Composition from hansenula polymorpha and application

Abstract

The disclosure provided herein relates to the use of methylotrophic yeast and derivatives thereof as sources of food additives and flavoring compounds.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 378,661, filed on October 6, 2022, and U.S. Provisional Application No. 63 / 512,850, filed on July 10, 2023, the entire contents of which are hereby incorporated by reference.

[0003] Reference to a sequence listing submitted electronically

[0004] The entire contents of the sequence listing submitted electronically (name: 5061_003PC02_Seqlisting_ST26.xml; size: 11,194 bytes; and creation date: October 6, 2023) together with this application are hereby incorporated by reference into this text. Technical field

[0005] The present invention relates to the use of Ogataea polymorpha as a food additive, flavoring, or as a component that can impart umami, savory, iron - like, and / or meat - like flavors to food compositions. Background art

[0006] The increasing demand for alternative, more sustainable foods that do not include animal derivatives for their preparation has presented the challenge of providing them with flavors and tastes acceptable to consumers. Imparting flavors to animal protein analogues is a challenge in the alternative food and snack food industries.

[0007] Yeast extracts have been widely used in the food industry to modulate desired flavors, textures, pH, colors, baking properties, chewiness, etc. Yeast extracts are commonly used as a major raw material, which is combined with reducing sugars, vitamins, and amino acids, as well as the Maillard reaction, to obtain products with unique flavors. The yeast commonly used for this purpose is Saccharomyces cerevisiae, followed by Cyberlindnera jadiini.

[0008] The invention disclosed herein relates to extracts, hydrolysates, and / or isolated molecules derived from Ogataea polymorpha as a novel and unique yeast for food applications.

[0009] This disclosure also relates to the production of recombinant heme proteins in methylotrophic yeasts such as Ogataea polymorpha.

[0010] Surprisingly, the inventors of the present invention have developed a method for efficiently obtaining recombinant heme proteins in Hansenula polymorpha without genetically manipulating the heme biosynthesis pathway (e.g., overexpression of enzymes involved in heme biosynthesis) and without adding exogenous heme to the culture. By the method of the present invention, the binding of heme to the produced recombinant heme protein is greater than 50%. Summary of the Invention

[0011] In some aspects, the present disclosure provides a Hansenula polymorpha extract comprising at least about 0.03% w / w of heme. In some aspects, the Hansenula polymorpha extract comprises about 0.03% w / w of heme. In some aspects, the Hansenula polymorpha extract comprises from about 0.03% to about 3.1% w / w of heme. In some aspects, the Hansenula polymorpha extract comprises 0.03% to 3.1% w / w of heme. In some aspects, the Hansenula polymorpha extract further comprises at least about 0.8 wt% of glutamic acid. In some aspects, the Hansenula polymorpha extract further comprises about 0.8 wt% of glutamic acid.

[0012] In some aspects, the Hansenula polymorpha extract further comprises at least about 0.007% w / w of 5'-ribonucleotide. In some aspects, the at least about 0.007% w / w of 5'-ribonucleotide is from 5'-GMP.

[0013] In some aspects, the Hansenula polymorpha extract comprises at least about 0.8% w / w of glutamic acid and at least about 0.007% w / w of 5'-ribonucleotide. In some aspects, the 5'-ribonucleotide is 5'-GMP.

[0014] In some aspects, the Hansenula polymorpha extract further comprises γ-glutamyl peptides. In some aspects, the γ-glutamyl peptides comprise glutathione.

[0015] In some aspects, the Hansenula polymorpha extract comprises at least about 0.8% w / w of glutamic acid, at least about 0.007% w / w of 5'-GMP, and at least about 0.2% w / w of glutathione.

[0016] In some aspects, the Hansenula polymorpha extract comprises at least about 0.03% - 3% w / w of heme, at least about 0.8% w / w of glutamic acid, at least about 0.007% w / w of 5'-GMP, and at least about 0.2% w / w of glutathione.

[0017] In some aspects, the Hansenula polymorpha extract comprises at least about 0.8% w / w of glutamic acid, at least about 0.007% w / w of 5'-GMP, and at least about 0.2% w / w of glutathione, and substantially does not contain heme content.

[0018] In some aspects, the Hansenula polymorpha extract also contains at least about 0.41% w / w histidine.

[0019] In some aspects, the Hansenula polymorpha is genetically modified to produce a recombinant heme protein. In some aspects, the heme protein is produced at least about 0.1% by weight of the total protein. In some aspects, the recombinant heme protein is selected from the group consisting of: hemoglobin, myoglobin, leghemoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c. In some aspects, the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

[0020] In some aspects, provided herein is a hydrolyzed Hansenula polymorpha extract that contains at least about 0.03% w / w heme.

[0021] In some aspects, provided herein is a hydrolyzed Hansenula polymorpha extract that contains from about 0.03% to about 3% w / w heme.

[0022] In some aspects, the hydrolyzed Hansenula polymorpha extract also contains at least about 0.8% w / w glutamic acid.

[0023] In some aspects, the hydrolyzed Hansenula polymorpha extract also contains 5'-ribonucleotides. In some aspects, the hydrolyzed Hansenula polymorpha contains at least about 0.007% w / w 5'-GMP.

[0024] In some aspects, the hydrolyzed Hansenula polymorpha extract also contains at least about 0.8% w / w glutamic acid and at least about 0.007% w / w 5'-ribonucleotides. In some aspects, the hydrolyzed Hansenula polymorpha contains at least about 0.007% w / w 5'-GMP.

[0025] In some aspects, the hydrolyzed Hansenula polymorpha extract also contains γ-glutamyl peptides. In some aspects, the γ-glutamyl peptide is glutathione. In some aspects, the Hansenula polymorpha extract contains at least about 0.2% w / w glutathione.

[0026] In some aspects, the hydrolyzed Hansenula polymorpha extract contains at least about 0.8% w / w glutamic acid, at least about 0.007% w / w 5'-GMP, and at least about 0.2% w / w glutathione.

[0027] In some aspects, the hydrolyzed Hansenula polymorpha extract also contains at least about 0.41% w / w histidine.

[0028] In some aspects, the hydrolyzed Hansenula polymorpha extract comprises at least about 0.8% w / w of glutamic acid, at least about 0.007% w / w of 5'GMP, and at least about 0.2% w / w of glutathione, and substantially does not contain heme content.

