent document 3] Gellissen, et al. New yeast expression platforms based on methylotrophic Ogataea polymorpHa and Pichia pastoris and on dimorpHic Arxula adeninivorans and Yarrowia lipolytica - a comparison, FEMS Yeast Res.5(11):1079-96 (Nov 2005) [Non-patent document 4] Sohn, et al., A family of telomere-associated autonomously replicating?sequences and their functions in targeted recombination in?Hansenula polymorpHa DL-1, J.Bacteriol.181:1005?1013 (1999)) [Non-Patent Document 5] speciesfungorum. org / Names / SynSpecies, asp?RecordID=362660, citing K, S. Shin, Y, K. Shin, J, H. Yoon & Y, H. Park, Int.J.Syst.Evol.Microbiol.51(6): 2168 (2001); Wick,, Tech.Bull.U, S.Dep.Agric.1029: 31 (1951) [Non-patent document 6] Morais & M, H.Maia, An.Esc.Sup.Quim.Univ.Recife 1: 16 (1959); Sect.Pays d'Amour Soc.Imp.Russe G?ogr.: 1 (1975) [Non-Patent Document 7] Ubiyvovk et al., Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast O.polymorphHa DL-1, BMC Biotechnol.11. 8 (2011) [Non-patent document 8] Scheidle et al., High-throughput screening of O.polymorphHa clones in the batch compared with the controlled-release fed-batch mode on a small scale, FEMS Yeast Research, 10(1), 83-92 (2009)) [Non-Patent Document 9] Sharma, M., et al, A review on microbial alkaline protease: an essential tool for various industrial approaches, Industrial Biotechnology, 15(2):69-78 (2019) [Non-Patent Document 10] Sharma, KMet al, Microbial alkaline proteases: Optimization of production parameters and their properties, Journal of Genetic Engineering and Biotechnology, 15(1):115-126 (2017), strem. com / uploads / technical_notes / 06-3115tech, pdf) [Non-Patent Document 11] Bakhsh, Allah, et al. "TraditiDNAl plant-based meat alternatives, current and a future perspective: A review" J.Agric.Life Sci 55: 1-10 (2021)

[0006] The present invention relates to hydrolyzed extracts and / or isolated molecules derived (ie, from) Ogataea polymorpha as novel and unique yeasts for food applications. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention also relates to the production of recombinant heme proteins in methylotrophic yeasts such as Ogataea polymorpha. [Means for solving the problem]

[0008] The present inventors have invented a method for efficiently obtaining recombinant heme proteins in Ogataea polymorpha. This method can be carried out without genetically manipulating the heme biosynthetic pathway of Ogataea polymorpha (e.g., overexpressing enzymes involved in heme biosynthesis) and without adding exogenous heme to the culture. The heme binding of the recombinant heme proteins produced by this method is 50% or higher.

[0009] In one embodiment, an Ogataea polymorpha extract is disclosed that contains at least about 0.03% w / w heme. In one embodiment, the Ogataea polymorpha extract contains at least about 0.03% w / w heme. In one embodiment, the Ogataea polymorpha extract contains at least about 0.03% w / w heme. In one embodiment, the Ogataea polymorpha extract contains at least about 0.03% w / w heme. In one embodiment, the Ogataea polymorpha extract contains at least about 0.8% glutamic acid. In one embodiment, the Ogataea polymorpha extract contains at least about 0.8% w / w glutamic acid.

[0010] In one embodiment, the Ogataea polymorphic fungus extract further contains 0.007% or more 5' ribonucleotides. In one embodiment, the 5' ribonucleotides are extracted from 5' GMP.

[0011] In one embodiment, the Ogataea polymorpha extract contains 0.8% w / w or more glutamic acid and 0.007% or more 5' ribonucleotides, hi one embodiment, the 5' ribonucleotides are 5' GMP.

[0012] In one embodiment, the Ogataea polymorpha extract comprises γ-glutamyl peptide, hi one embodiment, the γ-glutamyl peptide comprises glutathione.

[0013] In one embodiment, the Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid, at least 0.007% 5' ribonucleotides and at least 0.2% w / w glutathione.

[0014] In one embodiment, the Ogataea polymorpha extract contains at least 0.03-3% w / w heme, at least 0.8% w / w glutamic acid, at least 0.007% 5'GMP and at least 0.2% w / w glutathione.

[0015] In one embodiment, the Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid, at least 0.007% 5'GMP and at least 0.2% w / w glutathione, but no heme.

[0016] In one embodiment, the Ogataea polymorpha extract contains 0.14% w / w or more of histidine.

[0017] In one aspect, the Ogataea polymorpha extract is genetically modified to produce a recombinant heme protein, hi one aspect, the recombinant heme protein is produced at 0.1% w / w of the total protein weight. In one aspect, the recombinant heme protein is selected from the group consisting of hemoglobin, leghemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidases, cytochrome a, cytochrome b, and cytochrome c. In one aspect, the recombinant heme protein is an animal-derived or plant-derived protein.

[0018] In one embodiment, the Ogataea polymorpha extract contains at least 0.03% w / w heme.

[0019] In one aspect, the present invention is a hydrolyzed Ogataea polymorpha extract containing about 0.03% to 3.1% w / w heme.

[0020] In one aspect, the hydrolyzed Ogataea polymorpha extract contains greater than or equal to 0.8% w / w glutamic acid.

[0021] In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract comprises 5' ribonucleotides, hi one embodiment, the hydrolyzed Ogataea polymorphic fungus extract comprises 0.007% or more 5' GMP.

[0022] In one aspect, the hydrolyzed Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid and at least 0.007% 5' ribonucleotides, hi one aspect, the hydrolyzed Ogataea polymorpha extract contains at least 0.007% 5' GMP.

[0023] In one embodiment, the hydrolyzed Ogataea polymorpha extract contains gamma-glutamyl peptides. In one embodiment, the gamma-glutamyl peptide is glutathione. In one embodiment, the hydrolyzed Ogataea polymorpha extract contains greater than 0.2% w / w glutathione.

[0024] In one aspect, the hydrolyzed Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid, at least 0.007% 5'GMP, and at least 0.2% w / w glutathione.

[0025] In one embodiment, the hydrolyzed Ogataea polymorpha extract contains greater than or equal to 0.41% w / w histidine.

[0026] In one aspect, the hydrolyzed Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid, at least 0.007% 5'GMP, and at least 0.2% w / w glutathione, but no heme.

[0027] In one aspect, the hydrolyzed Ogataea polymorpha extract is extracted from a genetically modified Ogataea polymorpha fungus to produce a recombinant heme protein. In one aspect, the recombinant heme protein is produced at 0.1% w / w of the total protein weight. In one aspect, the recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c. In one aspect, the recombinant heme protein is an animal- or plant-derived protein.

[0028] In one aspect, the present specification discloses a flavor precursor mixture. The flavor precursor mixture comprises Ogataea pleomorpha biomass, proteins, amino acids, carbohydrates, and vitamins. In one aspect, the flavor precursor mixture further comprises a fat selected from the group consisting of vegetable fat, animal fat, microbial fat, and cell culture fat. In one aspect, the flavor precursor mixture comprises Ogataea pleomorpha biomass containing about 0.03% w / w or more heme. In one aspect, the Ogataea pleomorpha biomass is an extract. In one aspect, the Ogataea pleomorpha biomass is a hydrolyzed extract. In one aspect, the Ogataea pleomorpha biomass contains 0.8% w / w or more glutamic acid. In one aspect, the Ogataea pleomorpha biomass further comprises 5' ribonucleotides. In one embodiment, the Ogataea pleomorpha biomass further comprises glutamic acid at about 0.8% w / w or more, 5'GMP at about 0.007% w / w or more, and glutathione at about 0.2% w / w or more. In one embodiment, the Ogataea pleomorpha biomass further comprises histidine at about 0.41% w / w or more.

[0029] In one embodiment, the flavor precursor mixture includes Ogataea polymorpha fungus genetically modified to produce a recombinant heme protein. In one embodiment, the recombinant heme protein is produced at 0.1% w / w of the total protein weight. In one embodiment, the recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c. In one embodiment, the recombinant heme protein is an animal- or plant-derived protein.

[0030] In one embodiment, the flavor precursor mixture comprises about 1% w / w or more of Ogataea polymorpha, about 5% w / w or more of monosaccharides, and about 15% w / w or more of amino acids.

[0031] In one aspect, the present specification discloses a food product comprising Ogataea pleomorpha biomass. In one aspect, the Ogataea pleomorpha biomass is lysed biomass. In one aspect, the Ogataea pleomorpha biomass is partially or completely hydrolyzed biomass.

[0032] In one aspect, the food product contains 0.001% to 80% Ogataea pleomorpha biomass. In one aspect, the Ogataea pleomorpha biomass contains 0.03% w / w or more heme. In one aspect, the Ogataea pleomorpha biomass contains 0.8% w / w or more glutamic acid. In one aspect, the Ogataea pleomorpha biomass further contains 5' ribonucleotides. In one aspect, the Ogataea pleomorpha biomass contains about 0.8% w / w or more glutamic acid and further contains 5' ribonucleotides. In one aspect, the hydrolyzed Ogataea pleomorpha biomass contains 5' GMP. In one aspect, the hydrolyzed Ogataea pleomorpha biomass contains glutathione. In one aspect, the Ogataea pleomorpha biomass contains 1% w / w or more histidine.

[0033] In one embodiment, the food product does not contain any animal-derived ingredients.

[0034] In one aspect, the food product is selected from the group consisting of sauces, marinades, condiments, dressings, brines, broths, soups, tofu, tempeh, seitan, fermented vegetables, legumes, artificial / imitation meats, legume foods, cultured meat products, and dietary supplements.

[0035] In one aspect, the invention is a food product comprising a flavor precursor mixture comprising Ogataea pleomorpha biomass.

[0036] In one aspect, the present specification discloses a dietary supplement comprising Ogataea pleomorpha biomass containing at least about 0.03% w / w heme. In one aspect, the Ogataea pleomorpha biomass contains about 0.03% w / w heme. In one aspect, the Ogataea pleomorpha biomass contains about 0.03% to 3% w / w heme. In one aspect, the Ogataea pleomorpha biomass contains 0.03% to 3% w / w heme. In one aspect, the Ogataea pleomorpha biomass is a lysed biomass. In one aspect, the Ogataea pleomorpha biomass is hydrolyzed. In one aspect, the hydrolyzed Ogataea pleomorpha biomass contains amino acids, peptides, or polypeptides (having a size of 25, 20, 15, 10, 5, 2, 1, or 0.5 kDa or less). In one embodiment, the hydrolyzed Ogataea polymorpha biomass is further filtered to produce an insoluble fraction comprising carbohydrates, cell membrane components, or cell wall components, which are less than 25, 20, 15, 10, 5, 2, 1, or 0.5 kDa.

[0037] In one aspect, the present specification discloses a method for producing an Ogataea polymorphic fungus extract. The method includes the following steps. (A) Cultivating and growing Ogataea pleomorpha cells; (B) lysing the Ogataea pleomorpha cells; (C) adjusting the pH as needed; (D) A step of obtaining an Ogataea polymorpha extract by drying or concentration by removing water.

[0038] The method of the present invention further comprises step (E) after step (B). (E) A step of inactivating acidic phosphatase.

[0039] The method of the present invention further comprises step (F). (F) Hydrolysis of endogenous proteins and nucleic acids to obtain molecules of sizes less than 25, 20, 15, 10, 5, 2, 1, or 0.5 kDa.

[0040] The method of the present invention further comprises step (G). (G) Maintaining the temperature at one of 90°C, 85°C, 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, or 50°C.

[0041] The method of the present invention further comprises the following step (H): (H) Hydrolyzing the Ogataea pleomorpha extract to obtain a soluble fraction and an insoluble fraction. In one embodiment, the soluble portion comprises particles, hi one embodiment, the soluble portion comprises amino acids, peptides and / or polypeptides, the size of which is less than or equal to 100, 50, 25, 20, 15, 10, 5, 2, 1, or 0.5 kDa. In one embodiment, the undissolved portion comprises particles, hi one embodiment, the undissolved portion comprises carbohydrates, the size of which is less than or equal to 100, 50, 25, 20, 15, 10, 5, 2, 1, or 0.5 kDa.

[0042] In one aspect, the present specification discloses a method for preparing a seasoning / flavorant from Ogataea pleomorpha, the method comprising the steps of: (A) Obtaining Ogataea polymorpha biomass; (B) adding and mixing a source of carbohydrates, lipids, and amino acids to the Ogataea pleomorpha biomass; (C) A step of heat-treating the Ogataea pleomorpha biomas by exposing them to a temperature of 100°C to 130°C for 30 to 180 minutes, thereby producing a seasoning from the Ogataea pleomorpha biomas.

[0043] In one aspect, the Ogataea polymorphic fungus biomass is pre-lysed to obtain an extract. In one aspect, the Ogataea polymorphic fungus biomass is hydrolyzed. In one aspect, the hydrolyzed Ogataea polymorphic fungus biomass is filtered to separate a lysed portion and an unlysed portion. The lysed portion is used to obtain a seasoning. In one aspect, the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, oligosaccharides, and polysaccharides. In one aspect, the lipid is selected from the group consisting of animal fat, insect fat, fungal fat, vegetable fat, plant fat, synthetic fat, and microbial fat. In one aspect, the amino acid source is selected from the group consisting of natural proteins, partially hydrolyzed proteins, and free amino acids.

[0044] In one embodiment, the method of the present invention further comprises the step of (D) inducing Ogataea polymorpha cell lysis to obtain an extract. In one embodiment, the method of the present invention further comprises the step (E) of inducing proteolysis of the extract obtained in step (D). In one aspect, the method of the present invention further comprises the step of (F) inducing hydrolysis of nucleic acids in the extract. In one aspect, the method of the present invention further comprises the step of inducing the conversion of a (G) amino acid to glutamic acid.

[0045] In one aspect, the present specification discloses a method for producing a heme protein having 50% or more heme, the method comprising the steps of: (A) Providing a culture of transgenic Ogataea polymorpha fungus containing a nucleic acid encoding a heme protein operably linked to a methanol-inducible promoter; (B) Culturing the transgenic Ogataea polymorpha bacteria without adding exogenous heme to the culture. (C) A step of separating and purifying the heme protein.

[0046] In one aspect, the transgenic Ogataea polymorpha fungus does not contain an exogenous transcriptional activator or exogenous components of a heme biosynthetic pathway.

[0047] In one embodiment, the hemoprotein is selected from the group consisting of animal-derived hemoproteins and plant-derived hemoproteins.

[0048] In one aspect, the animal-derived heme protein is selected from the group consisting of bovine heme protein, porcine heme protein, ovine heme protein, equine heme protein, and caprine heme protein.

[0049] In one aspect, the recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0050] In one embodiment, the nucleic acid comprises a nucleic acid sequence having 70% or more homology (also referred to as "identity") to the nucleic acid sequence of SEQ ID NO: 1 (SEQ ID NO; 1).

[0051] In one embodiment, the nucleic acid is codon-optimized, and comprises a nucleic acid sequence having 70% or more homology to the nucleic acid sequence of SEQ ID NO:2.

[0052] In one embodiment, the nucleic acid comprises a nucleic acid sequence having 70% or more homology to the nucleic acid sequence of SEQ ID NO:1.

[0053] In one embodiment, the nucleic acid is codon-optimized. The nucleic acid comprises a nucleic acid sequence having 70% or more homology to the nucleic acid sequence of SEQ ID NO:2.

[0054] In one aspect, the present specification discloses a method for producing heme proteins from animals having a heme loading of 50% or more. In one aspect, the method comprises the following steps. (A) Introducing into Ogataea polymorpha a nucleic acid construct containing a promoter operably linked to a nucleic acid encoding an animal-derived heme protein. (B) culturing the Ogataea pleomorpha transgenic cells without adding exogenous heme to the culture. (C) A step of obtaining animal-derived heme proteins from the culture.

[0055] In one aspect, the transgenic Ogataea polymorpha fungus does not comprise an exogenous transcriptional activator of a heme biosynthetic pathway or an exogenous component of a heme biosynthetic pathway.

[0056] In one aspect, the animal-derived heme protein is selected from the group consisting of bovine heme protein, porcine heme protein, ovine heme protein, equine heme protein, and caprine heme protein.

[0057] In one aspect, the recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0058] In one aspect, the present specification discloses a method for producing recombinant bovine myoglobin having at least 50% heme-loaded recombinant bovine myoglobin, the method comprising the steps of: (A) introducing a nucleic acid encoding a recombinant bovine myoglobin operably linked to a promoter into an Ogataea polymorphic yeast cell; (B) culturing the Ogataea polymorpha fungus containing the nucleic acid encoding the recombinant bovine myoglobin to promote expression of the recombinant myoglobin without adding heme, and expressing the recombinant bovine myoglobin;

[0059] In one aspect, the transgenic Ogataea polymorpha fungus does not comprise an exogenous transcriptional activator of a heme biosynthetic pathway or an exogenous component of a heme biosynthetic pathway.

[0060] In one embodiment, the method of the present invention further comprises the step (C) of extracting and purifying the recombinant bovine myoglobin.

[0061] In one aspect, the present specification discloses a food product having bovine myoglobin protein or heme protein.

[0062] In one aspect, the present specification discloses a simulated meat food product comprising a lysate of cultured Ogataea pleomorpha.