[0029] In some aspects, the hydrolyzed Hansenula polymorpha extract is derived from genetically modified Hansenula polymorpha used for producing recombinant heme proteins. In some aspects, the heme protein is produced at at least 0.1% by weight of the total protein. In some aspects, 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 some aspects, the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

[0030] In some aspects, provided herein is a flavor precursor mixture comprising Hansenula polymorpha biomass, proteins, amino acids, carbohydrates, and vitamins. In some aspects, the flavor precursor mixture further comprises a fat selected from plant fat, animal fat, microbial fat, and cell culture fat. In some aspects, the flavor precursor mixture comprises Hansenula polymorpha biomass containing at least about 0.03% w / w heme. In some aspects, the Hansenula polymorpha biomass is an extract. In some aspects, the Hansenula polymorpha biomass is a hydrolyzed extract. In some aspects, the Hansenula polymorpha biomass further comprises at least about 0.8% w / w of glutamic acid. In some aspects, the Hansenula polymorpha biomass further comprises 5'-ribonucleotides. In some aspects, the Hansenula polymorpha biomass further comprises at least about 0.8% w / w of glutamic acid, at least about 0.007% w / w of 5'GMP, and at least about 0.2% w / w of glutathione. In some aspects, the Hansenula polymorpha biomass further comprises at least about 0.41% w / w of histidine.

[0031] In some aspects, the flavor precursor mixture comprises genetically modified Hansenula polymorpha used for producing recombinant heme proteins. In some aspects, the heme protein is produced at at least about 0.1% by weight of the total protein. In some aspects, 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 some aspects, the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

[0032] In some aspects, the flavor precursor mixture comprises at least about 1% w / w of Hanseniaspora polymorpha biomass, at least about 5% w / w of monosaccharides, and at least about 15% w / w of amino acids.

[0033] In some aspects, provided herein is a food product that comprises Hanseniaspora polymorpha biomass. In some aspects, the Hanseniaspora polymorpha biomass is lysed biomass. In some aspects, the lysed Hanseniaspora polymorpha biomass is partially or fully hydrolyzed biomass.

[0034] In some aspects, the food product disclosed herein comprises from about 0.001% to about 80% w / w of Hanseniaspora polymorpha biomass. In some aspects, the Hanseniaspora polymorpha biomass comprises at least about 0.03% w / w of heme. In some aspects, the Hanseniaspora polymorpha biomass further comprises at least about 0.8% w / w of glutamic acid. In some aspects, the Hanseniaspora polymorpha biomass further comprises 5'-ribonucleotides. In some aspects, the Hanseniaspora polymorpha biomass further comprises about 0.8% w / w of glutamic acid and 5'-ribonucleotides. In some aspects, the hydrolyzed Hanseniaspora polymorpha comprises 5'-GMP. In some aspects, the hydrolyzed Hanseniaspora polymorpha comprises glutathione. In some aspects, the Hanseniaspora polymorpha biomass further comprises at least about 1% w / w of histidine.

[0035] In some aspects, the food product is free of products of animal origin.

[0036] In some aspects, the food product is selected from the group consisting of: sauces, marinades, seasonings, dressings, brines, broths, soups, traditional protein foods (such as tofu, tempeh, seitan), fermented vegetables and legumes, meat analogs, meat products, extruded meat products, cultured meat products, and dietary supplements.

[0037] In some aspects, provided herein is a food product that comprises a flavor precursor mixture that comprises Hanseniaspora polymorpha biomass.

[0038] In some aspects, the present disclosure provides a dietary supplement comprising Hansenula polymorpha biomass containing at least about 0.03% w / w heme. In some aspects, the Hansenula polymorpha biomass comprises about 0.03% heme. In some aspects, the Hansenula polymorpha biomass comprises about 0.03% to about 3% w / w heme. In some aspects, the Hansenula polymorpha biomass comprises 0.03% to 3% w / w heme. In some aspects, the Hansenula polymorpha biomass is lysed biomass. In some aspects, the lysed Hansenula polymorpha biomass is further hydrolyzed. In some aspects, the hydrolyzed biomass is further filtered to obtain a soluble fraction comprising amino acids, peptides, polypeptides having a molecular weight less than about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa or about 0.5 kDa. In some aspects, the hydrolyzed biomass is further filtered to obtain an insoluble fraction comprising carbohydrates and / or cell membrane compounds and / or cell wall compounds having a molecular weight less than about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa or about 0.5 kDa.

[0039] In some aspects, the present disclosure provides a method for obtaining a Hansenula polymorpha extract, comprising: a) culturing and propagating Hansenula polymorpha cells; b) lysing the cells; c) adjusting the pH if necessary; and d) drying and / or concentrating by removing water to obtain a solid Hansenula polymorpha extract.

[0040] In some aspects, the method further comprises inactivating acid phosphatase after cell lysis.

[0041] In some aspects, the method comprises hydrolyzing endogenous proteins and nucleic acids to obtain a molecular size not exceeding about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa or about 0.5 kDa.

[0042] In some aspects, the method comprises maintaining the temperature below about 90 °C, about 85 °C, about 80 °C, about 75 °C, about 70 °C, about 65 °C, about 60 °C, about 55 °C or about 50 °C.

[0043] In some aspects, the method comprises filtering the hydrolyzed Hansenula polymorpha extract to obtain a soluble fraction and an insoluble fraction. In some aspects, the soluble fraction comprises particles. In some aspects, the soluble particles comprise amino acids, peptides and / or polypeptides having a molecular weight less than about 100 kDa, about 50 kDa, about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa, about 0.5 kDa.

[0044] In some aspects, the insoluble fraction comprises particles. In some aspects, the insoluble particles comprise carbohydrates, cell membrane compounds, and / or cell wall compounds having a size less than about 100 kDa, about 50 kDa, about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa, and / or about 0.5 kDa.

[0045] In some aspects, provided herein is a method for preparing a flavoring derived from Hansenula polymorpha, comprising: a) obtaining Hansenula polymorpha biomass; b) adding a carbohydrate, fat, and amino acid source to the Hansenula polymorpha biomass and mixing; c) subjecting the biomass to induced heat treatment at about 100 °C to about 130 °C for about 30 minutes to about 180 minutes to obtain a flavoring derived from Hansenula polymorpha. In some aspects, the Hansenula polymorpha biomass is pre-lysed to obtain an extract. In some aspects, the Hansenula polymorpha extract is further hydrolyzed. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to obtain a soluble fraction and an insoluble fraction, wherein the soluble fraction is used to obtain the flavoring. In some aspects, the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, oligosaccharides, and polysaccharides. In some aspects, the fat is selected from the group consisting of animal fats, insect fats, fungal fats, plant fats, synthetic fats, and microbial fats. In some aspects, the amino acid source is selected from the group consisting of natural proteins, partially hydrolyzed proteins, and free amino acids.

[0046] In some aspects, the method further comprises the step of inducing lysis of Hansenula polymorpha cells to obtain an extract. In some aspects, the method further includes the step of inducing proteolysis of the obtained extract. In some aspects, the method further includes the step of inducing nucleic acid hydrolysis of the extract. In some aspects, the method further includes the step of inducing the conversion of amino acids to glutamic acid.

[0047] In some aspects, provided herein is a method for producing a heme protein having a heme loading greater than about 50%. In some aspects, the method comprises the steps of: a. providing a culture of transgenic Hansenula polymorpha comprising a nucleic acid encoding a heme protein operably linked to a methanol-inducible promoter; b. culturing the transgenic Hansenula polymorpha without adding exogenous heme to the culture; and c. isolating and purifying the heme protein.

[0048] In some aspects, the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthetic pathway or an exogenous component of the heme biosynthetic pathway.

[0049] In some aspects, the heme protein is selected from the group consisting of: animal-derived heme proteins and plant-derived heme proteins.

[0050] In some aspects, the animal-derived heme proteins are selected from the group consisting of: bovine-derived heme proteins, porcine-derived heme proteins, ovine-derived heme proteins, equine-derived heme proteins, and caprine-derived heme proteins.

[0051] In some aspects, the animal-derived heme proteins are selected from the group consisting of: hemoglobin, myoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, lignin peroxidase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0052] In some aspects, the nucleic acid comprises a nucleic acid sequence having at least about 70% identity to the nucleic acid sequence of SEQ ID NO:1.

[0053] In some aspects, the nucleic acid is codon-optimized, wherein the nucleic acid comprises a nucleic acid sequence having at least about 70% identity to the nucleic acid sequence of SEQ ID NO:2.

[0054] In some aspects, the nucleic acid comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO:1.

[0055] In some aspects, the nucleic acid is codon-optimized, wherein the nucleic acid comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence of SEQ ID NO:2.

[0056] In some aspects, provided herein is a method for producing an animal-derived heme protein having a heme loading greater than about 50%. In some aspects, the method comprises: a. introducing a nucleic acid construct into a Hansenula polymorpha strain, wherein the nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding an animal-derived heme protein; b. culturing the Hansenula polymorpha transgenic cells without adding exogenous heme molecules to the culture; and c. isolating the animal heme protein from the culture.

[0057] In some aspects, the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthetic pathway or an exogenous component of the heme biosynthetic pathway.

[0058] In some aspects, the animal-derived heme proteins are selected from the group consisting of: bovine-derived heme proteins, porcine-derived heme proteins, ovine-derived heme proteins, equine-derived heme proteins, and caprine-derived heme proteins.

[0059] In some aspects, the animal - derived heme proteins are 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.

[0060] In some aspects, provided herein is a method for producing recombinant bovine myoglobin comprising greater than about 50% heme - loaded recombinant bovine myoglobin. In some aspects, the method comprises: a. introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Hansenula polymorpha yeast cells; b. culturing the Hansenula polymorpha containing the recombinant bovine myoglobin nucleic acid without adding heme to the culture to promote the expression of recombinant myoglobin, thereby expressing recombinant myoglobin.

[0061] In some aspects, the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthetic pathway or an exogenous component of the heme biosynthetic pathway.

[0062] In some aspects, the method further comprises extracting and purifying the recombinant bovine myoglobin.

[0063] In some aspects, provided herein is a food composition comprising any of the heme proteins disclosed herein or any of the bovine myoglobins disclosed herein.

[0064] In some aspects, provided herein is a meat - analogue food composition comprising a lysate of any of the cultured Hansenula polymorpha provided herein.

[0065] In some aspects, provided herein is a meat - analogue food composition comprising any of the heme proteins disclosed herein or any of the bovine myoglobins disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG.1. Optimizing medium parameters and process conditions for improving heme biosynthesis during Hansenula polymorpha culture. Figure 1A Shows the heme yield (per gram of cell dry weight) obtained from Hansenula polymorpha cultured on different carbon sources with and without methanol induction. Figure 1B Shows the heme yield (per gram of cell dry weight) obtained from Hansenula polymorpha cultured on different nitrogen sources with methanol induction and glycerol as the carbon source. Figure 1C Shows the heme yield (per gram of cell dry weight) obtained from Hansenula polymorpha cultured on the optimal carbon and nitrogen sources in a 150L - scale bioreactor volume during semi - pilot trials.

[0067] Figure 2 (FIG.2)。Figure 2 Indicates the expression plasmid pFPMT121 that has been used to generate recombinant production strains. pFPMT121 and its derivatives are used as circular plasmids for transforming Hansenula polymorpha strains RB11 and ALU3.

[0068] Figure 3 (FIG.3). Figure 3 Indicates the B14 plasmid, a derivative of pFPMT121 without an antibiotic resistance gene, which was used in this work to transform Hansenula polymorpha strain RB11.

[0069] Figure 4 (FIG.4). Figure 4 Indicates the plasmid map, which shows the synthetic gene (bovine myoglobin), the origin of replication (ColE1), the digested restriction enzyme sites (EcoRI and BamHI), and the antibiotic resistance gene (ampicillin AmpR).

[0070] Figure 5 (FIG.5). Figure 5 Indicates the final transformation vector, which contains a promoter element (FMDp), a codon-optimized bovine myoglobin gene (BtMG), and a transcription terminator (MOXt) for transforming Hansenula polymorpha strain RB11.

[0071] Figure 6 (FIG.6). Figure 6 Indicates the imaging of a sterile strain of an SDS-PAGE gel used for screening and selecting positive transformants by optical density analysis.

[0072] Figure 7 (FIG.7). Figure 7 Indicates the amino acid sequence similarity (CLUSTAL W alignment) between native bovine myoglobin and myoglobin expressed in Hansenula polymorpha strain RB11.

[0073] Figure 8 (FIG.8). Figure 8 Shows the SDS PAGE analysis of samples collected at different time intervals before and after methanol induction.

[0074] Figure 9 (FIG.9). Figure 9 Indicates the Western blot analysis of native bovine myoglobin and myoglobin expressed in Hansenula polymorpha strain RB11. Lanes 1 to 3 are standard myoglobin samples at concentrations of 0.0125%, 0.025%, and 0.05%, and lanes 5 to 8 are different dilutions (400x, 200x, 100x, and 50x) of recombinant myoglobin samples purified and concentrated by ultrafiltration.

[0075] Figure 10 (FIG.10). Figure 10 It is a photograph of Hansenula polymorpha yeast extract. Detailed implementation manners

[0076] The terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting.

[0077] It should be noted that, unless otherwise specified, the term "a / an" entity refers to one or more of the entities; for example, "nucleic acid sequence" should be understood to represent one or more nucleic acid sequences. Thus, the terms "a / an", "one or more", and "at least one" may be used interchangeably herein.