[0063] In one aspect, the present specification discloses a meat-like food product comprising bovine myoglobin protein or heme protein. [Brief explanation of the drawings]

[0064] [Figure 1A] To optimize the medium factors and treatment conditions for the heme biosynthesis pathway during the cultivation of Ogataea polymorphic strains, the heme yields obtained from Ogataea polymorphic strains cultured on various carbon sources with and without methanol induction are shown. [Figure 1B] Figure 1 shows the heme yield (per g dry weight of cells) obtained from Ogataea polymorpha cultured on different nitrogen sources with methanol induction and glycerol as the carbon source. [Figure 1C] Figure 1 shows the heme yield (per g dry weight of cells) obtained from Ogataea polymorpha cultivated on optimal carbon and nitrogen sources in a 150 L bioreactor during a quasi-trial. [Figure 2] The expression plasmid pFPMT121 used to generate the recombinant product strains is shown. pFPMT121 and its derivatives were used as circular plasmids for transformation of Ogataea polymorpha strains RB11 and ALU3. [Figure 3] This designates the B14 plasmid, a derivative of pFPMT121 that does not contain an antibiotic resistance gene, and was used in the present invention to transform Ogataea polymorpha strain RB11. [Figure 4] Plasmid map showing the synthetic gene (bovine myoglobin), origin of replication Col El, restriction enzyme sites for digestion (IEcoRI and BamHI), and antibiotic resistance gene (AmpR for ampicillin). [Figure 5] The final transformation vector contains the promoter element (FMDp), the codon-optimized bovine myoglobin gene (BtM6), and the transcription terminator (MOXt) used to transform Ogataea polymorpha strain RB11. [Figure 6]A sterile image of an SDS-PAGE gel is shown, which is used for screening and selection of positive transformants by densitometric analysis. [Figure 7] The amino acid sequence homology (CLUSTAL W alignment) between native bovine myoglobin and myoglobin expressed in Ogataea polymorpha RB11 is shown. [Figure 8] 1 depicts SDS PAGE analysis of specimens collected at different intervals before and after methanol induction. [Figure 9] Western blot analysis between native bovine myoglobin and myoglobin expressed in Ogataea polymorpha RB11. Columns 1-3 are standard myoglobin preparations at concentrations of 0.0125%, 0.025%, and 0.05%. Columns 5-8 are recombinant myoglobin preparations at different dilutions (400x, 20x, 100x, and 50x) purified and concentrated by ultrafiltration. [Figure 10] 1 is a photograph of an Ogataea polymorphic yeast extract. DETAILED DESCRIPTION OF THE INVENTION

[0065] The terms used in this specification will be explained first. The following explanation should not be construed as limiting. Unless otherwise specified, the number of devices or means can be singular or plural. For example, the term "nucleic acid sequence" includes one or more nucleic acid sequences.

[0066] The term "and / or" does not limit one of them. For example, "A and / or B" means "A" or "B" alone, but also "both A and B." "A, B and / or C" may include not only A, B, or C alone, but also two of A and B, two of A and C, two of B and C, or all of A, B, and C.

[0067] The terms "comprise," "have," and "contain" are synonymous unless otherwise specified. In this specification, "comprise A" and "have A" may include things other than A.

[0068] The term "about" is used herein in connection with numerical values, including a range of ±10% of the specified numerical value, except in the claims.

[0069] The term "at least" is used in reference to numerical values ​​and means "greater than or equal to." When used in terms of integers, the number of nucleotides in a nucleic acid molecule is an integer, so "at least 18" or "18 or more" of 21 nucleotides refers to 18, 19, 20, or 21 nucleotides. When used in terms of non-integers, "at least 5%" or "5% or more" also includes 5.0%, 5.1%, and 5.18%. Decimals are not taken into account.

[0070] Various aspects (embodiments) of the present invention are disclosed herein with range limitations. Numerical ranges include the numerical values ​​defining the range. When a numerical range is disclosed, it is intended to include the integers and fractional parts between the upper and lower limits of the numerical range. The upper and lower limits of a numerical range are independently included or excluded within the numerical range. Numerical ranges including both or either of the upper and lower limits are also included in the disclosure of the present invention. The description "range of 1 to 10" is a shorthand, but also includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and fractional parts thereof.

[0071] When a value is explicitly stated, values ​​that are approximately the same in amount / magnitude as the stated value are included in the disclosure of the invention. When a combination of devices / elements is disclosed, subcombinations of the components of the combination are also disclosed and are within the scope of the invention. When various elements or groups thereof are disclosed individually, each individual combination taken from them is also disclosed. When an element of the invention is disclosed as having multiple alternatives, the omission of one of the alternatives or the combination thereof still falls within the scope of the invention. The omission of multiple elements still falls within the scope of the invention.

[0072] Unless otherwise specified, all terms used herein have the same meaning as understood by a person skilled in the art (dictionary meaning).

[0073] This specification discloses multiple techniques / steps. These techniques / steps have individual advantages and can be employed in combination with one, several, or all of the other techniques / steps. This specification does not repeatedly list every possible combination of the individual steps. However, the specification and claims encompass all such combinations.

[0074] The term "percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences, nucleic acid sequences). "Percent identity" can be determined by aligning two sequences and introducing caps to maximize identity between the sequences. Alignments can be generated using conventional programs. Nucleotide sequence alignments can be performed using the blastn program with specified parameters. Amino acid sequence alignments can be performed using the blastp program with specified parameters. For more information, see the National Center for Biotechnology Information (NCBl): www.nlm.nih.gov.

[0075] The term "Ogataea pleomorpha biomass" refers to accumulated yeast or accumulated yeast cells, with or without the media culture used to grow and propagate the yeast.

[0076] The term "lysate" or "extract" refers to a mixture and / or solution of cell contents, cell wall remnants, and culture medium attached to the cell wall via attachment members that could not be removed prior to the lysis process in a cell lysis method.

[0077] The term "lysis" refers to the rupture of the plasma membrane and cell wall of a cell. Lysis can result in the escape of significant amounts of intracellular material into the extracellular space. Lysis can be accomplished using electrochemical, mechanical, permeabilization, thermal, enzymatic, chemical, electrical, radiological, viral, or microbial means. In one aspect, the methods described herein include performing the lysis of cells or microorganisms described herein to separate a chemical or mixture thereof from the contents of a bioreactor.

[0078] The present invention relates to the use of yeasts Ogataea polymorpha and compositions derived therefrom as flavorings in food products. Ogataea polymorpha or yeasts include Hansenula polymorpha, Hansenula angusta, Pichia angusta, Candida thermophila, Ogataea thermophila and Torulopsis methanothermo.

[0079] Ogataea polymorpha-derived compositions relate to Ogataea polymorpha cells, cell parts, cell fragments, and cell components (proteins, amino acids, vitamins, carbohydrates, minerals, lipids) and their use to obtain seasoning precursors, flavorings, and foods.

[0080] Flavors or seasonings, also known as flavorings, are food additives used to improve the taste, mouthfeel, or smell of food.

[0081] The term "yeast extract" refers to a product obtained from yeast lysis. In the present invention, "yeast extract" is derived from Ogataea polymorpha / yeast, and is therefore also referred to as "extract of Ogataea polymorpha." In one aspect, "extract of Ogataea polymorpha" refers to a soluble or insoluble extract of Ogataea polymorpha. In one aspect, "extract of Ogataea polymorpha" refers to a soluble and / or insoluble extract of Ogataea polymorpha in which the transgenic protein is still present. In one aspect, "extract of Ogataea polymorpha" refers to the soluble or insoluble residue of lysed Ogataea polymorpha from which the transgenic protein (e.g., myoglobin) has been completely or partially removed. In one aspect, "extract of Ogataea polymorpha" can be used as a flavor precursor or flavoring agent. In one aspect, "extract of Ogataea polymorpha" can be used as a dietary supplement.

[0082] In one aspect, the enzyme-mediated lysis that causes the disruption of the cell membrane also causes partial hydrolysis of certain cellular components, such as proteins, and in this case the term "extract of Ogataea pleomorpha" also refers to a partially or fully hydrolyzed extract of Ogataea pleomorpha.

[0083] The term "hydrolyzed yeast extract" refers to a yeast extract whose contents have undergone further hydrolysis of large molecules (e.g., nucleic acids, proteins, etc.). In one aspect, the polymers contained in Ogataea polymorpha cells are partially hydrolyzed. Partial hydrolysis refers to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hydrolysis. In one aspect, "hydrolyzed yeast extract" refers to the soluble or insoluble hydrolyzed residue of an extract of Ogataea polymorpha. In one aspect, "hydrolyzed yeast extract" refers to the soluble and / or insoluble hydrolyzed residue of an extract of Ogataea polymorpha in which the transgenic protein is still present. In one aspect, "hydrolyzed yeast extract" refers to the hydrolyzed remainder of lysed yeast biomass from which transgenic proteins (e.g., myoglobin) have been removed. In one aspect, hydrolyzed Ogataea polymorpha extract can be used as a flavor precursor or flavoring.

[0084] The term "glutamic derived compound" refers to a molecule containing a molecule resulting from the reaction of an amino acid, glutamic acid (e.g., glutamyl peptide, glutathione), its salt, and glutamine.

[0085] The term "glutamyl peptide" refers to a peptide containing multiple amino acid residues, at least one of which is glutamic acid, gamma-glutamic acid, or a salt thereof.

[0086] The terms "flavor composition" and "flavor precursor mixture" refer to a mixture of compositions capable of imparting a desirable flavor, aroma, sensation, and / or mouthfeel.

[0087] The terms "artificial meat," "meat substitute," "imitation meat," "meat analog," and "meat-like food" refer to foods that are not of animal origin, i.e., contain a significant amount of non-animal protein sources, but have the same or similar structure, texture, quality, appearance, and / or other characteristics as animal meat. Examples of animal meat include beef, pork, game, poultry (e.g., chicken, turkey, duck), and fish or seafood substitutes or analogs. The terms refer to meat-like foods that are pre-cooked or pre-embedded. These terms also encompass cultured meat products.

[0088] The terms "non-animal product" and "animal product-free" refer to the absence of materials / substances derived / obtained directly from animals (e.g., their organs, tissues, cells, proteins, nucleic acids). This definition does not apply to animal products (e.g., genes and / or amino acid sequences derived from sequencing animal genetic material or the corresponding proteins) produced in a non-animal host.

[0089] The term "holoprotein" refers to a conjugated functional protein that is bound to a ligand or prosthetic group.

[0090] The term "apoprotein" refers to a non-functional protein that is not bound to a ligand or protease group.

[0091] The term "heme protein" includes proteins that have the ability to attach heme protease groups to their structure. The term "heme protein" refers to essential components of animal flesh and / or animal proteins that provide color and flavor to plant-based meat products. "Myoglobin" and "hemoglobin" are considered heme proteins and are oxygen-binding proteins in animals. The term "heme protein" refers to heme-containing proteins. The term "containing" means that heme is bound to the protein via covalent or noncovalent bonds. The term "heme protein" refers to full-length proteins as well as their parts or variants.

[0092] The term "produce or produce" refers to the ability / function of a yeast (e.g., Ogataea pleomorpha) to express a protein of interest. In one embodiment, the protein of interest is transgenic / recombinant. In one embodiment, the protein of interest is isolated from the yeast.

[0093] The terms "cultivate" and "propagate" refer to growing yeast (e.g., Ogataea polymorpha) under appropriate conditions for the production of a desired end product (e.g., an animal heme protein).

[0094] The term "derived from" refers to the source of a flavorant, flavor precursor, or flavoring agent. For example, a flavorant derived from Ogataea polymorpha refers to a flavorant that includes a portion of Ogataea polymorpha (e.g., an animal heme protein isolated from the Ogataea polymorpha).

[0095] The terms "animal hemoprotein" and "animal-derived hemoprotein" refer to hemoproteins from animals, such as cows, pigs, sheep, horses, and goats, but do not include hemoproteins derived from humans. In one embodiment, the protein is isolated from yeast. In one embodiment, the term "animal hemoprotein" includes hemoproteins involved in oxygen transport or is selected from the group of hemoproteins listed below. Examples of hemoproteins include hemoglobin, myoglobin, neuroglobin, cytoglobin, and the following enzymes and hemoproteins: cytochrome P450s, cytochrome c oxidase, ligninase, catalase, and peroxidases, which are enzymes with substituted heme groups. This heme protein is involved in the electron transport chain, including cytochrome a, cytochrome b, and cytochrome c.

[0096] The term "plant-derived hemoprotein" refers to hemoproteins from monocotyledonous and dicotyledonous plants, including the following: Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn), Arabidopsis thaliana (Legume), Glycine max (soybean), Cicer arietinum (chickpea), Pisum sativum (bean), pHaseolus vulgaris (kidney beans) Vigna unguiculata (cowpea), Vigna radiata (Yaenari), Lupinus albus (lupin), Medicago sativa (alfalfa), Brassica napus (canola), Triticum sps. (wheat, wheat kernel, spelt), Gossypium hirsutum (cotton), Oryza sativa (rice), Zizania sps. (Willowgrass), Helianthus annuus (Sunflower), Beta vulgaris (sugar beet), Pennisetum glaucum (peanut barnyard millet), Chenopodium sp. (Cinchona), Sesamum sp. (sesame), Linum usitatissimum (Flax), Lactuca sativa (lettuce), Spinacia oleracea (spinach), Hordeum vulgare (barley).

[0097] The term "recombinant hemoprotein" refers to a protein obtained from a transgenic organism. The term "recombinant hemoprotein" refers to a protein encoded by an exogenous cDNA encoding a hemoprotein. The term "exogenous nucleic acid" is used interchangeably with "recombinant nucleic acid" and / or "heterologenous gene."

[0098] The terms "transgenic," "recombinant," "genetically engineered," and "genetically modified" mean that yeast (e.g., Ogataea variegata) has been transformed or transduced with a nucleic acid (recombinant sequence). The term "transformation" refers to the process by which a recombinant sequence is introduced and expressed in yeast cells using non-viral vectors.

[0099] The term "heme loading" refers to the amount of heme bound to an apo-heme recombinant protein. For example, according to Non-Patent Document 1, when porcine myoglobin expressed in Pichia pastoris is supplemented with 150 mg / L of exogenous heme in the culture medium (heme addition), resulting in 0.02 moles of heme per mole of myoglobin, this represents a heme loading of 22%. Theoretically, binding of 1 mole of myoglobin to 1 mole of heme results in 100% heme loading. The amount of heme protein bound to heme can be determined by known methods. For more information, see Non-Patent Document 2.

[0100] In one embodiment, heme molecules present in cells, culture medium, lysates, hydrolyzed lysates, and filtered hydrolysates are free, bound to, and / or captured in planar structures consisting of amino acids, peptides, oligopeptides, polypeptides, nucleotides, oligonucleotides, nucleic acids, and carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, and polysaccharides). Ogataea polymorpha

[0101] Methods for transforming Ogataea polymorpha are known in the art and include chemical transformation, electroporation, transduction, and biolistic particle delivery. For example, a plasmid applied for chemical transformation can be integrated or integrated into the yeast genome by homologous recombination. See non-patent literature 3 and 4 for more information.

[0102] Ogataea polymorpha has many aliases, including Candida thermopHila, Hansenula angusta, Hansenula polymorpha, Ogataea thermophile, and Trulopsis methanothermo. See Non-Patent Documents 5 and 6 for more information.

[0103] Ogataea polymorpha is considered a protein factory, a ubiquitous methylotrophic yeast, and this yeast is known as a model yeast (also called "strain") for peroxisome biology. However, the tight gene regulation (related to abiotic stress tolerance, oxidative status, methanol metabolism, heavy metal tolerance, and nitrate assimilation) defines this strain as a potential candidate for further understanding its role in food and feed applications. Methylotrophic yeasts can grow in extreme environments. Ogataea polymorpha tolerates temperatures above 45°C, and its ability to grow on methanol as both an energy and carbon source is enabled by a methanol utilization pathway shared by all known methylotrophic yeasts. Gene expression depends on a carbon source-dependent repression / de-repression / induction mechanism. This mechanism is conferred by the properties of methylotrophic yeast promoters. Specific promoters are repressed by glucose, derepressed by glycerol, and induced by methanol.

[0104] Methylotrophic yeast strains are well known and are described in Non-Patent Documents 7 and 8. In one embodiment, the carbon source used for the production of recombinant myoglobin is glycerol. However, since the promoter used is methanol-regulated, it is desirable to supplement the culture medium with methanol under these circumstances. The methanol requirement requires replacing the methanol-inducible promoter with a specific promoter that is constitutive or sugar-inducible. Therefore, culturing Ogataea polymorpha requires supplementing various culture media with glucose as the sole carbon source. Examples of culture media include glucose, glycerol, xylose, and cellobiose.

[0105] The term "cultivation" refers to the growth of recombinant Ogataea polymorpha (growth under optimal physical, chemical and operational parameters for cell growth and protein production).

[0106] In one aspect, Ogataea polymorphic yeast can be cultured under a variety of physical conditions, including maintaining the temperature of the surrounding medium at about 25°C to about 50°C and the pH of the medium at about 2.5 to 7.5 using an acid or alkaline solution.

[0107] In one aspect, Ogataea pleomorpha cells are grown in the presence of a carbon source, such as methanol, glycerol, glucose, galactose, fructose, sucrose, xylose, arabinose, or crude glycerol, recovered agricultural residue (lignocellulosic) hydrolysates, post-fermentation fiber and / or protein-rich residues and hydrolysates thereof, starch hydrolysates, floating plants, seaweed, or algae, biomass hydrolysates, and other renewable feedstocks.