[0078] In addition, as used herein, "and / or" will be regarded as explicitly disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to encompass "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0079] It should be understood that whenever an aspect is described herein in terms of "comprising", other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0080] The term "about" as used herein means approximately, roughly, around, or in its vicinity. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify numerical values above and below the stated value by, for example, a variance of 10%, higher or lower.

[0081] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can logically be included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant figures).

[0082] Throughout this disclosure, various aspects of the disclosure may be presented in a range format. A numerical range includes the numbers defining the range. In the case of reciting a range of values, it should be understood that each intermediate integer value and each fraction between the recited upper and lower limits of the range, as well as each sub-range between such values, are also specifically disclosed. The upper and lower limits of any range may be independently included within or excluded from the range, and each range that includes either limit, excludes both limits, or includes both limits is also covered by this disclosure. Thus, the ranges recited herein should be understood as shorthand for all values within the range, including the recited endpoints. For example, the range of 1 to 10 should be understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0083] In the case of explicitly reciting a value, it should be understood that values that are approximately the same amount or quantity as the recited value are also within the scope of this disclosure. In the case of disclosing a combination, each sub-combination of the elements of the combination is also specifically disclosed and is within the scope of this disclosure. Conversely, in the case of separately disclosing different elements or groups of elements, their combination is also disclosed. In the case where any element of this disclosure is disclosed as having multiple alternatives, the disclosed embodiments in which each alternative is separately excluded or excluded in any combination with other alternatives are also hereby disclosed; more than one element of this disclosure may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0084] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be further understood that terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and this disclosure, and will not be interpreted in an idealized or overly formal sense unless explicitly so defined herein.

[0085] In describing the present invention, it should be understood that numerous techniques and steps are disclosed. Each of these techniques has its respective advantages, and each can also be used in combination with one or more of the other disclosed techniques, or in some cases with all of the other disclosed techniques. Thus, for clarity, this description will avoid repeating every possible combination of the individual steps in an unnecessary manner. However, the specification and claims should be read with the understanding that such combinations are fully within the scope of the present invention and the claims.

[0086] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). The percent identity can be determined by aligning the two sequences and introducing gaps to maximize the identity between the sequences. Alignments can be generated using procedures known in the art. For the purposes herein, nucleotide sequence alignments can be performed with the blastn program suite under default parameters, and amino acid sequence alignments can be performed with the blastp program suite under default parameters (see the National Center for Biotechnology Information (NCBI) at world wide web ncbi.nlm.nih.gov).

[0087] As used herein, the term "Hansenula polymorpha biomass" refers to accumulated yeast or accumulated yeast cells, with or without the culture medium used for culturing and propagating the yeast.

[0088] The term "lysate" or "extract" refers to a culture medium containing a mixture and / or solution of cell contents, cell wall residues, and any medium components attached to the outside of the cell by specific binding, which have not been removed prior to the lysis process produced by any cell lysis method.

[0089] The term "lysis" refers to the rupture of the cytoplasmic membrane and, if present, the cell wall, such that a substantial amount of intracellular material can escape into the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal, enzymatic, chemical, electrical, radiation, viral, or microbial means. In some aspects, the methods described herein include lysing cells or microorganisms as described herein in order to separate chemicals or mixtures of chemicals from the contents of a bioreactor.

[0090] The invention disclosed herein relates to the use of the yeast Ogataea polymorpha and its derived compounds as flavorings and their application in food products. Ogataea polymorpha was previously known as Hansenula polymorpha, Hansenula angusta, Pichia angusta, Candida thermophila, Ogataea thermophila, and Torulopsis methanothermo.

[0091] As used herein, compounds derived from Ogataea polymorpha relate to Ogataea polymorpha cells, cell parts, cell fractions, cell components (proteins, amino acids, vitamins, carbohydrates, minerals, lipids), and their use for obtaining flavor precursors, flavorings, and food products.

[0092] As used herein, a flavoring (also referred to as a flavor or flavour) is a food additive used to improve the taste, texture, or odor of food.

[0093] The term "yeast extract" refers to a product obtained from yeast lysis. In the invention disclosed herein, the yeast extract is derived from Ogataea polymorpha and can thus be referred to as "Ogataea polymorpha extract". In some aspects, the term "Ogataea polymorpha extract" refers to soluble and / or insoluble Ogataea polymorpha extract. In some aspects, the term "Ogataea polymorpha extract" refers to soluble and / or insoluble Ogataea polymorpha extract that still contains transgenic proteins. In some aspects, the term "Ogataea polymorpha extract" refers to the soluble or insoluble residue of lysed Ogataea polymorpha biomass in which transgenic proteins (such as myoglobin) have been completely or partially removed. In some aspects, Ogataea polymorpha extract can be used as a flavor precursor or flavoring agent. In some aspects, Ogataea polymorpha extract can be used as a dietary supplement.

[0094] In some aspects, lysis mediated by enzymes that causes cell membrane rupture can also result in partial hydrolysis of some cell components (such as proteins). In these cases, the term "Ogataea polymorpha extract" also refers to partially or completely hydrolyzed Ogataea polymorpha biomass.

[0095] The term "hydrolyzed yeast extract" refers to a yeast extract that has been further subjected to hydrolysis of its contents, preferably hydrolysis of its macromolecules such as nucleic acids and proteins. In some aspects, the polymers contained in Hansenula polymorpha cells are partially hydrolyzed. Partial hydrolysis refers to hydrolysis of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In some aspects, "hydrolyzed yeast extract" refers to the soluble and / or insoluble hydrolysis residues of Hansenula polymorpha extract. In some aspects, "hydrolyzed yeast extract" refers to the soluble and / or insoluble hydrolysis residues of Hansenula polymorpha extract in which transgenic proteins are present or remain. In some aspects, "hydrolyzed yeast extract" refers to the hydrolysis residues of lysed yeast biomass from which transgenic proteins such as myoglobin have been removed prior to hydrolysis. In some aspects, the hydrolyzed Hansenula polymorpha extract can be used as a flavor precursor or flavorant.

[0096] The term "glutamate-derived compound" refers to a molecule containing the amino acid glutamate (e.g., glutamyl peptide, glutathione), its salts, and molecules obtained by the reaction of glutamate.

[0097] The term "glutamyl peptide" refers to a peptide containing two or more amino acid residues, wherein at least one amino acid residue is glutamate, γ-glutamic acid or its salt.

[0098] The terms "flavor composition" and "flavor precursor mixture" in the present disclosure refer to a mixture of compounds capable of imparting a desired flavor, aroma, sensation, and / or taste.

[0099] As used herein, the terms "cultured meat" or "meat substitute" or "meat analogue" or "meat-like product" or "meat food product" refer to a food product that is not derived from an animal, or contains a large amount of non-animal protein sources, but has a structure, texture, aesthetic quality, and / or other properties comparable or similar to animal meat, which includes livestock (e.g., beef, pork), game (e.g., venison), poultry (e.g., chicken, turkey, duck), and / or fish or seafood substitutes / analogues. The term refers to uncooked, cooked, and cooked meat food products. These terms also cover cultured meat products.

[0100] As used herein, the terms "non-animal product" or "animal product-free" refer to a product that does not contain any substances directly derived from animals, such as organs, tissues, cells, proteins, nucleic acids. This definition does not apply to animal products produced in non-animal hosts (e.g., genetic and / or amino acid sequences derived from animal genetic material or corresponding protein sequencing).

[0101] As used herein, the term "holoprotein" refers to a conjugated functional protein that binds to a ligand or prosthetic group.

[0102] As used herein, the term "apoprotein" refers to a non-functional protein that is not bound to a ligand or prosthetic group.

[0103] The term "heme protein" includes proteins that have the ability to bind a heme prosthetic group to their structure. As used herein, the term "heme protein" also refers to a key component of animal meat and / or animal protein and can provide color and flavor to plant-based meat products. Myoglobin and hemoglobin are considered heme proteins and are oxygen-binding proteins in animals. Additionally, as used herein, the term "heme protein" refers to a protein that contains heme, where the term "contains" means that the heme is linked to the protein by a covalent or non-covalent bond. As used herein, the term heme protein refers not only to full-length proteins but also to fragments or variants thereof.

[0104] As used herein, the term "produce" refers to the ability of yeast (e.g., Hansenula polymorpha) to express a protein of interest. In some aspects, the protein of interest is transgenic. In some aspects, the protein of interest is isolated from the yeast.

[0105] As used herein, the term "culture" or "propagate" refers to the growth of yeast (e.g., Hansenula polymorpha) under suitable conditions to produce a desired end product (e.g., animal heme protein).

[0106] As used herein, "derived from" refers to the source of a flavor, flavor precursor, or flavoring agent. For example, a flavor "derived from Hansenula polymorpha" refers to a flavor that includes one or more parts of Hansenula polymorpha (e.g., an animal heme protein isolated from said Hansenula polymorpha).

[0107] As used herein, the term "animal heme protein" or "animal-derived heme protein" refers to a heme protein that comes from an animal (such as cattle, pigs, sheep, horses, and goats). The term "animal-derived heme protein" or "animal heme protein" does not include human-derived heme proteins. According to some aspects of the present disclosure, animal heme proteins include and / or are selected from the group consisting of heme proteins involved in oxygen transport, such as hemoglobin, myoglobin, neuroglobin, and cytoglobin; enzymes having a heme prosthetic group, such as cytochrome P450s, cytochrome c oxidase, ligninase, catalase; and heme proteins involved in the electron transport chain, such as cytochrome a, cytochrome b, and cytochrome c.

[0108] As used herein, the term "plant-derived heme protein" refers to heme proteins from monocotyledonous or dicotyledonous plants such as Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn), Arabidopsis thaliana; legumes such as Glycine max (soybean), Cicer arietinum (chickpea or garbanzo bean), Pisum sativum (pea), Phaseolus vulgaris (common bean), Vigna unguiculata (cowpea), Vigna radiata (mung bean), Lupinus albus (lupine) or Medicago sativa (alfalfa); Brassica napus (canola); Triticum sps. (wheat, including wheat berries and spelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps. (wild rice); Helianthus annuus (sunflower); Beta vulgaris (beet); Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp. (sesame); Linum usitatissimum (flax); Lactuca sativa (lettuce); Spinacia oleracea (spinach); or Hordeum vulgare (barley).

[0109] As used herein, the term "recombinant heme protein" is a protein obtained from a genetically modified organism, wherein the recombinant protein is encoded by an exogenous cDNA encoding a heme protein. As used herein, the term "exogenous nucleic acid" may be used interchangeably herein with "recombinant nucleic acid" and / or "heterologous gene".

[0110] The terms "transgenic", "recombinant", "genetically engineered", "genetically modified", "modified" as related to yeast (e.g., Hansenula polymorpha) mean that the yeast has been transformed or transduced with one or more nucleic acids (recombinant sequences). The term "transformation" refers to the process of introducing and expressing recombinant sequences in yeast cells using a non-viral vector.

[0111] In addition, as used herein, the term "heme loading" refers to the amount of heme bound to an apomyoglobin recombinant protein. For example, Zhang et al. expressed porcine myoglobin in Pichia pastoris and supplemented the culture medium with 150 mg / L of exogenous heme and obtained 0.22 moles of heme per mole of myoglobin, which represents a 22% heme loading. (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)). In theory, one mole of myoglobin can bind one mole of heme, which represents 100% heme loading. The amount of heme-bound hemeprotein can be determined by any known method, such as those described by Hopp, M.T. et al., Heme Determination and Quantification Methods and their Suitability for Practical Applications and Everyday Use. Anal. Chem. 92, 14, 9429–9440 (2020).

[0112] According to one aspect of the disclosure, the heme moiety present in cells, culture medium, lysates, hydrolysates, and filtered hydrolysates can be free or bound to amino acids, peptides, oligopeptides, polypeptides, nucleotides, oligonucleotides, nucleic acids, carbohydrates (such as monosaccharides, disaccharides, oligosaccharides, polysaccharides), and / or trapped in lipid structures.

[0113] Hansenula polymorpha.

[0114] Methods for transforming Ogataea polymorpha are well known in the art and include chemical transformation, electroporation, transduction, and biolistic particle delivery. For example, plasmids for chemical transformation are incorporated or integrated into the yeast genome by homologous recombination (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); 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)).

[0115] Ogataea polymorpha has many alternative names in the art, such as Candida thermophila, Hansenula angusta, Ogataea polymorpha, Hansenula thermophila, and Globospora methanophila. (See 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); Morais & M.H. Maia, An. Esc. Sup. Quim. Univ. Recife 1:16 (1959); G. Péter, Tornai - Leh., K.S. Shin & Dlauchy, FEMS Yeast Res. 7(3):495 (2007); and Urakami, Trav. Sous - Sect. - Khiakta, Sect. Pays d'Amour Soc. Imp. Russe Géogr.:1 (1975)).