[0108] In one embodiment, Ogataea polymorpha is grown supplemented with a nitrogen source, such as ammonium phosphate, ammonium sulfate, animal, plant, or fungal meat extracts, milk hydrolysate, corn protein hydrolysate, soy protein hydrolysate, pea protein hydrolysate, rice protein hydrolysate, corn peptone, soy peptone, potato protein hydrolysate, yeast hydrolysate, bacterial hydrolysate, fungal hydrolysate, and malt grain extract.

[0109] In one embodiment, growth of Ogataea polymorphic yeast is supplemented with macro- and micro-nutrients, vitamins, and minerals when grown on synthetic or chemically defined media.

[0110] In one embodiment, the optimal carbon and nitrogen sources for improved yeast biomass growth and heme production are glycerol and corn protein hydrolysate powder.

[0111] A medium optimized for the growth and expression of recombinant heme proteins in genetically engineered Ogataea polymorpha includes, for example, the following requirements (1) to (7): (1) Pre-innoculum (YPD) medium: 2% soy peptone, 1% yeast extract, 2% glucose, 2% agar per plate, 100 mg / L adenine. (2)Preparation instructions: Each material is weighed separately according to the media preparation protocol. The individual components are placed in a mixing container and water is added to reach the weight specified in the media preparation protocol. Once all components are completely dissolved, the pH and conductivity are measured and recorded in the media preparation protocol. The suspension is then sterilized by autoclaving (20 minutes at 121°C). (3) Fermentation or Production Medium (SYN 6): 1.32 g / L ammonium hydrogen phosphate; 3 g / L magnesium sulfate, 3.32 g / L potassium chloride; 0.33g / L sodium chloride, 20g / L glycerol, 20mL (100x calcium chloride), 20 mL of 100x solution of diatomaceous elements (6.65 g / L EDTA, 6.65 g / L ammonium ferrous sulfate, 0.55 g / L copper sulfate; 2 g / L zinc sulfide; 2.65 g / L manganese sulfide), 20 mL of 100x vitamin solution (0.04 g / L D-biotin, 13.35 g / L thiamine hydrochloride), 20 mL of trace element 100x solution (65 mg / L nickel sulfate; 65 g / L cobalt chloride, 65 g / L boric acid, 65 g / L potassium iodide, 65 g / L sodium molybdate). (4)Preparation instructions: Each material is weighed separately according to the media preparation protocol. The individual components are placed in a mixing container and water is added to reach the weight noted in the media preparation protocol. Once all components are completely dissolved, the pH and conductivity are measured and recorded in the media preparation protocol. (5) After sterilization in an autoclave, add the following mixed solution to the reaction vessel. 20 mL of microelement solution, 20mL of vitamin solution, 20 mL of calcium chloride solution, 20 mL of trace element solution. (6)Culture: The pre-inoculum is added to the production medium and cultured at 37°C, pH 4.8, with an agitation speed of 200-1400 rpm and a dissolved oxygen concentration (DO) maintained at 30%. (7) 10% Structrol J673 is used as an antifoam to prevent foam buildup or formation, and the pH is maintained at 4.8 by the addition of 12.5% ​​alkali (25% ammonia solution) and 28% acid (85% sulfuric acid). For inhibition, 75% w / v glycerol is fed to the reaction vessel.

[0112] The term "parameters" refers to the concentrations of chemical components such as carbon, nitrogen, metal and non-metal sources, and physical conditions such as pH, temperature, dissolved oxygen level, incubation time, agitation speed (rpm), and aeration / ventilation.

[0113] In one embodiment, the culture is initiated in batch mode with 2% w / v glycerol as the carbon source and is transitioned to fed-batch mode with a linear feed rate of 2-6 g / L / h once the concentration of glycerol in the production medium is below 10 g / L. During cultivation, the carbon source supply rate and dissolved oxygen concentration (DO) in the reactor are adjusted. For example, throughout cultivation, the dissolved oxygen concentration (DO) is maintained at 20-40%, and the agitation speed is maintained at maximum. Regarding physical parameters, the temperature is maintained at 37°C and the pH is maintained at 5.5.

[0114] In one aspect, downstream processing of the yeast cell biomass harvested from the reactor vessel using centrifugation, sedimentation, filtration, or other mechanical means is the basis for the production of flavor, aroma, and nutritional components.

[0115] In one aspect, the present invention relates to the use of Ogataea polymorpha cells to produce extracts, hydrolyzed extracts, and purified extracts, which further produce flavor components. According to the present invention, the biomass, extracts, hydrolyzed extracts (referred to as "hydrolyzed extracts"), and / or specific components from the hydrolyzed extracts, Maillard reaction precursor mixtures, Maillard reaction products, and flavors derived from Ogataea polymorpha are also referred to as "Ogataea polymorpha-derived products." In one aspect, Ogataea polymorpha biomass is used directly, wet or dry, to produce flavors, nutritional additives, or food products. In one aspect, Ogataea polymorpha biomass is dissolved to obtain an extract, which is then used to produce a flavor or food product. In one aspect, Ogataea polymorpha extract is hydrolyzed and used in a flavor or food product. In one aspect, yeast extract or hydrolyzed yeast extract is filtered and a specific fraction is concentrated. In one aspect, the flavor or fragrance is a food product. In one embodiment, flavors and aromas impart a characteristic flavor to a food product. In one embodiment, Ogataea polymorpha biomass is exposed to an extraction solvent to concentrate and purify specific soluble components. Examples of soluble components include heme, nucleotides, and amino acids.

[0116] In one aspect, the invention is a genetically modified Ogataea polymorpha fungus that produces a recombinant heme protein.

[0117] In one aspect, the present invention is a genetically engineered Ogataea polymorpha fungus that produces a recombinant heme protein. The recombinant heme protein is isolated from the cell lysate by filtration, precipitation, or other known separation methods. In one aspect, the remaining components are hydrolyzed to produce a hydrolyzed yeast extract and its specific fractions.

[0118] The terms "transgenic," "recombinant," "genetically engineered," and "genetically modified" mean that yeast (e.g., Ogataea variegata) has been transformed or transduced with a nucleic acid (recombinant sequence). The term "transformation" refers to the process by which a recombinant sequence is introduced and expressed in yeast cells using a non-viral vector.

[0119] The term "regulatory element" includes promoter elements that affect RNA polymerase binding. The term "regulatory element" refers to a DNA segment that controls the start point and frequency of transcription (RNA synthesis) of a gene under the control of the promoter element in a host organism. Promoter elements useful in carrying out the methods of the present invention include: formate dehydrogenase (FMD) (SEQ ID NO: 3), Methanol oxidase (MOX) (SEQ ID NO:4) Alcohol oxidase I (AOX1), glyceraldehyde-3-phosphate dehydrogenase (GAP), Alcohol dehydrogenase (ADH1), Translation elongation factor (TEF1), Hexokinase (GLK), glucose-6-phosphate isomerase (GPI), Fructose-1,6-bisphosphate aldolase (FBA), Triosephosphate isomerase (TRI), phosphoglycerate mutase (PGM), pyruvate kinase (PYK), pyruvate dehydrogenase (PDH), isocitrate lyase (ICL1), L-rhamanoate dehydratase (LRA3), L-2-keto-3-deoxyrhamnonate (LRA4), glycosylphosphatidylinositol (GPI) anchored protein (GCW14), Maltase (MAL), dihydroxyacetone synthase (DAS), Alcohol dehydrogenase (ADH2), 6-phosphogluconate dehydrogenase (PGD), Transaldolase (TAL), ribulose phosphate epimerase (RPE), catalase (CAT), superoxide dismutase (SOD), trehalose-6-phoshate (TPS1), Plasma membrane ATpase pump (PMA1) promoters from O. polymorpha MOX, AOX1, GAP1 promoters from Pichia pastoris ( MOX, AOX1 and GAP1 promoters from Pichia pastoris )ADH1, ADH1, pyruvate decarboxylase (PDC1), GAP1 Glycerol update protein (GUP1 & GUP2) promoters from S. cerevisiae.

[0120] The term "transcription terminator" refers to a DNA fragment containing a signal structure for RNA polymerase, which terminates transcription. An example of a terminator element that can be used is the MOX (SEQ ID NO:4), amine oxidase (AMO), or phosphate starvation (PHO1) terminator from Ogataea polymorpha.

[0121] In one embodiment, the nucleic acid construct comprises the FMD promoter (SEQ ID NO:3).

[0122] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence homology to SEQ ID NO:3: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0123] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence homology to SEQ ID NO:3: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0124] In one embodiment, the nucleic acid construct comprises the MOX promoter (SEQ ID NO: 4).

[0125] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence identity to SEQ ID NO:4: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0126] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence identity to SEQ ID NO:4: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0127] In one embodiment, the nucleic acid construct comprises a marker. In one aspect, the marker comprises a supplemental marker or a dominant marker. In one aspect, the supplemental nutritional marker is: Sc LEU2* (Leucine auxotrophy), Sc URA3 (Uracil auxotrophy) (SEQ ID NO: 5), Hp URA3 (Uracil auxotrophy), Hp ADE11 (adenine auxotrophy), Hp MET6 (Methionine auxotrophy), Hp LEU2 (Leucine auxotrophy), Hp AUR1 (Aureobasidin auxotrophy), Hp IMH3 (inosine monophosphate dehydrogenase auxotrophy). In one embodiment, the dominant marker is: Sh-ble (Zeocin resistance), Sn-nat1 (Nourseothricin resistance), Kp-hph (Hygromycin B resistance), Tn-KanMX (G418 / Geneticin resistance), Sv-Pat (Bialaphos resistance). In one aspect, the nucleic acid construct comprises: Sc URA3 (Uracil auxotrophy) (SEQ ID NO: 5) marker.

[0128] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence homology to SEQ ID NO:5: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0129] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence having the following percentage of sequence homology to SEQ ID NO:5: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0130] "Transformation" is a term that describes the technique of transferring exogenous DNA into a given cell, resulting in a recombinant or genetically modified cell. Yeast transformation protocols, particularly those for transforming Ogataea polymorpha, are known in the art and include chemical transformation, electroporation, transduction, and biological particle delivery. See non-patent literature 3 and 4.

[0131] In one embodiment, Ogataea polymorpha expressing a recombinant myoglobin gene is cultured under known media components, physical parameters, and operating parameters.

[0132] In one embodiment, Ogataea polymorpha expressing a recombinant myoglobin gene is grown in a synthetic or chemically defined medium containing predetermined substances, such as carbon sources, nitrogen sources, vitamins, minerals, macro- and micronutrients, and metal supplements. Operating parameters are maintained for higher biomass and higher protein expression.

[0133] In one aspect, recombinant Ogataea polymorpha expressing the myoglobin gene will be grown in a complex medium containing corn protein hydrolysate to evaluate its cost-effectiveness and commercial feasibility. In one aspect, the inclusion of corn protein hydrolysate powder as a nitrogen source will supplement the production medium with vitamins, minerals, and trace elements required when synthetic media are used.

[0134] The term "downstream" includes procedures performed for the isolation of yeast biomass from whole cells, cell lysis, and the isolation and purification of heme-containing proteins, specifically myoglobin, since the protein of interest is intracellular.

[0135] In one aspect, the downstream process for recovering recombinant myoglobin from Ogataea polymorpha comprises steps (i), (ii), and (iii). (i) Cell Washing: Microbial cell biomass produced through microbial reactor culture is harvested via a bucket centrifuge (630 rpm). The cell pellet is suspended in deionized water (and may also be resuspended in buffer, salt solution, or an appropriate surfactant) and centrifuged at 100-8000 g (RCF). This process / step is repeated 1-4 times. At each step, a known volume of specimen / sample is collected to obtain quantitative / qualitative characteristics of the microbial cell, metabolic, and other characteristic composition. After the final cell washing step, the cells are suspended in a lysis buffer (containing monobasic phosphate, dibasic phosphate, and / or water) to disrupt the cells. In one embodiment, cell washing and recovery can be performed using conventional methods, including various filtration methods, continuous or conventional centrifugation, and sedimentation. In one embodiment, the buffer used for cell lysis can be a known standard buffer. These standard buffers include phosphate, Tris, borate, citrate, acetate, glycine, or diethanolamine. In one aspect, the spent medium is maintained with the biomass without washing. (ii) Cell disruption: Microbial cells of wild-type Ogataea polymorphic yeast or recombinant Ogataea polymorphic yeast can be lysed using various methods. Examples of methods include suspending the cells in a lysis buffer and then subjecting them to physical (bead milling), chemical (acid, alkali, or hydrogen peroxide), enzymatic (degradative enzymes), and high-throughput (high-pressure homogenization or hydrodynamic cavitation) methods. These techniques are adopted based on the product and preference. For example, using recombinant Ogataea polymorphic yeast expressing myoglobin, the yeast cell suspension was passed through a Dyno Mill 1–7 times. During milling, the suspension was kept below 55°C to preserve the structure and functionality of the protein and heme groups for the next step. After cell disruption, the suspension was centrifuged at 100–8,000 RCF for 60 minutes. The suspension was then collected and stored frozen at below −10°C. (iii) Protein renaturation and purification: Final recovery and purification of myoglobin protein from cell lysates is obtained through successive steps of micro- and ultrafiltration modules with cut-off membranes ranging from 1 μm to 1 kDa.

[0136] In one aspect, the invention is a method for producing a heme-bound holoprotein. In one embodiment, the method comprises the following steps (A), (B), and (C). (A) culturing a recombinant Ogataea polymorpha strain with a homologous or heterologous nucleic acid sequence of a gene encoding the whole heme biosynthetic pathway or a rate-limiting gene operably linked to a methanol-inducible or constitutive promoter; (B) culturing the recombinant Ogataea polymorphic yeast overexpressing heme biosynthesis without adding exogenous heme to the culture medium. (C) Heme protein isolation and purification steps. In one embodiment, expression of a recombinant animal-derived heme protein is induced. In one embodiment, after culturing and inducing expression of the recombinant animal-derived heme protein, the cells are harvested and spray-dried. In one embodiment, after culturing and inducing expression of the recombinant animal-derived heme protein, the cells are harvested, lysed, and spray-dried. In one embodiment, after culturing and inducing expression of the recombinant animal-derived heme protein, the cells are harvested, lysed, their contents hydrolyzed, and spray-dried.

[0137] In one aspect, the invention is a method for producing a recombinant animal derived heme protein. In one embodiment, the method of the present invention comprises the following steps (A), (B), and (C): (A) Steps for introducing the nucleic acid construct into Ogataea polymorphic yeast. The nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding a heme protein of animal origin. (B) culturing cells of the Ogataea polymorphic yeast. This step is performed without overexpressing the heme biosynthetic pathway or supplementing the culture medium with heme molecules. (C) Isolation of animal-derived heme proteins from culture medium or yeast cells based on intracellular or extracellular expression. In one embodiment, expression of a recombinant animal-derived heme protein is induced. In one embodiment, expression of a recombinant animal-derived heme protein is cultured and induced, followed by harvesting and spray drying the cells. In one embodiment, expression of a recombinant animal-derived heme protein is cultured and induced, followed by harvesting, lysing, and spray drying the cells. In one embodiment, expression of a recombinant animal-derived heme protein is cultured and induced, followed by harvesting, lysing, and hydrolyzing the contents of the cells and spray drying the cells.

[0138] In one aspect, the method produces a heme protein comprising a holoprotein with the following percent heme loading: At least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, at least about 85%, at least about 90%, or 100%. In one embodiment, heme loading on proteins can be estimated through the proposed HPLC quantitative analysis or calorimetric adsorption method.

[0139] In one aspect, the method produces a heme protein comprising the following percentage (%) of heme-loaded holoprotein: At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 100%.

[0140] In one aspect, the method produces a heme protein comprising the following percentage (%) of heme-loaded holoprotein: About 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or 100%.

[0141] In one aspect, the present invention is a method for producing recombinant bovine myoglobin. In one embodiment, the method of the present invention comprises the following steps (A) and (B): (A) Introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Ogataea polymorphic yeast. (B) culturing the Ogataea polymorphic yeast containing the recombinant bovine myoglobin nucleic acid; This step (B) promotes expression of the recombinant myoglobin, but without overexpression of the entire heme biosynthetic pathway and without exogenous supplementation of heme to the culture medium. In one embodiment, the method of the present invention further comprises the following steps (C) and (D): (C) extracting and purifying the recombinant bovine myoglobin. (D) Further processing of all remaining fractions to produce a hydrolyzed yeast extract. In one aspect, the heme biosynthetic pathway in Ogataea pleomorpha may be modified or heme may be exogenously supplemented to the culture medium.

[0142] In one aspect, the present invention provides a method for producing a heme protein containing greater than 50% heme. In one aspect, the method comprises the following steps: (A) preparing a heme protein containing more than 50% heme; (A) Preparing a recombinant Ogataea polymorphic yeast containing a nucleic acid encoding a heme protein operably linked to a methanol-inducible promoter. (B) Culturing the recombinant Ogataea polymorphic yeast without exogenous supplementation of heme to the culture medium. (C) Extracting and purifying the heme-protein complex.

[0143] In one aspect, the invention is a method for producing a heme protein having a heme loading of: At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%.

[0144] In one aspect, the invention is a method for producing a heme protein having a heme loading of: About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%.

[0145] In one aspect, the invention is a method for producing a heme protein having a heme loading of: At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

[0146] In one aspect, the present invention is a method for producing an animal-derived heme protein having a heme load of 50% or greater. In one embodiment, the method of the present invention comprises the following steps (A), (B), and (C): (A) Step of introducing the nucleic acid construct into an Ogataea polymorphic strain (yeast). The nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding a heme protein of animal origin. (B) Culturing cells of the Ogataea polymorphic strain. This step (B) is carried out without supplementing the culture medium with exogenous heme molecules (i.e., without supplementing heme molecules from outside). (C) isolating animal-derived proteins from the culture.