[0116] Hansenula polymorpha is considered a protein factory, a ubiquitous unconventional methylotrophic yeast, and is well-known as a model yeast strain for peroxisome biology. However, its abnormal gene regulation related to abiotic stress tolerance, oxidative environment, methanol metabolism, heavy metal resistance, and nitrate assimilation defines this strain as a strong candidate for further understanding its role in food and feed applications. Methylotrophic yeasts can grow in extreme environments. Hansenula polymorpha can tolerate temperatures of 45 °C and higher, and the ability of the strain to grow on methanol as the sole energy and carbon source is achieved through the methanol utilization pathway common to all known methylotrophic yeasts. Gene expression is affected by a carbon source-dependent repression / derepression / induction mechanism, which is conferred by the nature of the methylotrophic yeast promoter. There are specific promoters that are repressed by glucose, derepressed by glycerol, and induced by methanol.

[0117] The cultivation of methylotrophic yeasts is known in the art (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. polymorpha DL-1, BMC Biotechnol. 11, 8 (2011); Scheidle et al., High-throughput screening of O. polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale, FEMS Yeast Research, 10(1), 83 - 92 (2009)). In some aspects, the carbon source used to produce recombinant myoglobin is glycerol. However, since the promoter used is methanol-regulated, in these cases, it may be necessary to supplement the medium with methanol. The methanol requirement can be replaced by substituting the methanol-inducible promoter with a constitutive or sugar-inducible specific promoter. Thus, Hansenula polymorpha can be cultured by supplementing various substrates such as glucose, glycerol, xylose, and cellobiose as the sole carbon source.

[0118] The term "cultivation" as used herein refers to growing a recombinant Hansenula polymorpha strain under optimal physical, chemical, and operating parameters for high cell growth and protein production.

[0119] In some aspects, Hansenula polymorpha yeast cells can be cultured under different physical conditions, where an acid or base solution can be used to maintain 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 about 7.5.

[0120] In some aspects, Hansenula polymorpha yeast cells can grow in the presence of one or more of the following carbon sources: methanol, glycerol, glucose, galactose, fructose, sucrose, xylose, arabinose, or crude glycerol, hydrolysates of post-harvest agricultural residues (lignocellulose), post-fermentation residues rich in fiber and / or protein and their hydrolysates, starch hydrolysates, duckweed, seaweed, or algal biomass hydrolysates, and other renewable raw materials.

[0121] In some aspects, Hansenula polymorpha yeast cells can be grown by supplementing with one or more of the following nitrogen sources: ammonium phosphate; ammonium sulfate; animal, plant, and fungal hydrolysates such as meat extract; hydrolyzed milk protein; hydrolyzed corn protein; hydrolyzed soy protein; hydrolyzed pea protein; hydrolyzed rice protein; corn peptone; soy peptone; hydrolyzed potato protein; yeast hydrolysate; bacterial hydrolysate; fungal hydrolysate; and malt extract.

[0122] In some aspects, when growing on a synthetic or chemically defined medium, the growth of Hansenula polymorpha yeast biomass is supplemented with macronutrients, micronutrients, vitamins, and minerals.

[0123] In some aspects, the optimal carbon and nitrogen sources for improving yeast biomass growth and heme production are glycerol and hydrolyzed corn protein powder.

[0124] A medium optimized for culturing and expressing recombinant heme proteins in genetically modified Hansenula polymorpha can contain, for example:

[0125] (1) Pre-inoculation (YPD) medium: 2% soy peptone; 1% yeast extract; 2% glucose; 2% agar for plates; 100 mg / l adenine.

[0126] (2) Preparation instructions: Weigh each substance separately according to the medium preparation protocol. Place the individual components in a mixing container and add water until the weight recorded in the medium preparation protocol is reached. When all components are completely dissolved, measure the pH and conductivity and record them in the medium preparation protocol. In addition, the suspension is sterilized in an autoclave (20 minutes, 121°C).

[0127] (3) Fermentation or production medium (SYN6): 13.32 g / L ammonium dihydrogen phosphate; 3 g / L magnesium sulfate; 3.32 g / L potassium chloride; 0.33 g / L sodium chloride; 20 g / L glycerol; 20 mL (100x calcium chloride); 20 mL trace element 100x solution (6.65 g / L EDTA; 6.65 g / L ammonium ferrous sulfate; 0.55 g / L copper sulfate; 2 g / L zinc sulfate; 2.65 g / L manganese sulfate); 20 mL vitamin 100x solution (0.04 g / L D-biotin; 13.35 g / L thiamine hydrochloride); 20 mL 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).

[0128] (4) Preparation instructions: Weigh each substance separately according to the medium preparation protocol. Place the individual components into a mixing vessel and add water until the weight recorded in the medium preparation protocol is reached. Once all components are completely dissolved, measure the pH and conductivity and record them in the medium preparation protocol.

[0129] (5) After autoclaving, add the following stock solutions to the reactor: 20 mL trace element solution; 20 mL vitamin solution; 20 mL calcium chloride solution; 20 mL trace element solution.

[0130] (6) Cultivation: Once the pre-inoculum is added to the production medium, cultivation is carried out at 37 °C, pH 4.8, with the dissolved oxygen concentration maintained at 30%, and the stirrer speed between 200 - 1400 rpm.

[0131] (7) 10% Structol J 673 is used as an antifoaming agent to prevent any foam deposition or formation; the pH is maintained at 4.8 by adding 12.5% base (25% ammonia solution) and 28% acid (85% phosphoric acid) solution. For pressure reduction, 75% w / v glycerol is fed into the reactor.

[0132] As used herein, the term "parameter" refers to the concentration of chemical components (such as carbon, nitrogen, metal, and non-metal sources), physical conditions (such as pH, temperature, dissolved oxygen level), and operating parameters (such as incubation time, agitation (revolutions per minute (RPM)), aeration), etc.

[0133] In some aspects, cultivation is started in batch mode with 2% w / v glycerol as the carbon source and, once the glycerol concentration in the production medium is less than 10 g / L, is switched to fed-batch mode at a linear feed rate of 2 - 6 g / L / h. During cultivation, the substrate feed rate and the dissolved oxygen (DO) concentration in the reactor are adjusted such that a DO measurement of 20% - 40% is maintained throughout the incubation time at maximum agitation speed. Throughout the cultivation process, the physical parameters are maintained at 37 °C and pH 5.5.

[0134] In some aspects, downstream processing of the yeast cell biomass harvested from the bioreactor using centrifugation, sedimentation, filtration or other mechanical means is fundamental to generating flavor, aroma and nutritional components.