[0147] In one aspect, the method produces animal-derived heme proteins having the following percent heme load: At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%.

[0148] In one aspect, the method produces animal-derived heme proteins having the following percent heme load: About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%.

[0149] In one aspect, the method produces animal-derived heme proteins having the following percent heme loading: At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%.

[0150] In one aspect, the present invention is a method for producing recombinant bovine myoglobin that contains at least 50% heme-loaded recombinant bovine myoglobin. In one embodiment, the method of the present invention comprises steps (A) and (B). (A) A step of introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Ogataea polymorphic strain (yeast) cells. (B) Cultivating the Ogataea polymorpha strain containing the recombinant bovine myoglobin nucleic acid. The step (B) is carried out without supplementing the culture medium with heme molecules to promote and express recombinant myoglobin. In one embodiment, the method of the present invention further comprises step (C). (C) A step of isolating and purifying recombinant bovine myoglobin from the culture.

[0151] In one embodiment, the method of the present invention produces recombinant bovine myoglobin that contains at least 50% heme-loaded recombinant bovine myoglobin. In one aspect, the method of the present invention produces recombinant myoglobin having the following percentage of heme-loaded recombinant bovine myoglobin: At least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or 100%.

[0152] In one aspect, the method of the present invention produces recombinant myoglobin having the following percentage of heme-loaded recombinant bovine myoglobin: About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%.

[0153] In one aspect, the method of the present invention produces recombinant myoglobin having the following percentage of heme-loaded recombinant bovine myoglobin: At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%.

[0154] In one aspect, the recombinant Ogataea polymorpha does not contain an exogenous transcriptional activator of a heme biosynthetic pathway or an exogenous component of a heme biosynthetic pathway.

[0155] In one embodiment, the heme protein is selected from the group consisting of animal-derived heme proteins and plant-derived heme proteins.

[0156] In one aspect, the animal-derived heme protein is selected from the group consisting of bovine heme protein, porcine heme protein, ovine heme protein, equine heme protein, and caprine heme protein.

[0157] In one aspect, the recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0158] In one embodiment, the nucleic acid comprises a nucleic acid sequence having 70% or more homology to the nucleic acid sequence of SEQ ID NO:1.

[0159] In one embodiment, the nucleic acid comprises a nucleic acid sequence having the following percentage of sequence identity to the nucleic acid sequence of SEQ ID NO:1: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0160] In one embodiment, the nucleic acid comprises a nucleic acid sequence having the following percentage of sequence identity to the nucleic acid sequence of SEQ ID NO:1: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0161] In one embodiment, the myoglobin has an amino acid sequence corresponding to SEQ ID NO:6. In one embodiment, the amino acid sequence has the following percentage of sequence homology to the amino acid sequence of SEQ ID NO:6: At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. In one embodiment, the amino acid sequence has the following percentage of sequence homology to the amino acid sequence of SEQ ID NO:6: At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0162] In one aspect, the invention is a food composition comprising a cultured heme protein disclosed herein or a bovine myoglobin protein disclosed herein.

[0163] In one aspect, the invention is a meat analog comprising a lysate of the cultured Ogataea pleomorpha bacteria disclosed herein.

[0164] In one aspect, the invention is a food composition comprising a cultured heme protein disclosed herein or a bovine myoglobin protein disclosed herein.

[0165] The recombinant heme protein is isolated and used to prepare / produce food compositions. In one aspect, the recombinant heme protein produced by the engineered Ogataea polymorpha fungus is used in whole, in part, or with modifications. In one aspect, the modifications include a predetermined process. Examples of predetermined processes include solubilization, precipitation, partial or complete hydrolysis, cross-linking, emulsification, texturization, cooking, extrusion, high-shear mixing, Klett cell production, and reaction (including nitrate-based curing). In one aspect, examples of meat and meat analogs produced include ground meat, sliced ​​meat, meat analogs such as chunks, cubes, and stakes, 3D-formed burgers, fillets, balls, links, finger-links, sticks, slabs, nuggets, pies, sliced ​​meats, bonbons, rings, sausages, ham, and reconstituted or coated meat-like products. Extract from Ogataea polymorpha

[0166] In one aspect, the present invention is a cell extract derived from a culture of Ogataea pleomorpha, a methylotrophic yeast that is a good source of molecules for the food and nutrition industries.

[0167] In one aspect, the Ogataea polymorpha fungus is obtained from a genetically engineered Ogataea polymorpha fungus that produces a recombinant heme protein. The recombinant heme protein is a recombinant plant heme protein, a recombinant bacterial heme protein, a recombinant fungal heme protein, or a recombinant animal heme protein. In one aspect, the recombinant protein is selected from the group consisting of hemeglobin, myoglobin, hemeglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, lychninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0168] In one embodiment, the Ogataea polymorpha extract contains the following percentages of heme: at least 0.03 % w / w, at least 0.05 % w / w, at least 0.1 % w / w, at least 0.15 % w / w, at least 0.2% w / w, at least 0.225 % w / w, at least 0.25 % w / w, at least 0.275% w / w, at least 0.30% w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6 % w / w, at least 0.7% w / w, at least 0.8 % w / w, at least 0.9 % w / w, at least 1.0% w / w, at least 1.1 % w / w, at least 1.2% w / w, at least 1.3 % w / w, at least 1.4 % w / w, at least 1.5% w / w, at least 1.6 % w / w, at least 1.7% w / w, at least 1.8 % w / w, at least 1.9% w / w, at least 2.0 % w / w, at least 2.1% w / w, at least 2.2% w / w, at least 2.3 % w / w, at least 2.4% w / w, at least 2.5 % w / w, at least 2.6 % w / w, at least 2.7% w / w, at least 2.8 % w / w, at least 2.9% w / w, at least 3.0 % w / w, at least 3.1% w / w.

[0169] In one embodiment, the Ogataea polymorpha extract contains the following percentages of heme: About 0.03% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.225% w / w, about 0.25% w / w, about 0.275% w / w, about 0.30% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1.0% w / w, about 1.1% w / w, about 1.2% w / w, about 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8 %w / w, approximately 1.9%w / w, approximately 2.0%w / w, approximately 2.1%w / w, 2.2%w / w, approximately 2.3%w / w, approximately 2.4%w / w, approximately 2.5%w / w, approximately 2.6%w / w, approximately 2.7%w / w, approximately 2.8%w / w, approximately 2.9%w / w, 3.0 %w / w, 3.1% w / w.

[0170] In one embodiment, the Ogataea polymorpha extract contains heme in the following percentage ranges: About 0.03% w / w to about 3.1% w / w, about 0.1% w / w to about 3.1% w / w, about 0.5% w / w to about 3.1% w / w, about 1% w / w to about 3.1% w / w, about 1.5% w / w to about 3.1% w / w, about 2% w / w to about 3.1% w / w, about 2.5% w / w to about 3.1% w / w, about 0.1% w / w to about 3% w / w, about 0.1% w / w to about 2.5% w / w, about 0.1% w / w to about 2% w / w, about 0.1% w / w to about 1.5% w / w, about 0.1% w / w to about 1% w / w, and about 0.1% w / w to about 0.5% w / w.

[0171] In one embodiment, the Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid and 0.2% glutathione.

[0172] In one embodiment, the Ogataea polymorpha extract contains the following percentages of glutamic acid: At least 0.8% w / w, at least 0.9% w / w, at least 1% w / w, at least 1.5% w / w, at least 2% w / w, at least 2.5% w / w, at least 3% w / w, at least 3.5% w / w, or at least 4% w / w.

[0173] In one embodiment, the Ogataea polymorpha extract contains the following percentages of glutamic acid: About 0.8% w / w, about 0.9% w / w, about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, or about 4% w / w.

[0174] In one embodiment, the Ogataea polymorpha extract contains glutamic acid in the following percentage ranges: About 0.8% w / w to about 4% w / w, about 1% w / w to about 4% w / w, about 1.5% w / w to about 4% w / w, about 2% w / w to about 4% w / w, about 2.5% w / w to about 4% w / w, about 3% w / w to about 4% w / w, about 3.5% w / w to about 4% w / w, about 0.8% w / w to about 3.5% w / w, about 0.8% w / w to about 3% w / w, about 0.8% w / w to about 2.5% w / w, about 0.8% w / w to about 2% w / w, about 0.8% w / w to about 1.5% w / w, about 0.8% w / w to about 1% w / w.

[0175] In one embodiment, the Ogataea polymorpha extract contains glutathione in the following percentages: At least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1%.

[0176] In one embodiment, the Ogataea polymorpha extract contains glutathione in the following percentages: About 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%.

[0177] In one embodiment, the Ogataea polymorpha extract contains glutathione in the following percentage ranges: About 0.2% w / w to about 1% w / w, about 0.5% w / w to about 1% w / w, about 0.7% w / w to about 1% w / w, about 0.2% w / w to about 0.5% w / w.

[0178] In one embodiment, the Ogataea polymorphic fungus extract is rich in 5' ribonucleotides. An example of a 5' ribonucleotide is 5' GMP or 5' IMP. In one embodiment, the Ogataea polymorphic fungus extract contains 0.007% or more 5' ribonucleotides. In one embodiment, the Ogataea polymorphic fungus extract contains 0.007% or more 5' GMP.

[0179] In one embodiment, the Ogataea polymorphic fungus extract contains the following percentages of 5' ribonucleotides: At least 0.005%, at least 0.006%, at least 0.007%, at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05%.

[0180] In one embodiment, the Ogataea polymorphic fungus extract contains the following percentages of 5' ribonucleotides: About 0.005%, about 0.006%, about 0.007%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%.

[0181] In one embodiment, the Ogataea polymorphic fungus extract contains 5' ribonucleotides in the following percentage ranges: About 0.005% to about 0.05%, about 0.007% to about 0.05%, about 0.01% to about 0.05%, about 0.02% to about 0.05%, about 0.03% to about 0.05%, about 0.04% to about 0.05%, about 0.007% to about 0.04%, about 0.007% to about 0.03%, about 0.007% to about 0.02%, about 0.007% to about 0.01%.

[0182] In one embodiment, the Ogataea polymorpha extract contains the following percentages of 5'GMP: At least 0.005%, at least 0.006%, at least 0.007%, at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05%.

[0183] In one embodiment, the Ogataea polymorpha extract contains the following percentages of 5'GMP: About 0.005%, about 0.006%, about 0.007%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%.

[0184] In one embodiment, the Ogataea polymorpha extract contains 5'GMP in the following percentage ranges: About 0.005% to about 0.05%, about 0.007% to about 0.05%, about 0.01% to about 0.05%, about 0.02% to about 0.05%, about 0.03% to about 0.05%, about 0.04% to about 0.05%, about 0.007% to about 0.04%, about 0.007% to about 0.03%, about 0.007% to about 0.02%, about 0.007% to about 0.01%.

[0185] In one aspect, the Ogataea polymorphic fungus extract contains the following percentages of 5'IMP: At least 0.005%, at least 0.006%, at least 0.007%, at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05%.

[0186] In one aspect, the Ogataea polymorphic fungus extract contains the following percentages of 5'IMP: About 0.005%, about 0.006%, about 0.007%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%.

[0187] In one aspect, the Ogataea polymorphic fungus extract contains 5'IMP in the following percentage ranges: About 0.005% to about 0.05%, about 0.007% to about 0.05%, about 0.01% to about 0.05%, about 0.02% to about 0.05%, about 0.03% to about 0.05%, about 0.04% to about 0.05%, about 0.007% to about 0.04%, about 0.007% to about 0.03%, about 0.007% to about 0.02%, about 0.007% to about 0.01%.

[0188] In one embodiment, the Ogataea polymorpha extract contains at least 0.03% w / w heme, 0.8% glutamic acid, and 0.2% glutathione.

[0189] In one embodiment, the Ogataea polymorpha extract contains at least 0.03% w / w heme, 0.8% w / w glutamic acid, 0.2% glutathione, and 0.007% 5' ribonucleotides (5' IMP and / or 5' GMP).

[0190] In one embodiment, the Ogataea polymorpha extract contains at least 0.03% w / w heme, 0.8% w / w glutamic acid, 0.2% glutathione, and 0.007% 5'GMP.

[0191] In one embodiment, the Ogataea polymorpha extract contains at least 0.03% w / w heme and at least 0.007% 5'GMP.

[0192] In one embodiment, the Ogataea polymorpha extract contains at least 0.8% w / w glutamic acid, 0.2% glutathione, and 0.007% 5' ribonucleotides, but no heme.

[0193] In one embodiment, the Ogataea polymorpha extract contains at least about 0.8% w / w glutamic acid and about 0.2% glutathione.

[0194] In one aspect, the Ogataea polymorphic fungus extract is rich in 5' ribonucleotides. An example of a 5' ribonucleotide is 5' GMP or 5' IMP. In one aspect, the Ogataea polymorphic fungus extract contains 0.007% 5' ribonucleotides. In one aspect, the Ogataea polymorphic fungus extract contains 0.007% 5' GMP.

[0195] In one embodiment, the Ogataea polymorpha extract comprises about 0.03% w / w heme, about 0.8% w / w glutamic acid, and about 0.2% w / w glutathione.

[0196] In one aspect, the Ogataea polymorpha extract contains at least about 0.03% w / w heme, about 0.8% w / w glutamic acid, about 0.2% w / w glutathione, and about 0.007% 5' ribonucleotides (5' IMP and / or 5' GMP).

[0197] In one embodiment, the Ogataea polymorpha extract contains about 0.03% w / w heme, about 0.8% w / w glutamic acid, about 0.2% w / w glutathione, and about 0.007% 5'GMP.

[0198] In one embodiment, the Ogataea polymorpha extract contains about 0.03% w / w heme and about 0.007% 5'GMP.

[0199] In one embodiment, the Ogataea polymorpha extract contains about 0.8% w / w glutamic acid, about 0.2% glutathione, and about 0.007% 5' ribonucleotides, but no heme.

[0200] In one aspect, the method of the present invention for obtaining Ogataea polymorphic fungi comprises: (i) culturing and growing Ogataea pleomorpha cells; (ii) optionally washing and harvesting the cell; (iii) Lysing the cells Step (iii) can be carried out by a method known in the art. Examples of such methods include autolysis, physical lysis, mechanical lysis, chemical lysis (mediated by acid or alkaline solutions), enzymatic lysis, microbial lysis, or a combination thereof.

[0201] In one embodiment, after step (i) above, the cells may be washed and harvested to produce Ogataea pleomorpha biomass.

[0202] In one embodiment, after culturing and growing Ogataea pleomorpha, the cells are harvested in the culture medium, or in a portion of the culture medium, to produce Ogataea pleomorpha.

[0203] In one embodiment, after dissolution, the biomass is spray dried to produce solid Ogataea pleomorpha. In one embodiment, after dissolution, the extract is spray dried to produce solid Ogataea pleomorpha.

[0204] In one embodiment, the temperature is kept low to obtain a heme-rich extract. This is because the instability of the heme molecule and its rapid degradation at high temperatures is avoided. In one embodiment, the temperature of the process for obtaining a heme-rich (heme-rich) Ogataea polymorphic extract is below 70°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 40°C and 70°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 45°C and 60°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 48°C and 58°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 49°C and 57°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 50°C and 55°C. If a thermal pasteurization step is required, the psychrophilic step involves heating to a temperature of 90°C or above for a period of 1 to 100 seconds.

[0205] In one embodiment, the temperature of the method of the present invention for obtaining an extract of Ogataea polymorpha is temperature independent, since the heme has previously been isolated and separated from the biomass.

[0206] In one embodiment, the temperature of the method of the present invention for obtaining an extract of Ogataea polymorpha is temperature independent, as the resulting extract is substantially free of heme.

[0207] In one aspect, the Ogataea pleomorpha biomass comprises at least 0.03% w / w heme per dry cell weight.

[0208] In one embodiment, the Ogataea polymorpha extract comprises at least 0.03% w / w heme per dry cell weight.

[0209] In one embodiment, the Ogataea pleomorpha biomass comprises about 0.03% w / w heme per dry cell weight.

[0210] In one embodiment, the Ogataea polymorpha extract contains about 0.03% w / w heme per dry cell weight.

[0211] In one aspect, to obtain an Ogataea polymorph extract having a high glutamic acid derived composition, the proteins of the Ogataea polymorph extract are hydrolyzed and the enzymatic conversion of glutamate to glutamic acid using glutaminase is further induced.

[0212] In one embodiment, to obtain Ogataea polymorpha strains with high 5' ribonucleotide content, nucleic acids are preferably hydrolyzed with nucleases or chemicals. In one embodiment, the addition of enzymes such as 5' adenylic deaminase is necessary to generate 5' IMP through the conversion of 5' AMP to 5' IMP. To achieve maximum 5' ribonucleotide content, it is important to inactivate endogenous acid, neutral, or alkaline phosphatases in Ogataea polymorpha strains. Endogenous phosphatases can hydrolyze phosphate groups from nucleosides to produce nucleotides. Nucleosides that produce nucleotides are unrelated to flavor. In one embodiment, Ogataea polymorpha strains are enriched in 5' GMP and / or 5' IMP.

[0213] In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract and / or a glutamic acid-derived 5'-ribonucleotide-rich composition is 70°C or higher. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 40°C and 70°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 45°C and 60°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 48°C and 58°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 49°C and 57°C. In one embodiment, the temperature of the process for obtaining a heme-rich Ogataea polymorphic extract is between 50°C and 55°C. If a thermal pasteurization step is required, the pycno-sterilization step involves heating at a temperature of 90°C or higher for a period of 1 to 100 seconds.

[0214] In one embodiment, the method of the present invention for obtaining a hydrolyzed Ogataea polymorpha extract is temperature independent, since heme is previously isolated and separated from the extract.

[0215] In one aspect, the method of the present invention for obtaining a hydrolyzed Ogataea polymorpha extract is temperature independent, as the resulting extract is substantially heme-free.

[0216] In one embodiment, lysis of Ogataea pleomorpha cells can be achieved in an alkaline pH range of 7-12, 7.5-10.5, 8-10, or 8.5-9.0, which not only achieves cell lysis but also allows for deactivation of endogenous acid phosphatases and maintains a viscosity low enough for proper agitation during the process.

[0217] In one embodiment, lysis of Ogataea pleomorpha at alkaline pH is grown in a lysis yeast cocktail containing the following four enzymes: proteases (proteases active in the neutral to alkaline pH range); Glucanase (active in the neutral to alkaline pH range), mannanase (mannanase active in the neutral to alkaline pH range), Chitinase (a chitinase active in the neutral to alkaline pH range). Some of the known commercially available pre-lysis enzymes are Alcalase, Esparase, Savinase, Neutrase, Kitalase, Zymolyase, Lyticase, Glusulase, and Promode, of which Alcalase, Esparase, Savinase, Neutrase, and Promode are registered trademarks. In this regard, please refer to Non-Patent Document 9 and Non-Patent Document 10.

[0218] In one embodiment, hydrolysis using an alkaline lysis mixture can be further pursued by adding other proteases (protease) and adjusting the pH of the medium to suit the activity of the selected protease. After lowering the pH to neutral or even more acidic (pH 4-7), the action of neutral or acidic proteases can be performed. In one embodiment, proteolysis can be carried out with a mixture / mixture of proteases. Commercially available mixtures / mixtures of proteases include Protease A, Protease P, ProteAX, Flavourzyme, Umamizyme, Papain, Bromelain, Protamex, Neutrase, Sumizyme BNP-L. Among these, Protease A, Flavourzyme, Protamex, Umamizyme, and Neutrase are registered trademarks.

[0219] In one embodiment, after proteolysis, the hydrolyzed extract of Ogataea polymorpha contains many small peptides (less than 1.0 kDa).

[0220] In one embodiment, after proteolysis, conversion of glutamine to glutamic acid or its salts can be used to increase the amount of glutamic acid content (known as umami). In one embodiment, this conversion can be carried out using glutaminase, such as L-glutaminase from Escherichia coli, glutaminase from Bacillus amyloliquefaciens, glutaminase from Bacillus licheniformis, protein-glutaminase (PG), or glutaminase from Chryseobacterium proteolyticum.

[0221] In one embodiment, the hydrolyzed extract of Ogataea polymorpha is further treated with a deaminase containing at least 0.8% glutamic acid.

[0222] In one embodiment, the hydrolyzed extract of Ogataea polymorpha is further treated with a deaminase containing at least 0.8% glutathione.

[0223] In one embodiment, nucleic acid hydrolysis and AMP conversion to IMP can occur after or before proteolysis. flavor precursor mixture

[0224] Flavor precursor mixtures are mixtures of compositions characterized by adding flavors, tastes, and aromas to foods that are released by cooking the mixture or by the Maillard reaction.

[0225] In one embodiment, the flavor precursor mixture comprises an extract of Ogataea polymorpha.

[0226] In one embodiment, the flavor precursor mixture comprises a hydrolyzed Ogataea polymorpha extract.

[0227] In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 30 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 25 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 20 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 15 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 10 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 5 kDa or less. In one embodiment, the hydrolyzed Ogataea polymorphic fungus extract is filtered to retain soluble molecules having a size of 3 kDa or less. In one aspect, the hydrolyzed Ogataea polymorphic extract is filtered to retain soluble molecules having a size of 2 kDa or less. In one aspect, the hydrolyzed Ogataea polymorphic extract is filtered to retain soluble molecules having a size of 1 kDa or less. In one aspect, the hydrolyzed Ogataea polymorphic extract is filtered to retain soluble molecules having a size of 0.5 kDa or less. In one aspect, the hydrolyzed Ogataea polymorphic extract is filtered to retain insoluble molecules.

[0228] In one embodiment, the Ogataea pleomorpha biomass, extract and / or hydrolyzed extract of the flavor precursor mixture comprises the following percentages of heme: at least 0.03 % w / w, at least 0.05 % w / w, at least 0.1 % w / w, at least 0.15 % w / w, at least 0.2 % w / w, at least 0.225 % w / w, at least 0.25 % w / w, at least 0.275% w / w, at least 0.30% w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6% w / w, at least 0.7% w / w, at least 0.8% w / w, at least 0.9% w / w, at least 1.0% w / w, at least 1.1% w / w, at least 1.2% w / w, at least 1.3 % w / w, at least 1.4% w / w, at least 1.5% w / w, at least 1.6% w / w, at least 1.7% w / w, at least 1.8% w / w, at least 1.9% w / w, at least 2.0% w / w, at least 2.1% w / w, at least 2.2% w / w, at least 2.3 % w / w, at least 2.4% w / w, at least 2.5% w / w, at least 2.6% w / w, at least 2.7% w / w, at least 2.8% w / w, at least 2.9% w / w, at least 3.0% w / w, at least 3.1% w / w.

[0229] In one embodiment, the Ogataea pleomorpha biomass, extract and / or hydrolyzed extract of the flavor precursor mixture comprises the following percentages of heme: About 0.03% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.225% w / w, about 0.25% w / w, about 0.275% w / w, about 0.30% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1.0% w / w, about 1.1% w / w, about 1.2% w / w, about 1.3 %w / w, about 1.4%w / w, about 1.5%w / w, about 1.6%w / w, about 1.7%w / w, about 1.8%w / w, about 1.9%w / w, about 2.0%w / w, about 2.1%w / w, about 2.2%w / w, about 2.3 %w / w, about 2.4%w / w, about 2.5%w / w, about 2.6%w / w, about 2.7%w / w, about 2.8%w / w, about 2.9%w / w, about 3.0%w / w, about 3.1%w / w.

[0230] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises at least 0.8% w / w glutamic acid. In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises at least 0.2% w / w glutathione. In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises at least 0.007% 5'GMP. In one aspect, the Ogataea polymorpha biomass, extract and / or hydrolyzed extract of the flavor precursor mixture comprises at least 0.03% w / w heme, at least 0.8% w / w glutamic acid, at least 0.2% w / w glutathione, and at least 0.007% 5'GMP.

[0231] In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises about 0.8% w / w glutamic acid. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises about 0.2% w / w glutathione. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises about 0.007% 5'GMP. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in the flavor precursor mixture comprises about 0.03% w / w heme, about 0.8% w / w glutamic acid, about 0.2% w / w glutathione, and about 0.007% 5'GMP.

[0232] In one embodiment, to obtain a flavour precursor mixture, the content of biomass, extract and / or hydrolysed extract of Ogataea pleomorpha in the mixture is at least 0.01%. In one embodiment, to obtain a flavor precursor mixture, the content of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in this mixture is in the following percentages: at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59 %, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90%.

[0233] In one embodiment, to obtain a flavor precursor mixture, the content of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in this mixture is in the following percentages: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46 %, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90%.

[0234] In one embodiment, to obtain a flavor precursor mixture, the content of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in this mixture is: Between about 1% and about 90%, between about 1% and about 80%, between about 1% and about 70%, between about 1% and about 60%, between about 1% and about 50%, between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 1% and about 10%, between about 1% and about 5%, between about 5% and about 90%, between about 10% and about 90%, between about 20% and about 90%, between about 20% and about 72%, between about 30% and about 90%, between about 40% and about 90%, between about 50% and about 90%, between about 60% and about 90%, between about 70% and about 90%, between about 80% and about 90%.

[0235] In one aspect, the flavor precursor mixture of Ogataea pleomorpha biomass, extract, and / or hydrolyzed extract comprises monosaccharides, such as pentose and hexose, and a reducing sugar selected from the group consisting of rhamnose, arabinose, ribose, dextrose, glucose, glucosamine, glucose, fructose, steviol, and synthetic sweeteners.

[0236] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha comprises the following percentages of monosaccharides: at least 0.1 % w / w, at least 0.2% w / w, at least 0.3 % w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6 % w / w, at least 0.7% w / w, at least 0.8% w / w, at least 0.9 % w / w, at least 1 % w / w, at least 1.5% w / w, at least 2% w / w, at least 2.5% w / w, at least 3 % w / w, at least 4% w / w, at least 5% w / w, at least 6% w / w, at least 7 % w / w, at least 8 % w / w, at least 9 % w / w, at least 10% w / w, at least 11 % w / w, at least 12% w / w, at least 13 % w / w, at least 14 % w / w, at least 15% w / w, at least 20 % w / w, at least 25 % w / w, at least 30 % w / w, or at least 35% w / w.

[0237] In one embodiment, the flavour precursor mixture of biomass, extract and / or hydrolysed extract of Ogataea pleomorpha comprises the following percentages of monosaccharides: About 0.1% w / w, about 0.2% w / w, about 0.3% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 20% w / w, about 25% w / w, about 30 % w / w, or about 35% w / w.

[0238] In one embodiment, the content of monosaccharides in the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha is as follows: Between about 0.1% and about 35%, between about 0.1% and about 30%, between about 0.1% and about 25%, between about 0.1% and about 20%, between about 0.1% and about 15%, between about 0.1% and about 10%, between about 0.1% and about 5%, between about 0.1% and about 1%, between about 1% and about 35%, between about 5% and about 35%, between about 10% and about 35%, between about 15% and about 35%, between about 20% and about 35%, between about 25% and about 35%, between about 30% and about 35%.

[0239] In one embodiment, the flavor precursor mixture of the Ogataea pleomorpha biomass, extract and / or hydrolyzed extract comprises disaccharides, examples of which are sucrose, lactose, and maltose.

[0240] In one embodiment, the flavor precursor mixture of the biomass, extract, and / or hydrolyzed extract of Ogataea polymorpha comprises amino acids. Examples of amino acids include alanine, alkynine, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamine, and mixtures thereof. In one embodiment, the flavor precursor mixture comprises di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nano-, and decapeptides. In one embodiment, the flavor precursor mixture comprises oligopeptides. In one embodiment, the flavor precursor mixture of the biomass, extract, and / or hydrolyzed extract of Ogataea polymorpha comprises glutathione.

[0241] In one aspect, the biomass, extract and / or hydrolyzed extracted flavor precursor biomass contains at least 0.4% w / w histidine.

[0242] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha contains the following percentages of histidine: At least 0.4% w / w, at least 0.5% w / w, at least 0.6% w / w, at least 0.7% w / w, at least 0.8% w / w, at least 0.9% w / w, at least 1% w / w, at least 1.5% w / w, at least 2% w / w, at least 2.5% w / w, or at least 3% w / w.

[0243] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha contains the following percentages of histidine: About 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, or about 3% w / w.

[0244] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha contains the following percentages of histidine: Between about 0.4% and about 3%, between about 0.5% and about 3%, between about 1% and about 3%, between about 1.5% and about 3%, between about 2% and about 3%, between about 2.5% and about 3%, between about 0.4% and about 2.5%, between about 0.4% and about 2%, between about 0.4% and about 1.5%, between about 0.4% and about 1%.

[0245] In one embodiment, the flavour precursor mixture of biomass, extract and / or hydrolysed extract of Ogataea pleomorpha comprises at least 15% w / w amino acids.

[0246] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha comprises the following percentages of amino acids: at least 0.1 % w / w, at least 0.2% w / w, at least 0.3 % w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6 % w / w, at least 0.7% w / w, at least 0.8 % w / w, at least 0.9% w / w, at least 1 % w / w, at least 1.5% w / w, at least 2 % w / w, at least 2.5% w / w, at least 3 % w / w, at least 4 % w / w, at least 5 % w / w, at least 6 % w / w, at least 7 % w / w, at least 8 % w / w, at least 9 % w / w, at least 10 % w / w, at least 11 % w / w, at least 12 % w / w, at least 13 % w / w, at least 14 % w / w, or at least 15 % w / w.

[0247] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha comprises the following percentages of amino acids: About 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, or about 15% w / w.

[0248] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha contains the following percentages of histidine: Between about 0.1% and about 15%, between about 0.5% and about 15%, between about 1% and about 15%, between about 5% and about 15%, between about 10% and about 15%, between about 0.1% and about 10%, between about 0.1% and about 5%, between about 0.1% and about 4%, between about 0.1% and about 3%, between about 0.1% and about 1%.

[0249] In one embodiment, the flavor precursor mixture of Ogataea pleomorpha biomass, extract and / or hydrolyzed extract comprises vitamins. Examples of vitamins include vitamin A, vitamin C, vitamin D, vitamin E, vitamin B, vitamin K, and mixtures thereof. In one embodiment, the vitamin is a B complex vitamin. In one embodiment, the vitamin is vitamin B1.

[0250] In one embodiment, the flavor precursor mixture of Ogataea pleomorpha biomass, extract and / or hydrolyzed extract comprises extracts from Saccharomyces cerevisiae, Cyberlindnera jadinii, seaweed kelp and / or mushroom.

[0251] In one embodiment, the flavor precursor mixture of biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha further comprises a fat. An example of a fat is a vegetable oil or a fatty acid. In one embodiment, the fatty acid is a saturated fatty acid. In one embodiment, the fatty acid is an unsaturated fatty acid. In one embodiment, the fatty acid is a mixture of saturated and unsaturated fatty acids. In one embodiment, the fat is an edible vegetable oil. Examples of edible vegetable oils are olive oil, canola oil, sunflower oil, soybean oil, safflower oil, chia oil, rapeseed oil, peanut oil, linoleic oil, coconut oil, palm oil, and mixtures thereof.

[0252] In one embodiment, the vegetable fat is a saturated fatty acid. Examples of saturated fatty acids are lauric acid, myristic acid, permic acid, and stearic acid.

[0253] In one embodiment, the vegetable fat is a monounsaturated fatty acid. An example of a monounsaturated fatty acid is: Myristoleic acid, Palmitoleic acid, cis-Vaccenic acid, Vaccenic acid Paullinic acid, Oleic acid Elaidic acid, 11-Eicosenoic acid, Erucic acid, Brassidic acid, Nervonic acid, Sapienic acid Gadoleic acid, Petroselinic acid,

[0254] In one embodiment, the vegetable fat is a free polyunsaturated fatty acid (PUFA). An example of a free polyunsaturated fatty acid is: Hexadecatrienoic acid (HTA), Alpha-linolenic acid (ALA), Stearidonic acid (SDA), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA), Eicosapentaenoic acid (EPA, Timnodonic acid), Heneicosapentaenoic acid (HPA), Dosapentaenoic acid (DPA, Clupanodonic acid), Docosahexaenoic acid (DHA, Cervonic acid), Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid), Linoleic acid (LA), Gamma-linolenic acid (GLA), Eicosadienoic acid), Dihomo-gamma-linolenic acid (DGLA), Arachidonic acid (AA), Docosadienoic acid), Adrenic acid (AdA), Docosapentaenoic acid (DPA), Tetracosatetraenoic acid, Tetracosapentaenoic acid.

[0255] In one embodiment, the fat is a mixture of saturated fatty acids, monosaturated fatty acids, polyunsaturated fats (PUFAs).

[0256] In one embodiment, the fat is a mixture of vegetable oil and cultured fat, which is a culture of adipocytes.

[0257] In one embodiment, the flavor precursor mixture of Ogataea polymorpha biomass, extract, and / or hydrolyzed extract further comprises a polypeptide, protein, or protein fragment (hereinafter simply referred to as "protein fragment"). In one embodiment, the protein fragment is of plant origin. In one embodiment, the protein fragment is of fungal origin. In one embodiment, the protein fragment is of animal origin. In one embodiment, the protein fragment is a recombinant protein. In one embodiment, the protein fragment is a recombinant hemoprotein. In one embodiment, the protein fragment is a recombinant animal hemoprotein.

[0258] In one embodiment, the flavor precursor mixture of Ogataea polymorpha biomass, extract, and / or hydrolyzed extract further comprises a carrier / carrying material. The carrier / carrying material is a polysaccharide. Examples of polysaccharides include starch, modified starch, carrageenan, acacia gum, gum arabic, carboxymethylcellulose, chitosan, and mixtures thereof. Another carrier / carrying material is Saccharomyces cerevisiae extract and salts thereof. Maillard Reaction Products (MRP)

[0259] In one aspect, the invention is a method for producing a flavor composition derived from a flavor precursor mixture.

[0260] In one embodiment, the method of the present invention involves culturing a flavor precursor mixture under predetermined conditions of temperature and moisture content to obtain a flavor composition.

[0261] In one embodiment, the flavor precursor mixture is added to a food matrix and the flavor is developed during cooking.

[0262] The Maillard Reaction (MR) process is a well-known process, and Maillard reaction products (MRPs) are products derived from MR. MRPs are flavor compositions that can be added to foods. When an MRP is combined with another MRP (obtained using other types of amino acids or vegetable protein hydrolysates), the flavor is improved.

[0263] In one embodiment, a flavor precursor mixture of an extract and / or hydrolyzed extract of Ogataea pleomorpha is thermally reacted to obtain MRPs derived from Ogataea pleomorpha.