[0135] In some aspects, the present disclosure provides the use of Hansenula polymorpha cells to produce extracts, hydrolysates and / or purified extracts for further use in the production of flavorings. Within the scope of the present disclosure, biomass, extracts, hydrolysates and / or specific fractions of hydrolysates, Maillard reaction precursor mixtures, Maillard reaction products and flavorings derived from Hansenula polymorpha are also referred to as "Hansenula polymorpha-derived products". In some aspects, Hansenula polymorpha biomass is used directly, in wet or dry form, in the production of flavorings, nutritional additives or food products. In some aspects, Hansenula polymorpha biomass is lysed to obtain an extract which is used in the production of flavorings or food products. In some aspects, the Hansenula polymorpha extract is hydrolyzed for further use in flavorings or food products. In some aspects, a filtration step is performed on the yeast extract or hydrolyzed yeast extract to concentrate specific fractions. In some aspects, the flavoring is a food product. In some aspects, the flavoring is used to impart a characteristic flavor to a food product. In some aspects, Hansenula polymorpha biomass is exposed to solvent extraction to concentrate and purify specific solvent-soluble components such as heme, nucleotides and / or amino acids.

[0136] In some aspects, the present disclosure provides Hansenula polymorpha that has been genetically modified to produce recombinant heme protein.

[0137] In some aspects, the present disclosure provides Hansenula polymorpha that has been genetically modified to produce recombinant heme protein, wherein the recombinant heme protein has been removed from the cell lysate by filtration, precipitation or any other known separation method. In some aspects, the remaining components are hydrolyzed to produce a hydrolyzed yeast extract and its specific fractions.

[0138] The terms "transgenic", "recombinant", "genetically engineered", "genetically modified", "modified" in relation to yeast (e.g., Hansenula polymorpha) mean that the yeast has been transformed or transduced with one or more exogenous nucleic acids (recombinant sequences). The term "transformation" refers to the process of introducing and expressing recombinant sequences in yeast cells by using non-viral vectors.

[0139] The term "regulatory element" includes promoters that affect RNA polymerase binding. As used herein, a "promoter" refers to a DNA fragment that controls the start point and start frequency of transcription (RNA synthesis) of a gene located under the control of a promoter element in a host organism. Promoter elements that may be considered for use in practicing the methods disclosed herein 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), triose phosphate isomerase (TRI), phosphoglycerate mutase (PGM), pyruvate kinase (PYK), pyruvate dehydrogenase (PDH), isocitrate lyase (ICL1), L-rhamnulose dehydrogenase (LRA3), L-2-keto-3-deoxy-rhamnulate (LRA4), glycosylphosphatidylinositol (GPI)-anchored protein (GCW14), maltase (MAL), dihydroxyacetone synthase (DAS), alcohol dehydrogenase (ADH2), 6-phosphogluconate dehydrogenase (PGD), transaldolase (TAL), ribulose-5-phosphate epimerase (RPE), catalase (CAT), superoxide dismutase (SOD), trehalose-6-phosphate (TPS1), and the plasma membrane ATPase pump (PMA1) promoter from Hansenula polymorpha, the MOX, AOX1, and GAP1 promoters from Pichia pastoris, and the ADH1, pyruvate decarboxylase (PDC1), GAP1, and glycerol uptake proteins (GUP1 and GUP2) from Saccharomyces cerevisiae.

[0140] As used herein, a "transcription terminator" refers to a DNA fragment containing an RNA polymerase signaling structure that causes transcription to terminate. Examples of available terminator elements are MOX (SEQ ID NO:4), amine oxidase (AMO), or the phosphate starvation (PHO1) terminator from Hansenula polymorpha.

[0141] In some aspects, the nucleic acid construct comprises the FMD promoter (SEQ ID NO:3).

[0142] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:3.

[0143] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:3.

[0144] In some aspects, the nucleic acid construct comprises the MOX promoter (SEQ ID NO:4).

[0145] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:4.

[0146] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:4.

[0147] In some aspects, the nucleic acid construct comprises a marker. In some aspects, the marker is a auxotrophic marker or a dominant marker. In some aspects, the auxotrophic marker can be: Sc LEU2* (leucine auxotroph), ScURA3 (uracil auxotroph) (SEQ ID NO:5), Hp URA3 (uracil auxotroph), Hp ADE11 (adenine auxotroph), Hp MET6 (methionine auxotroph), Hp LEU2 (leucine auxotroph), Hp AUR1 (pimaricin auxotroph) or Hp IMH3 (inosine monophosphate dehydrogenase auxotroph). In some aspects, the dominant marker can be: Sh-ble (bleomycin resistance), Sn-nat1 (nourseothricin resistance), Kp-hph (hygromycin B resistance), Tn-KanMX (G418 / geneticin resistance) or Sv-Pat (bialaphos resistance). In some aspects, the nucleic acid construct comprises the ScURA3 (uracil auxotroph) (SEQ ID NO:5) marker.

[0148] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:5.

[0149] In some aspects, the nucleic acid construct comprises a nucleic acid sequence having 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% sequence identity to SEQ ID NO:5.

[0150] "Transformation" is the term that explains the technology of transferring exogenous DNA into selected cells to generate recombinant or genetically modified cells. Yeast strain transformation protocols, especially for Hansenula polymorpha, are well known in the art and include the spheroplast method, chemical transformation, electroporation, transduction, the glass bead method, and biolistic particle delivery (Gellissen et al., New yeast expression platforms based on methylotrophic O. polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison, FEMS Yeast Res 5(11):1079-96 (Nov 2005); 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)).

[0151] In some aspects, Hansenula polymorpha expressing the recombinant myoglobin gene is cultured under known culture medium components, physical, and operating parameters.

[0152] In some aspects, Hansenula polymorpha expressing the recombinant myoglobin gene is cultured under synthetic or chemically defined culture medium components, which include a carbon source, a nitrogen source, vitamins, minerals, macro- and micronutrients, and metal supplements. Operating parameters are maintained to obtain higher biomass and higher protein expression.

[0153] In some aspects, recombinant Hansenula polymorpha expressing the myoglobin gene is grown in a complex medium containing corn protein hydrolysate to achieve a cost-effective and commercially feasible method. In some aspects, including corn protein hydrolysate powder as a nitrogen source also supplements the production medium with the necessary vitamins, minerals, and trace elements that must be added when using a synthetic medium.

[0154] As used herein, the term "downstream" includes unit procedures for separating yeast biomass, cell lysis, and separating and purifying the heme-containing protein (specifically myoglobin from whole cells), as the protein of interest is intracellular.