[0264] In one embodiment, the temperature of the MR operation is between about 85°C and 200°C, more preferably between about 90°C and 160°C, and most preferably between about 95°C and 140°C. In one embodiment, the heating time of the MR operation is between about 10 minutes and 60 minutes, between about 60 minutes and 120 minutes, between about 120 minutes and 900 minutes, between about 180 minutes and 720 minutes, between about 240 minutes and 600 minutes, or between about 300 minutes and 540 minutes. In one embodiment, the pressure of the MR operation is between about 0.1 bar and 10 bar, between about 0.25 bar and 7.5 bar, between about 0.4 bar and 5 bar, between about 0.5 bar and 3 bar, between about 0.75 bar and 2.5 bar, or between about 1 bar and 2 bar. In one embodiment, the pH of the MR operation is about 0.5-11, about 1-10.5, about 2-10, about 3-9, about 4-8.5, or about 5-8.

[0265] In one embodiment, the temperature of the MR operation is between 85°C and 200°C, more preferably between 90°C and 160°C, and most preferably between 95°C and 140°C. In one embodiment, the heating time of the MR operation is between 10 minutes and 60 minutes, between 60 minutes and 1200 minutes, between 120 minutes and 900 minutes, between 180 minutes and 720 minutes, between 240 minutes and 600 minutes, or between 300 minutes and 540 minutes. In one embodiment, the pressure of the MR operation is between 0.1 bar and 10 bar, between 0.25 bar and 7.5 bar, between 0.4 bar and 5 bar, between 0.5 bar and 3 bar, between 0.75 bar and 2.5 bar, or between 1 bar and 2 bar. In one embodiment, the pH of the MR operation is 0.5-11, or 1-10.5, 2-10.3-9, 4-8.5, or 5-8.

[0266] In one embodiment, the flavor composition (MRP) obtained after the Maillard reaction is dried on or mixed with a carrier (e.g., maltodextrin) by known methods (e.g., spray drying, vacuum drying, freeze drying, etc.). In one embodiment, other flavorings can be added to the MRP. In one embodiment, the MRP derived from the Ogataea polymorph is preferably supplemented with a spiro-1,2,4-trithiolane composition. The spiro composition is 3,5-bis(2'-methyltetrahydrofuran-3')spiro-1,2,4-trithiolane. In one embodiment, the amount of the spiro-1,2,4-trithiolane composition is 1% to 50% of the spiro composition per 100 g of the MRP composition. In one embodiment, the amount of the spiro-1,2,4-trithiolane composition is between about 5% and about 40%, between about 10% and about 35%, or between about 15% and about 30% of the spiro composition per 100 g of the MRP composition.

[0267] In one aspect, the MRPs derived from Ogataea polymorpha include other sources of MSG (e.g., isolated from Corynebacterium glutamicum or other natural sources). In one aspect, the MRPs derived from Ogataea polymorpha include the addition of other sources of IMP and / or GMP. In one aspect, the MRPs derived from Ogataea polymorpha include the addition of mushroom extract and / or other sources of nutrients or flavor peptides derived from other natural sources. Food products

[0268] In one aspect, the present invention is a food product containing a cell component or cell fraction of Ogataea pleomorpha.

[0269] In one aspect, the present invention is a food product containing an extract of Ogataea polymorpha.

[0270] In one aspect, the invention is a food product containing hydrolyzed Ogataea pleomorpha.

[0271] In one aspect, the invention is a food product containing a flavor precursor mixture comprising a biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha.

[0272] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the food product contains the following percentages of heme: Contains at least 0.03% w / w, at least 0.05% w / w, at least 0.1% w / w, at least 0.15% w / w, at least 0.2% w / w, at least 0.225% w / w, at least 0.25% w / w, at least 0.275% w / w, at least 0.30% w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6% w / w, at least 0.7% w / w, at least 0.8% w / w, at least 0.9% w / w, at least 1.0% w / w, at least 1.1% w / w, at least 1.2% w / w. at least 1.3 % w / w, at least 1.4 % w / w, at least 1.5 % w / w, at least 1.6 % w / w, at least 1.7% w / w, at least 1.8% w / w, at least 1.9 % w / w, at least 2.0% w / w, at least 2.1% w / w, at least 2.2% w / w, at least 2.3 % w / w, at least 2.4 % w / w, at least 2.5 % w / w, at least 2.6 % w / w, at least 2.7% w / w, at least 2.8% w / w, at least 2.9% w / w, at least 3.0 % w / w, or at least 3.1% w / w.

[0273] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the food product contains the following percentages of heme: Including about 0.03% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.225% w / w, about 0.25% w / w, about 0.275% w / w, about 0.30% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1.0% w / w, about 1.1% w / w, and about 1.2% w / w. About 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8% w / w, about 1.9% w / w, about 2.0% w / w, about 2.1% w / w, about 2.2% w / w, about 2.3% w / w, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, about 2.7% w / w, about 2.8% w / w, about 2.9% w / w, about 3.0% w / w, or about 3.1% w / w.

[0274] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains at least 0.8% w / w glutamic acid. In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains at least 0.2% w / w glutathione. In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains at least 0.007% 5'GMP. In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains at least 0.03% w / w heme, 0.8% w / w glutamic acid, 0.2% w / w glutathione, and 0.007% 5'GMP.

[0275] In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains about 0.8% w / w glutamic acid. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains about 0.2% w / w glutathione. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains about 0.007% 5'GMP. In one aspect, the biomass, extract, and / or hydrolyzed extract of Ogataea pleomorpha in a food product contains 0.03% w / w heme, 0.8% w / w glutamic acid, 0.2% w / w glutathione, and 0.007% 5'GMP.

[0276] In one aspect, the present invention is a food product comprising MRPs derived from Ogataea pleomorpha.

[0277] In one embodiment, the food product of the present invention is a solid or liquid (drink). The food product of the present invention is a seasoning, baked goods, protein supplement, vitamin and mineral supplement, meat product, or meat substitute (meat analog). The meat product or meat substitute can be a meat sausage replica, a meat pate replica, a ground meat replica, or a muscle replica. The seasoning is a sauce. The meat substitute does not contain anything directly derived from an animal. The meat substitute may contain a small amount of direct animal derivatives. A product derived directly from an animal is defined as a product isolated in whole or in part from an animal, isolated protein, isolated tissue, isolated muscle, or isolated cell. Recombinant proteins produced in non-animal hosts are not considered directly derived animal products. Recombinant nucleic acids encoding animal proteins are not considered directly derived animal products.

[0278] A sauce is a liquid or semi-liquid preparation that accompanies food as a condiment. The composition of the sauces of the present invention will vary depending on the intended use.

[0279] Tofu is a traditional food made from soybean protein. Its manufacturing process generally consists of preparing a milk by soaking and grinding the soybeans, boiling the mixture, and filtering out any remaining particles. The soybeans are then coagulated and pressed into a solid form. Thus, tofu is considered a plant-based protein-rich food.

[0280] Tempeh is a traditional Indonesian protein-rich food made from fermented and cooked soybeans (e.g., similar to rice, millet, and Rhizopus oligsporus cultures). In this regard, see Non-Patent Document 11.

[0281] Seitan is a traditional food product made from wheat flour. It is produced by activating and washing the starch. Alternatively, seitan can be obtained from commercially available essential wheat crust and produced by adding water to the hydrate. This may be followed by a heat treatment to cook the product. The resulting product has an inexpensive texture and can be added as an ingredient to a variety of dishes.

[0282] Patent Document 1 discloses a plant-based meat analog. This meat analog is produced by sequentially mixing methylcellulose into an ice / water mixture, then mixing modified gluten and highly soluble vegetable protein in water, gently heating to gel, and adding oil dropwise to form an emulsion phase, producing modified starch. Patent Document 2 discloses a method for making vegetable protein meat analogs. This method involves sequentially mixing methylcellulose into an ice / water mixture to form a cream, and then mixing it with modified gluten. The vegetable protein has high solubility in water, but gels upon gentle heat treatment. It also discloses oils for making emulsion bases, and modified food starches and flavoring ingredients for forming flavored emulsion bases. Patent Document 3 discloses a meat analog containing vegetable protein, a sugar and sulfur composition, and a heme-containing protein. Patent Document 4 discloses a meat replica containing muscle, fat, and connective tissue replicas. The muscle replica consists of a replica of muscle formed by asymmetric fibers, such as spun or extruded fibers. The fat replica contains a gel with fat droplets suspended therein. The fat is vegetable oil and the gel is vegetable protein. The connective tissue replica contains approximately 50% protein by total weight and approximately 50% liquid weight, with low fat and polysaccharide components. The protein is prolamin.

[0283] In one aspect, compositions derived from Ogataea polymorpha can be used as dietary supplements to address deficiencies in minerals (e.g., iron), amino acids, vitamins, and nucleotides in human food or animal diets.

[0284] Dietary supplements refer to products intended for consumption with food that contain "dietary ingredients" intended to supplement the diet. Dietary supplements containing compositions derived from Ogataea pleomorpha come in many forms, including pills, tablets, capsules, gummies, softgels, liquids, and powders.

[0285] In one aspect, a dietary supplement comprising a composition derived from Ogataea pleomorpha can be used as a dietary supplement composition for treating anemia and other iron deficiency-related disorders. The dietary supplement may comprise dead cells of Ogataea pleomorpha, whether the cells are live or dead. The dietary supplement may comprise lysed cells of Ogataea pleomorpha. The dietary supplement may comprise a solution obtained from filtration of a hydrolyzed extract of Ogataea pleomorpha. The dietary supplement may comprise an insoluble fraction obtained from filtration of a hydrolyzed extract of Ogataea pleomorpha.

[0286] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the dietary supplement comprises the following percentages of heme: Contains at least 0.03% w / w, at least 0.05% w / w, at least 0.1% w / w, at least 0.15% w / w, at least 0.2% w / w, at least 0.225% w / w, at least 0.25% w / w, at least 0.275% w / w, at least 0.30% w / w, at least 0.4% w / w, at least 0.5% w / w, at least 0.6% w / w, at least 0.7% w / w, at least 0.8% w / w, at least 0.9% w / w, at least 1.0% w / w, at least 1.1% w / w, at least 1.2% w / w. at least 1.3 % w / w, at least 1.4% w / w, at least 1.5% w / w, at least 1.6% w / w, at least 1.7% w / w, at least 1.8 % w / w, at least 1.9% w / w, at least 2.0% w / w, at least 2.1% w / w, at least 2.2% w / w, at least 2.3 % w / w, at least 2.4% w / w, at least 2.5 % w / w, at least 2.6% w / w, at least 2.7% w / w, at least 2.8 % w / w, at least 2.9 % w / w, at least 3.0% w / w, or at least 3.1% w / w.

[0287] In one aspect, the biomass, extract and / or hydrolyzed extract of Ogataea pleomorpha in the dietary supplement comprises the following percentages of heme: Including about 0.03% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.225% w / w, about 0.25% w / w, about 0.275% w / w, about 0.30% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1.0% w / w, about 1.1% w / w, and about 1.2% w / w. About 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8% w / w, about 1.9% w / w, about 2.0% w / w, about 2.1% w / w, about 2.2% w / w, about 2.3% w / w, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, about 2.7% w / w, about 2.8% w / w, about 2.9% w / w, about 3.0% w / w, or about 3.1% w / w.

[0288] In one embodiment, the composition derived from Ogataea pleomorpha may be spread (coated) on the surface of a food product. In one embodiment, the composition derived from Ogataea pleomorpha may be solubilized or suspended and then injected or mixed into the food product. In one embodiment, a spray-dried composition derived from Ogataea pleomorpha may be sprinkled on the food product. Experimental Example Experimental example 1: Optimization of medium for growth of Ogataea polymorpha and improvement of heme biosynthesis

[0289] 1.1 Carbon and Nitrogen Source Selection: To determine a cost-effective and commercially viable medium for growth and heme biosynthesis of Ogataea polymorpha using yeast, a one-factor screening and optimization was conducted using various carbon and nitrogen sources. These experiments were conducted in batch mode in 500 mL Erlenmeyer shake flasks with a 100 mL working volume. Carbon sources tested were glucose, fructose, pure glycerol, and crude glycerol. Nitrogen sources tested were synthetic medium (SYN6), corn steep liquor, and corn protein hydrolysate. After optimization, the medium components and operating parameters were validated in a 150 L bioreactor in fed-batch mode fermentation.

[0290] 1.2 Fermentation conditions: YPD agar plates (2% glucose) were inoculated with frozen cells from a glycerol cell bank culture and grown for 45-65 hours at 30-37°C. Seed cultures were inoculated from fresh YPD agar plates into YPD medium (2% w / v glucose) and grown overnight (20-25 hours, d=2.5 cm, agitation speed 180 rpm, temperature 30-37°C).

[0291] In a bioreactor, cultivation was initiated in batch mode with a carbon (glycerol) concentration of 2% w / v. Additional carbon (glycerol) was continuously added in fed-batch mode. The conditions were as follows: duration 35–40 h, seed feed rate 2–6 g / h. Medium seed, aeration, and centrifugation speed (rpm) were adjusted to maintain dissolved oxygen and a DO2 between 20% and 40% at maximum agitation speed. During fermentation, pH was set and maintained at 4.5–5.0. The modified medium consisted of 12.5% ​​(v / v) ammonia solution and 28% (v / v) phosphoric acid solution. Antifoam (10% (w / v) PEG 6000) was added appropriately using an AF controller. After 40 h of cultivation, 0.5% v / v methanol was added as an inducer of oxidative stress, and the culture was continued for another 24 h, after which the culture was harvested.

[0292] 1.3 Cell lysis and heme extraction: Cells were washed 3–4 times using sequential centrifugation and resuspension to remove insoluble deposits on the cell surface, extracellular deposits, or medium components. After the final wash, cells were resuspended in 20 mM NaOH PBS buffer and disrupted. Either a Dyno mill or a high-pressure homogenizer and / or sonication was used for cell lysis / disruption. After cell disruption, the suspension was centrifuged to remove insoluble particles, and the supernatant was stored at −20°C for later use. Heme was extracted with 4 volumes of acidic acetone, incubated at −20°C for 20 minutes, centrifuged, and the sample was diluted 10x with acidic acetonitrile for quantitative analysis using HPLC.

[0293] 1.4 Quantitative analysis of heme: An HPLC method was developed using a gradient flow of (A) acetonitrile and (B) 0.1% acetic acid water as the mobile phase under the following conditions: Stationary phase: Luna 5uM, C18(2) 100A, 250x4.6 mm column. Flow rate: 0.5mL / min. Column pressure: 50-60 Kgf / cmxcm. Detector: SPD-M20A diode string detector. Wavelength: 406nm. Hemin stock: Dissolve 41 mg of hemin (2.5 mM) in 25 mL of 20 mM NaOH in PBS. Hemin working stock: Dilute the hemin stock 50 times (50 μM) using acidic acetonitrile (80% acetonitrile containing 20% ​​1.6 M HCl). A pink or red suspension (pH < 5) results. HPLC Standards: Further samples were prepared using a 50 μM working stock by diluting to the required concentration using acidified acetonitrile.

[0294] 1.5 Results: Figure 1A shows the heme yield (per g of cell dry weight) obtained from Ogataea polymorpha cultured on various carbon sources with or without methanol induction. Due to its reducing properties, glucose is the best carbon source for the growth and development of any microorganism. Consequently, the highest heme yield was 0.39 mg / g CDW (0.039% w / w) and the highest cell dry weight (CDL) was 43.12 g / L. The heme yield in pure glycerol with methanol induction performed after 24 hours was observed to be the highest at 0.53 mg / g CDW (0.053% w / w). Further experiments were performed using glycerol as the preferred carbon source.

[0295] Figure 1B shows heme production as a function of nitrogen source. When synthetic medium (SYN 6) was replaced with a complex medium containing corn protein hydrolysate powder, the heme yield increased fourfold (15 vs. 60 mg / L) (0.053 vs. 0.225% w / v). With 2% w / v CPH powder, a yield of 2.25 mg heme per gram of cell dry weight was observed.

[0296] Figure 1C shows the heme quantification analysis for the scale-up of the experimental process to a volume of 150 L. Optimal media parameters and process conditions were determined in flask bench-scale experiments, and a maximum heme yield of 30 mg / g of cell dry weight (3% w / w) was observed.

[0297] 1.6 Nutritional Summary: Samples from two different batches were evaluated using various techniques (e.g., standard ISO, AOAC, ELISA, spectrophotometry, gas chromatography (GC), high-performance liquid chromatography (HPLC), and mass spectrophotometry (MS)) for various factors (microbiological parameters, heavy metals, carbohydrates, total protein, dietary fiber, sugars, vitamins, minerals, amino acids, carcinogens, and aromatic volatiles) that may affect the nutritional value of the product as listed in Table 1. Example 2. Genetically modified Ogataea polymorpha expressing recombinant myoglobin: 2.1 Selection, codon optimization, and synthesis of myoglobin genes

[0298] The myoglobin (MG) gene from Bos taurus was codon-optimized (SEQ ID NO:2) for expression in Ogataea polymorpha and synthesized at Thermo Fisher Science. Gene optimization increases the chances of efficient expression in the target host and also facilitates cloning by removing unnecessary restriction sites. 2.2 Host transformation and selection

[0299] The Ogataea polymorphic fungi used were RB11 (ura3) and ALU3 (adel, leu2, ura3); Obtained from Artes Biotechnology, GmbH.

[0300] pFPMT121 and its derivatives were used as circular plasmids for the transformation of strains RB11 and ALU3. B14 (Figure 5) and pFPMT121 derivatives are plasmids that do not contain antibiotic resistance genes and were used for the transformation of Ogataea polymorpha RB11 in this study.

[0301] For the expression construction of plasmid B14-BtMG (Figure 6), plasmid DNA provided by Thermo Fisher Scientific / Gene Technologies (Figure 7) was digested with the restriction enzymes EcoRI and BamHI to obtain a 480-bp gene (myoglobin) fragment. This fragment contains the optimized nucleotide sequence encoding bovine myoglobin (BtMG). After restriction enzyme electrophoresis, the fragment was purified from an agarose gel. The vector plasmid B14 was digested with EcoRI and BamHI, and the linearized plasmid was gel-purified. Annealing of the EcoRI fragment to the promoter element (FMDp) of the heterologous gene and ligation of the BamHI site to the terminator element (MOXt) of the heterologous gene were performed.

[0302] Strains RB11 and ALU3 of Ogataea polymorpha were transformed using a yeast electroporation protocol. 36RB11 and 72ALU3 transformants were screened using minimal (selective) or complex (non-selective) media to achieve stable integration of the plasmid. The stain-free imaging technique utilizes polyacrylamide gels containing a unique trihalo compound, which undergoes short-term photoactivation to directly fluoresce proteins in the gel. This allows immediate visualization of proteins at any time during electrophoresis and Western blotting. This trihalo compound is covalently linked to tryptophan residues, which enhance their fluorescence when exposed to ultraviolet light, enabling detection of as little as 10–25 ng of protein.

[0303] The myoglobin expression level was more than 10-fold higher in the RB11 strain (10-16%) than in the ALU3 strain (3-6%). The sample pool RB11 / b14-BtMB#35 produced extremely high expression, approximately 16.3% of total protein, as shown in lane 12 of Figure 6. After successful construction of the recombinant plasmid and transformation of Ogataea polymorpha, the plasmid and genome of the transformant were sequenced. The resulting polynucleotides were translated into open reading frames using the Expasy Tool (ExPAsy), and the resulting full-length 3'-5' ORF was compared with the bovine myoglobin sequence using the CLUSTAL multi-sequence alignment (MSA) tool. The sequence of the myoglobin gene integrated into the Ogataea polymorpha chromosome was observed to have 100% similarity to the bovine myoglobin gene (Figure 7). 2.3 Production of recombinant myoglobin protein in a benchtop biological incubator

[0304] A producer strain of Ogataea polymorpha was selected from 36 sample pools, RB11 / b14-BtMB#35, and cultured in a 3L bioreactor for protein production and further characterization. The strain was grown in fed-batch mode with an initial glycerol feed of 2% and derepressed by feeding 2-6 g / L of glycerol per hour for 48 hours. Once the culture reached steady state, protein synthesis was induced by adding 1% methanol and grown for 24 hours.

[0305] The cell biomass was harvested and lysed using a bead mill to obtain a lysate containing myoglobin protein, which was subjected to SDS-PAGE and quantitatively analyzed by densitometric signal.

[0306] Sequence analysis confirmed that the expression of the 154-amino acid polypeptide chain was similar to that of bovine myoglobin. Densitometric analysis showed that Ogataea polymorpha RB11 / BtMG produced 1.6 g / L of myoglobin (approximately 10-15% of the total protein), with a conversion yield of 20 mg / g of cell dry weight. Furthermore, heme loading information is important for determining protein functionality. Therefore, the heme quantification assay was optimized using a heme assay kit (Sigma-Aldrich Heme Assay Kit for 250 Colorimeter Tests (sigmaaldorich.com)). This colorimetric assay was performed using commercially available myoglobin (left) (animal-derived) and Moolec myoglobin (right), resulting in concentrations of 200 μM (8.6 mg / mL) and 30 μM (1.6 mg / mL), respectively. 2.4 Production of recombinant myoglobin protein in a pre-production scale bioreactor (10 L)

[0307] Production was initiated in a Sartorius stainless steel fermentor (C-DCU 15-L). It was performed as a batch with 2% glycerol as the sole carbon source. The fed-batch mode of fermentation was initiated 12 hours later, when the glycerol concentration in the production medium reached 10 g / L or less. Linear glycerol feeding was performed at a feed rate of 2–6 g / L per hour for 12–50 hours. The medium feed rate and dissolved oxygen (DO) concentration in the reactor were adjusted to maintain a DO level of 20–40% throughout the cultivation period at maximum agitation speed. The pH was set and maintained at 4.8 throughout the fermentation. The modified medium consisted of a 20% v / v ammonia solution and a 20% v / v phosphoric acid solution. Antifoam (10% w / v Structol J 673) was added appropriately using an AF controller. After 50 hours of cultivation, methanol induction was performed by adding methanol in a batch mode. The execution procedure is as follows: At the start and after 5 hours: add 1.0% v / v methanol sol, respectively. After 8, 11, 14, and 17 hours: Add 0.5% v / v methanol sol at each time point (Figure 8). 2.5 Separation and purification of myoglobin from the harvested cell biomass

[0308] The resulting microbial cell biomass was harvested by bucket centrifugation. The cell pellet was suspended in softened water and further centrifuged. This step was performed three or more times. At each step, a known volume of sample was collected and quantitative and qualitative analysis of the microbial cells, metabolic products, and other characteristic composition characteristics were performed. After a final washing step, the cells were resuspended in lysis buffer for cell disruption.

[0309] Microbial cells were lysed by passing the suspension from step 1 through a Dyno Mill three times. During milling, the suspension was warmed to 30°C. After cell disruption, the suspension was centrifuged at 6362 RCF for 60 minutes. The supernatant was then collected and frozen at -20°C.

[0310] Here, the expressed myoglobin is intracellular, and therefore the microbial biomass harvested after cultivation was processed through subsequent steps of separation and purification. 2.5.1 Cell washing

[0311] The resulting microbial cell biomass was harvested using a bucket centrifuge (630 RS). The cell pellet was suspended in soft water and further centrifuged at 6362 RCF. This process was repeated three more times. At each step, a known volume of sample was collected and quantitative / qualitative analysis of the microbial cells, metabolic products, and other characteristic compositional characteristics was performed. After a final washing step, the cells were resuspended in lysis buffer and disrupted. 2.5.2 Quantitative Analysis of Microbial Growth

[0312] 5 mL of each sample was weighed and filled with approximately 45 mL of deionized water. The suspension was centrifuged for 10 minutes at 3333 g and for 5 minutes at 5000 g. The pellets were transferred to a glass bottle of the specified weight. The mixture was then incubated overnight at 93°C and the following morning at 105°C for 5 hours. Finally, the dry weight was calculated in g / kg. 2.5.3 Cell disruption

[0313] Microbial cells were lysed by passing the suspension from step 1 through a Dyno Mill three times. During milling, the suspension was warmed to 30°C. After cell disruption, the suspension was centrifuged at 6362 RCF for 60 minutes. The suspension was then collected and frozen at -20°C. 2.5.4 Protein Recovery and Purification

[0314] The cell lysate was subjected to microfiltration and ultrafiltration processes by passing it through membranes with different cutoffs ranging from 5 kDa to 800 kDa. 2.5.5 Qualitative analysis of myoglobin

[0315] Samples collected during methanol induction and downstream processing were quantitatively analyzed for myoglobin by SDS-PAGE. Sequence analysis confirmed that the expression of the 154 amino acid polypeptide chain was similar to bovine myoglobin. Densitometry analysis revealed that Ogataea polymorpha RB11 / BtMG produced 1.6 g / L of myoglobin (approximately 10-15% of the total protein), with a conversion yield of 20 mg / g of cell dry weight.

[0316] The protein banding pattern by SDS PAGE analysis (Figure 8) showed a characteristic 17 kDa appearance after methanol induction (lane T7) that persisted until the end of induction (lane T end). Similarly, samples obtained at the end of each downstream process step were qualitatively analyzed for myoglobin protein.

[0317] Western blot analysis (Figure 9) revealed a myoglobin-specific band of approximately 17 kDa in the final sample. 2.5.6 Quantitative analysis of heme

[0318] Furthermore, quantitative analysis of recombinant myoglobin produced by the engineered Ogataea polymorphic strains revealed that heme loading was essential for determining protein functionality. Therefore, a heme quantitative analysis assay was optimized using the previously described heme assay kit.

[0319] The procedure for the heme assay is as follows. (A) According to the manufacturer's instructions, 50 μl of purified myoglobin was mixed with 200 μl of reagent and incubated at room temperature for 5 minutes. (B) After incubation, the absorbance of the specimen was measured using a spectrophotometer and recorded at 400 nm. (C) Simultaneously, a linear standard graph was generated using the heme calibrator at varying dilutions. According to the instructions above, this heme calibrator corresponds to 62.5 μM heme with a linear detection range of 0.6-125 μM.

[0320] The optical density reading was 0.148, which corresponds to 300.95 μM heme, which is equivalent to 501.58 μM myoglobin with a 60% heme load. Table 2

[0321] Furthermore, samples from the 5 kDa filter device were quantitatively analyzed for myoglobin concentration and heme loading, revealing a myoglobin concentration of 30-40 g / L (60-70% pure) and a heme loading of 50-60% on apomyoglobin. 2.6 Production of recombinant myoglobin in Pichia pastoris

[0322] To provide further details supporting the effectiveness of Ogataea polymorpha as a suitable host for the production of heme-containing proteins, the myoglobin (MG) gene from Bos taurus was codon-optimized to obtain expression in Pichia pastris. The myoglobin gene was expressed under the AOX1 promoter and terminator sequences.

[0323] Active transformants obtained after transformation and confirmation of the myoglobin gene were cultured in yeast extract, peptone medium, and dextrose (YPD) medium for expression analysis / studies.

[0324] One colony of the active transformant from the plate screened by PCR identification was inoculated into 10 ml of YPD liquid medium.

[0325] After 24 hours, the culture medium was milky white, and 10 ml of the culture medium was inoculated into 1 L of YPD medium (1% inoculation amount), followed by cultivation at 28°C and 230 rpm.

[0326] Preparation of methanol for induction: Hemin was dissolved in 0.2 M NaOH. The concentration of hemin was 100 mM. It was then diluted 10 times (10-fold) with methanol and sterile filtered to obtain 10 mM hemin in methanol.

[0327] After 50 dilutions (approximately 24 hours), when the bacterial OD600 reached 0.6-0.7, the culture was poured into two sterile, chilled 500 ml tubes and centrifuged at 6000 rpm for 5 minutes at 4°C on an ultraclean bench. The supernatant was poured off, and the yeast was resuspended in 900 ml of YP medium. The resuspension (resuspension system) was poured back into a 5 L conical flask. 100 ml of sterile 1 M phosphate buffer (working concentration of phosphate: 0.1 M) was added, and 10 ml of methanol containing 10 mM hemin was added to the resuspension to initiate induction. The working concentration of methanol was 1%, and the working concentration of hemin was 0.1 mM.

[0328] 10 ml of methanol containing 10 mM hemin was added to the resuspension every 24 hours.

[0329] Samples were collected daily and analyzed by TCA enrichment electrophoresis. The first methanol addition was counted as time zero (0). After approximately 48 hours (induction times vary depending on the protein), the culture medium was poured into two 500 mL centrifuge tubes and centrifuged at 6000 rpm for 10 minutes. The supernatant was collected and frozen at -20°C for further use.

[0330] As mentioned above, no heme loading of myoglobin protein was observed without exogenous heme addition. After exogenous heme addition, myoglobin with up to 22% heme loading was observed. Experimental Example 3 - Ogataea polymorpha cell lysis

[0331] Frozen Ogataea polymorpha biomass was thawed at room temperature and then diluted with tap water to a concentration of approximately 30% dry matter (analyzed using a Mettler Toledo moisture analyzer HB 431). Two conditions (samples) were established: a control biomass at pH 5.2 and a biomass at pH 8.5 (adjusted to pH 8.5 by adding 33% NaOH). The latter was combined with 2.5% Alcatase enzyme (Novozymes, 2.4 L, pure (2.4 AU-A / g)). Both biomass solutions were incubated at 50°C for 4 hours with continuous shaking. The pH of the Alcatase sample (pH 8.5 biomass) was maintained at approximately 8.5 by adding 33% NaOH by volume during the process. Two samples were collected every hour and stored at -10°C for later analysis. Example 4 - Enzymatic hydrolysis

[0332] Four hours after inoculation, the control biomass was terminated for analysis. The Alcalase enzyme-treated biomass was first maintained at pH 5.5 with 5% hydrochloric acid, after which the first enzyme was added (Step 1). The first enzyme mixture contained RP-1G (0.16% aminoenzyme, a 5' phosphodiesterase that hydrolyzes RNA into 5' nucleotides) and Deamizyme (0.067% aminoenzyme, converting the nucleotide 5' GMP to 5' IMP). The nucleotides 5' GMP and 5' IMP are known flavor enhancers. The mixture was incubated at 50°C for 5 hours with continuous shaking.

[0333] The solution was then set to pH 7.0 with 33% NaOH. A second yeast mixture was then added (Step 2). The second enzyme mixture was: *Protana UBoost: Protana UBoost (0.17% Novozymes, glutaminase (produces glutaminate from glutamine)), *Protana Prime: Protana Prime (0.17% Novozymes, an exo-peptidase mix that releases free amino acids) *ProteAXH (0.17% amino enzyme, expresses protease and peptide activity) is. These enzymes are known to enhance umami flavor and were incubated for 12 hours at 50° C. with continuous shaking. Samples were collected at the end of each enzymatic digestion / digestion step.

[0334] To determine that the dissolution and hydrolysis processes were not degrading the heme, samples were taken at each step and the heme concentration was determined by the method used in Example 1 (1.4). This is shown in Table 3. Experimental Example 5 Microfiltration and Ultrafiltration

[0335] The hydrolyzed products were first separated by microfiltration. An 800 kDa membrane was placed in a filtration system with a SANI membrane (Vibro-Lab 3500). The hydrolyzed solution was then pumped through this membrane device in combination with continuous vibration. The retentate (insoluble fraction) consisted of large polysaccharides (β-glucans, glucose polymers that form the yeast cell wall) and the permeate, a clear, dark brown solution representing the intercellular compartments of yeast cells. The 800 kDa permeate (soluble fraction) was then used for ultrafiltration using a 1 kDa membrane. This resulted in the production of soluble small peptides and nucleotides in the permeate. Experimental Example 6 Quantitative analysis of GMP

[0336] The concentration of flavor-enhancing GMP was determined using the spray-dried fraction of yeast hydrolysate obtained after filtration (after Example 5). This was performed using an isocratic or gradient HPLC elution method. The gradient elution was performed using (A) 0.5% orthophosphoric acid and (B) 100% methanol as the mobile phase, on a Gemini Phenyl C6 reversed-phase column with a 5 μm diameter, 110 A diameter, and 50 x 4.6 mm diameter. The conditions were a flow rate of 0.5 mL / min, column pressure of 50-60 kgf / cm x cm, and detector: SPD-M20A, with a diode array detector wavelength of 254 nm.

[0337] Results: A mean concentration of 0.007% w / w of GMP per g of Ogataea polymorphic yeast hydrolysate was observed. Experimental Example 7: Quantitative analysis of glutathione

[0338] The yeast hydrolysate obtained after filtration was subjected to positive electrospray nebulization for the analysis of glutathione concentration, a umami flavor component, using a UPLC-QTOF with a C18 column. Gradient elution was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as the mobile phase, with an Acquity UPLC BEH C18, 50 x 2.1 mm column as the stationary phase. The flow rate was maintained at 0.25 mL / min, and the MS scan range was 100-2000 m / z.

[0339] Results: A mean concentration of 11.62% w / w oxidized glutathione and 0.29% w / w reduced glutathione per gram of spray-dried yeast product was observed. Experimental Example 8: Flavoring derived from Ogataea polymorpha

[0340] 8.1- Flavoring 1: Mixture 1 was prepared by mixing 370 g of Ogataea polymorphic yeast extract (20%-24% ds) as a protein source, 16 g of aminocysteine, methionine, glutamine, and chrysin, 3-5 g of vitamin B1, 40 g of a reducing carbohydrate source (d-xylose, reverse osmosis, arabinose, rhamnose, and glucose), 80 g of water, and 8-10 g of 33% caustic alkali in a high-shear mixer. Mixture 1 was poured into a Parr high-pressure stirred reactor. The slurry (from Mixture 1) was heated to 120°C in 30 minutes. After reaching 120°C, the slurry was held at this temperature for 60 minutes (a 3 bar pressure increase was observed during this time). After reaction, the batch was cooled to 50°C in 30 minutes. The resulting liquid was mixed with a predetermined amount of carrier and then spray-dried or vacuum-dried, or left as a liquid.

[0341] 8.2- Flavoring 2: Mixture 2 was prepared by mixing 200 g of 20%-24% ds Ogataea polymorpha extract, 25 g of aminolysine, arginine, histidine (cysteine), 5 g of taste enhancers IMP / GMP, 15 g of lactic acid, succinic acid, and tartaric acid (lactic acid), 120 g of reducing carbohydrate source (glucose, arabinose), and 37 g of 33% caustic alkali in a high-shear mixer. Mixture 2 was poured into a Parr high-pressure stirred reactor. The slurry (from Mixture 2) was heated to 120°C in 30 minutes. After reaching 120°C, the slurry was held at this temperature for 30 minutes. After reaction, the batch was cooled to 50°C in 30 minutes. The resulting liquid was either used directly or mixed with a predetermined amount of carrier, and then spray-dried or vacuum-dried.

[0342] 1.3- Flavoring 3: A cell culture of genetically modified Ogataea polymorpha expressing myoglobin was lysed with 0.1% papain at 65°C for 2 hours. It was then lysed with 0.05% Protease A Amino 2SD (Amino Enzymes Inc.) for 24 hours at 50°C. 580 g of the genetically modified Ogataea polymorpha extract was mixed with 15.8 g of yeast extract, 60 g of gum acacia, 120 g of water, 14 g of amino acids (Gly, Ala, Cys, Met), 0.35 g of vitamin B1, 3.6 g of reducing sugars (d-xylose, reverse osmosis, arabinose, rhamnose), and 7.2 g of sodium phosphate buffer to obtain Mixture 3. This mixture 3 was heat treated at 120°C for 30 minutes at a pressure of 1-2.5 bar and a pH of 6.5, resulting in a Maillard reaction product (Flavor 3).

[0343] 8.4 - Flavoring 4: 750 g of the Maillard reaction product of flavoring 3 was mixed with 255 g maltodextrin, 45 g gum acacia, 380 g water and spray dried. 280 g of this spray-dried mixture was further blended with other ingredients: 75 g Furaneol, 90 g Yeast Extract KU 012, 100 g Yeast Extract KA 65, 160 g of organic acids (lactic acid, malic acid, citric acid, tartaric acid, succinic acid, and acetic acid), 160 g of aminoglycosides (aminoglycine, arabinose, cysteine, glutamine, and methionine), and 200 g of salt. The resulting flavoring was tasted at 0.4%. Further tasting with 0.2% salt in lukewarm water yielded a distinctive meaty flavor with a metallic, bloody aftertaste.

[0344] Experimental Example 9: Food Application 9.1: Sauce: Soy sauce 10%, Flavoring 1: 5%, Salt: 19%, Water: 56%, Beef Flavor Top Note: 0.5%, Spice Extract (Peppers, Chili, Garlic, Onion): 0.1%, Taste Enhancer IMP / GMP / MSG (MSG: Monosodium Glutamine): 3.5%, Tomato Paste: 1%, Molasses: 3%, Flavoring 2: 1%, Xanthan Gum: 0.3% were mixed to obtain a mixture. The mixture was heated to 80°C and packed hot into containers. The product had a mellow Korean-style beef flavor.

[0345] 9.2: Food matrices: Based on known techniques, several food matrices have been developed. Cells, extracts, hydrolyzed extracts and flavoring agents of Ogataea pleomorpha have been incorporated into these food matrices. The list of foods and major ingredients is shown in Tables 4-6.

[0346] The present invention employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, recombinant biology, microbiology, recombinant DNA, and immunology, which are well known in the art.

[0347] The documents disclosed in the specification may also be referenced for understanding the present invention. The sequence listing is shown below.

Claims

1. Contains heme at least 0.03% w / w An extract of Ogataea polymorpHa characterized by:

2. The Ogataea polymorpha extract contains 0.03% to 3.1% w / w of heme. The Ogataea polymorphic fungus extract according to claim 1.

3. The Ogataea polymorpha extract contains 0.8% w / w or more of glutamic acid.

3. The Ogataea polymorphic fungus extract according to claim 1 or 2.

4. The Ogataea polymorpha extract further contains 0.007% w / w or more of 5' ribonucleotides. The Ogataea polymorphic fungus extract according to any one of claims 1 to 3.

5. The 5' ribonucleotide is 5' GMP The Ogataea polymorphic fungus extract according to claim 4.

6. The Ogataea polymorpha extract contains glutamic acid at 0.8% w / w or more and 5' ribonucleotides at 0.007% w / w or more. Also includes The Ogataea polymorphic fungus extract according to any one of claims 1 to 5.

7. The 5' ribonucleotide is 5' GMP The Ogataea polymorphic fungus extract according to claim 6.

8. The Ogataea polymorpha extract further contains 0.2% w / w or more of glutathione. The Ogataea polymorphic fungus extract according to any one of claims 1 to 7.

9. The Ogataea polymorpha extract contains 0.4% w / w or more of histidine. The Ogataea polymorphic fungus extract according to any one of claims 1 to 8.

10. the Ogataea polymorpha fungus is genetically modified to produce a recombinant heme protein; The recombinant heme protein is produced at a rate of 0.1% w / w or more of the total protein. The Ogataea polymorphic fungus extract according to any one of claims 1 to 9.

11. The recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c. The Ogataea polymorphic fungus extract according to claim 10.

12. The recombinant heme protein is an animal-derived protein or a plant-derived protein. The Ogataea polymorphic fungus extract according to claim 10.

13. Contains 0.03% w / w or more of heme A hydrolyzed Ogataea polymorpha extract characterized in that:

14. The hydrolyzed Ogataea polymorpha extract contains 0.03% w / w to 3.1% w / w of heme.

14. The hydrolyzed Ogataea polymorpha extract of claim 13.

15. The hydrolyzed Ogataea polymorpha extract further contains at least 0.8% w / w glutamic acid.

15. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13-14.

16. The hydrolyzed Ogataea polymorpha extract further contains 0.007% w / w or more 5' ribonucleotides.

16. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13 to 15.

17. The 5' ribonucleotide is 5' GMP 17. The hydrolyzed Ogataea polymorpha extract of claim 16.

18. The hydrolyzed Ogataea polymorpha extract contains glutamic acid at 0.8% w / w or more and 5' ribonucleotides at 0.007% or more.

18. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13 to 17.

19. The 5' ribonucleotide is 5' GMP 19. The hydrolyzed Ogataea polymorpha extract of claim 18.

20. The hydrolyzed Ogataea polymorpha extract further contains 0.4% w / w or more of histidine.

20. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13 to 19.

21. The hydrolyzed Ogataea polymorpha extract further contains at least 0.2% w / w glutathione.

21. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13 to 20.

22. the Ogataea polymorpha fungus is genetically modified to produce a recombinant heme protein; The recombinant heme protein is produced at a rate of 0.1% w / w or more of the total protein.

22. Hydrolyzed Ogataea polymorpha extract according to any one of claims 13 to 21.

23. The recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

23. The hydrolyzed Ogataea polymorpha extract of claim 22.

24. The recombinant heme protein is an animal-derived protein or a plant-derived protein.

23. The hydrolyzed Ogataea polymorpha extract of claim 22.

25. Biomass of Ogataea polymorpha, including proteins, amino acids, carbohydrates, and vitamins A flavor precursor mixture comprising:

26. The Ogataea polymorpha fungus contains 0.03% w / w or more of heme.

26. The flavor precursor mixture of claim 25.

27. The Ogataea polymorpha biomass is an extract A flavour precursor mixture according to any one of claims 25-26.

28. The Ogataea polymorpha biomass is a carbohydrate extract 28. The flavor precursor mixture of claim 27.

29. The Ogataea polymorpha extract further contains 0.8% w / w or more glutamic acid. A flavour precursor mixture according to any one of claims 25 to 28.

30. The Ogataea polymorphic fungus further contains 0.007% or more 5' ribonucleotides.

30. A flavour precursor mixture according to any one of claims 25 to 29.

31. The Ogataea polymorphic fungus further contains 0.8% w / w or more of glutamic acid and 0.007% w / w or more of 5' ribonucleotides. A flavour precursor mixture according to any one of claims 25 to 30.

32. The 5' ribonucleotide is 5' GMP 31. The flavor precursor mixture of claim 30.

33. The 5' ribonucleotide is 5' GMP 32. The flavor precursor mixture of claim 31 .

34. The Ogataea polymorpha fungus further contains 0.2% w / w or more glutathione.

34. A flavor precursor mixture according to any one of claims 25 to 33.

35. The Ogataea polymorpha fungus further contains 0.4% w / w or more of histidine. A flavour precursor mixture according to any one of claims 25 to 34.

36. the Ogataea polymorpha fungus is genetically modified to produce a recombinant heme protein; The recombinant heme protein is produced at a rate of 0.1% w / w or more of the total protein.

36. A flavor precursor mixture according to any one of claims 25 to 35.

37. The recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

37. The flavor precursor mixture of claim 36.

38. The recombinant heme protein is an animal-derived protein or a plant-derived protein.

37. The flavor precursor mixture of claim 36.

39. The flavor precursor mixture further contains 1% w / w or more of Ogataea polymorpha biomass, 5% w / w or more of monosaccharides, and 15% w / w or more of amino acids.

39. A flavor precursor mixture according to any one of claims 25 to 38.

40. Contains biomass of Ogataea polymorpha A food characterized by:

41. The Ogataea polymorpha biomass is a dissolved biomass.

41. The food product of claim 40.

42. The lysed Ogataea pleomorpha biomass is hydrolyzed biomass.

42. The food product of claim 41 .

43. The food product contains 0.001% w / w to 80% w / w of Ogataea pleomorpha biomass.

43. The food product according to any one of claims 41 to 42.

44. The Ogataea polymorpha fungus further contains 0.03% w / w or more of heme.

44. The food product of any one of claims 40 to 43.

45. The Ogataea polymorpha fungus contains 0.03% w / w to 3.1% w / w of heme.

44. The food product of any one of claims 40 to 43.

46. The Ogataea polymorpha fungus further contains 0.8% w / w or more glutamic acid.

46. ​​A food product according to any one of claims 40 to 45.

47. The Ogataea polymorphic fungus further contains 0.007% or more 5' ribonucleotides.

47. A food product according to any one of claims 40 to 46.

48. The Ogataea polymorphic fungus contains glutamic acid at 0.8% w / w or more and 5' ribonucleotides at 0.007% w / w or more. Also includes 47. The food product of any one of claims 41 to 46.

49. The 5' ribonucleotide is 5' GMP 48. The food product of claim 47.

50. The 5' ribonucleotide is 5' GMP 49. The food product of claim 48.

51. The Ogataea polymorpha fungus further contains 0.4% w / w or more of histidine.

51. A food product according to any one of claims 40 to 50.

52. The Ogataea polymorpha fungus further contains 0.2% w / w or more glutathione.

52. The food product of any one of claims 40 to 51.

53. The food does not include food of animal origin.

53. A food product according to any one of claims 40 to 52.

54. The food product is selected from the group consisting of sauces, marinades, condiments, dressings, brines, broths, soups, tofu, tempeh, seitan, fermented vegetables, legumes, artificial meats, legume foods, cultured meat products, and dietary supplements.

54. A food product according to any one of claims 40 to 53.

55. 40. A method for producing a flavor precursor mixture comprising the steps of:

40. The food product of any one of claims 25 to 39.

56. 1. A method for producing an Ogataea polymorpha extract, comprising: (A) culturing and growing Ogataea pleomorpha cells; (B) lysing the Ogataea pleomorpha cells; (C) adjusting the pH as needed; (D) Obtaining an Ogataea polymorpha extract by drying or concentrating by removing water. have A method for producing an extract of Ogataea polymorpha, comprising:

57. After step (B), (E) Inactivating Acid Phosphatase Further having 57. The method of claim 56.

58. In step (E), the pH is increased from 5 to 14.

58. The method of claim 57.

59. (F) Hydrolysis of endogenous proteins and nucleic acids to 30 kDa Further having 57. The method of claim 56.

60. (G) Maintaining the temperature at 60°C or less Further having 57. The method of claim 56.

61. Includes hydrolysis of proteins and nucleic acids up to 25 kDa, 20 kDa, 15 kDa, 10 kDa, 5 kDa, 2 kDa, 1 kDa, and 0.5 kDa 57. The method of claim 56.

62. A method for preparing a seasoning / flavoring from Ogataea pleomorpha, comprising: (A) obtaining Ogataea pleomorpha biomass; (B) adding and mixing a source of carbohydrates, lipids, and amino acids to the Ogataea pleomorpha biomass; (C) inducing a heat treatment by applying a temperature between 100°C and 130°C for 30 to 180 minutes to the Ogataea pleomorpha biomass to produce a seasoning from the Ogataea pleomorpha; have Method for preparing seasonings / flavorings from Ogataea polymorpha.

63. The carbohydrate is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, oligosaccharides, and polysaccharides.

63. The method of claim 62.

64. The lipid is selected from the group consisting of animal fat, insect fat, fungal fat, vegetable fat, plant fat, hard fat, and microbial fat.

64. The method according to any one of claims 62-63.

65. The source of the amino acids is selected from the group consisting of natural proteins, partially hydrolyzed proteins, and free amino acids.

65. The method of any of claims 62-64.

66. (D) Inducing Ogataea polymorpha cell lysis to obtain an extract Further having 66. The method of any one of claims 62-65.

67. (E) Inducing proteolysis of the extract obtained in step (D). Further having 67. The method of claim 66.

68. (F) inducing hydrolysis of nucleic acids in the extract Further having 67. The method of claim 66.

69. (G) Inducing the conversion of amino acids to glutamic acid Further having 69. The method of any one of claims 62-68.

70. Contains Ogataea polymorpha biomass containing 0.03% w / w or more of heme A nutritional supplement characterized by:

71. Ogataea polymorpha biomass contains 0.03% w / w to 3.1% w / w of heme.

71. The method of claim 70.

72. The Ogataea polymorpha biomass is a dissolved biomass.

72. The method according to any one of claims 70-71.

73. The dissolved biomass is further hydrolyzed 73. The method of claim 72.

74. The molten fraction is obtained after filtering the hydrolyzed biomass.

74. The method of claim 73.

75. The molten portion contains particles of 100 kDa or less.

75. The method of claim 74.

76. The molten portion contains particles of 50 kDa or less.

75. The method of claim 74.

77. The molten portion contains particles of 5 kDa or less.

75. The method of claim 74.

78. The molten portion contains particles of 1 kDa or less.

75. The method of claim 74.

79. The molten portion contains particles of 0.5 kDa or less.

75. The method of claim 74.

80. 1. A method for producing a hemoprotein having at least 50% heme w / w, comprising: (A) providing a culture of transgenic Ogataea polymorpha fungus containing a nucleic acid encoding a heme protein operably linked to a promoter; (B) culturing the transgenic Ogataea pleomorpha bacteria without adding exogenous heme to the culture; (C) A step of separating and purifying the heme protein have A method for producing heme proteins containing 50% or more w / w heme.

81. The promoter is a methanol-inducible promoter.

81. The method of claim 80.

82. The transgenic Ogataea polymorpha does not contain exogenous transcriptional activators of the heme biosynthetic pathway or exogenous components of the heme biosynthetic pathway.

82. The method according to any one of claims 80-81.

83. The heme protein is selected from the group consisting of animal-derived heme proteins and plant-derived heme proteins.

83. The method of any of claims 80-82.

84. The animal-derived heme protein is selected from the group consisting of bovine heme protein, porcine heme protein, ovine heme protein, equine heme protein, and caprine heme protein.

84. The method of claim 83.

85. The recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

85. The method according to any one of claims 83-84.

86. The nucleic acid comprises a nucleic acid sequence having 70% or more w / w homology to the nucleic acid sequence of SEQ ID NO:

1.

86. The method of any one of claims 80-85.

87. 1. A method for producing a heme protein containing 50% or more heme by weight, comprising: (A) introducing into Ogataea polymorpha a nucleic acid construct comprising a promoter operably linked to a nucleic acid encoding an animal-derived heme protein; (B) obtaining a culture of transgenic Ogataea pleomorpha without adding exogenous heme to said culture; (C) obtaining animal-derived heme proteins from the culture. have A method for producing heme proteins containing 50% or more w / w heme.

88. The promoter is a methanol-inducible promoter.

88. The method of claim 87.

89. The transgenic Ogataea polymorpha does not contain exogenous transcriptional activators of the heme biosynthetic pathway or exogenous components of the heme biosynthetic pathway.

89. The method according to any one of claims 87-88.

90. The animal-derived heme protein is selected from the group consisting of bovine heme protein, porcine heme protein, ovine heme protein, equine heme protein, and caprine heme protein.

90. The method of any one of claims 87-89.

91. The recombinant heme protein is selected from the group consisting of hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

91. The method of any of claims 87-90.

92. The nucleic acid comprises a nucleic acid sequence having 70% or more w / w homology to the nucleic acid sequence of SEQ ID NO:

1.

92. The method of any of claims 87-91.

93. 1. A method for producing recombinant bovine myoglobin having at least 50% w / w of recombinant bovine myoglobin with heme, comprising: (A) introducing a nucleic acid encoding a recombinant bovine myoglobin operably linked to a promoter into an Ogataea polymorpha yeast; (B) culturing the Ogataea polymorpha strain containing the recombinant bovine myoglobin nucleic acid to promote expression of the recombinant myoglobin without the addition of heme. have A method for producing recombinant bovine myoglobin having at least 50% w / w of recombinant bovine myoglobin with heme.

94. The promoter is a methanol-inducible promoter.

94. The method of claim 93.

95. The transgenic Ogataea polymorpha does not contain exogenous transcriptional activators of the heme biosynthetic pathway or exogenous components of the heme biosynthetic pathway.

95. The method according to any one of claims 93-94.

96. (C) extracting and purifying the recombinant bovine myoglobin. Further having 96. The method of any of claims 93-95.

97. The nucleic acid comprises a nucleic acid sequence having 70% or more homology to the nucleic acid sequence of SEQ ID NO:

1.

97. The method of any of claims 93-96.

98. A hemoprotein produced by the method of claim 80 or 87, or a bovine myoglobin produced by the method of claim 93. Contains A food characterized by:

99. A lysate of cultured Ogataea pleomorpha produced by the method of claims 80, 87 or 93. Contains A food composition characterized by:

100. A hemoprotein produced by the method of claim 80 or 87, or a bovine myoglobin produced by the method of claim 93. Contains A meat-like food characterized by:

Images