[0155] In some aspects, the downstream process for recovering recombinant myoglobin from Hansenula polymorpha biomass may include steps (i) to (iii):

[0156] (i) Cell washing: The microbial cell biomass produced by bioreactor cultivation is harvested by a basket centrifuge (630RS). The cell pellet is resuspended in deionized water (it can also be resuspended in a buffer, salt solution, or a suitable surfactant) and centrifuged at a force of 100 - 8000g or relative centrifugal force (RCF). This process is repeated 1 - 4 times, and samples of a known volume are collected at each step for the qualitative and quantitative characterization of microbial cells, metabolites, and other characteristic compounds. After the final washing step, the cells are resuspended in a lysis buffer (dihydrogen phosphate; hydrogen phosphate and / or water) for cell disruption. In some aspects, cell washing and recovery can be achieved by standard and well-known methods such as various forms of filtration and centrifugation-based methods such as continuous centrifugation or decantation. In some aspects, the cell lysis buffer can be changed to a standard and well-known buffer that contains compounds such as phosphate, tris, borate, citrate, acetate, glycine, or diethanolamine. In some aspects, the spent medium is preserved with the biomass without washing.

[0157] (ii) Cell disruption: Wild-type or recombinant microbial cells can be suspended in a lysis buffer and disrupted using physical (bead milling), chemical (acid, base, or hydrogen peroxide), enzymatic (lysozyme), and advanced high-throughput methods (high-pressure homogenization or hydrodynamic cavitation). These techniques are employed based on the product and preference. For example, for recombinant Hansenula polymorpha expressing myoglobin, the yeast cell suspension is passed through a Dyno mill 1 - 7 times. During milling, the suspension is maintained at or below 55 °C to preserve the structure and function of the protein and heme groups for further steps. After cell disruption, the suspension is centrifuged at 100 - 8000 RCF for 60 minutes. Thereafter, the supernatant is collected and can be stored frozen at -10 °C or lower.

[0158] (iii) Protein recovery and purification: The final recovery and purification of myoglobin from the cell lysate are obtained through consecutive steps using microfiltration and ultrafiltration modules with membranes having a molecular weight cut-off of 1 μm to 1 kDa.

[0159] In some aspects, provided herein are methods of describing the production of a holoprotein that binds heme. In some aspects, the method comprises the steps of: a) culturing a transgenic Hansenula polymorpha yeast having a homologous or heterologous nucleic acid sequence of a rate-limiting gene or a gene sequence encoding an entire heme biosynthetic pathway operably linked to a methanol-inducible or constitutive promoter; b) culturing the transgenic Hansenula polymorpha yeast overexpressing heme biosynthesis in a medium without addition of exogenous heme; and optionally c) isolating and purifying the heme protein. In some aspects, the expression of a recombinant animal-derived heme protein is induced. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested and spray-dried. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested, lysed and spray-dried. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested, lysed, their contents are hydrolyzed and spray-dried.

[0160] In some aspects, provided herein are methods of producing a recombinant animal-derived heme protein. In some aspects, the method comprises a) introducing a nucleic acid construct into a Hansenula polymorpha yeast strain, wherein the nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding an animal-derived heme protein; b) culturing the transgenic Hansenula polymorpha yeast cells in a medium without overexpressing the heme biosynthetic pathway or exogenously supplementing heme molecules; and optionally c) isolating the animal heme protein from the culture medium or yeast cells based on intracellular or extracellular expression. In some aspects, the expression of a recombinant animal-derived heme protein is induced. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested and spray-dried. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested, lysed and spray-dried. In some aspects, after culturing and inducing the expression of the recombinant animal-derived heme protein, the cells are harvested, lysed, their contents are hydrolyzed and spray-dried.

[0161] In some aspects, the method produces a heme protein having a holoprotein with 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%, or at least about 85%, or at least about 90%, or 100% heme loading. In some aspects, the heme loading on the protein can be estimated by the HPLC quantification or colorimetric adsorption method of the present invention.

[0162] In some aspects, the method produces a heme protein of a holoprotein loaded with heme that has 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%, or at least 80%, or at least 85%, or at least 90%, or 100% heme.

[0163] In some aspects, the method produces a heme protein of a holoprotein loaded with heme that has 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%, or about 80%, or about 85%, or about 90%, or 100% heme.

[0164] In some aspects, provided herein is a method for producing recombinant bovine myoglobin, the method comprising: a) introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Hansenula polymorpha yeast cells; b) culturing the Hansenula polymorpha containing the recombinant bovine myoglobin nucleic acid to promote the expression of recombinant myoglobin without overexpressing the entire heme biosynthetic pathway and without exogenously supplementing heme to the culture medium. In some aspects, the method further comprises extracting and purifying the recombinant bovine myoglobin and subjecting all remaining fractions to further processing to produce a hydrolyzed yeast extract. In some aspects, the heme biosynthetic pathway in Hansenula polymorpha can be modified or exogenous heme can be supplemented to the culture medium.

[0165] In some aspects, provided herein are methods for producing a heme protein having a heme loading greater than 50%. In some aspects, the method comprises the steps of: a. providing a culture of transgenic Hansenula polymorpha containing a nucleic acid encoding a heme protein operably linked to a methanol-inducible promoter; b. culturing the transgenic Hansenula polymorpha without adding exogenous heme to the culture; and c. isolating and purifying the heme protein.

[0166] In some aspects, the method produces a heme protein having a heme loading greater than 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%.

[0167] In some aspects, the method produces a heme protein having a heme loading greater than about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%.

[0168] In some aspects, the method produces heme proteins with a heme loading greater than at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

[0169] In some aspects, provided herein is a method for producing an animal-derived heme protein with a heme loading greater than 50%. In some aspects, the method comprises: a. introducing a nucleic acid construct into a strain of Hansenula polymorpha, wherein the nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding an animal-derived heme protein; b. culturing the transgenic Hansenula polymorpha cells without adding exogenous heme molecules to the culture; and c. isolating the animal heme protein from the culture.

[0170] In some aspects, the method produces an animal-derived heme protein with a heme loading greater than 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%.

[0171] In some aspects, the method produces an animal-derived heme protein with a heme loading greater than about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%.

[0172] In some aspects, the method produces an animal-derived heme protein with a heme loading greater than at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

[0173] In some aspects, provided herein is a method for producing recombinant bovine myoglobin comprising recombinant bovine myoglobin carrying greater than 50% heme. In some aspects, the method comprises: a. introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Hansenula polymorpha yeast cells; b. culturing the Hansenula polymorpha containing the recombinant bovine myoglobin nucleic acid without adding heme to the culture to promote the expression of recombinant myoglobin, thereby expressing recombinant myoglobin. In some aspects, the method further comprises extracting and purifying the recombinant bovine myoglobin.

[0174] In some aspects, the method produces recombinant myoglobin comprising recombinant bovine myoglobin carrying greater than 50% heme. In some aspects, the method produces recombinant myoglobin comprising 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%, or at least about 85%, or at least about 90%, or 100% recombinant bovine myoglobin carrying heme.

[0175] In some aspects, the method produces recombinant myoglobin comprising about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80% heme-bearing recombinant bovine myoglobin.

[0176] In some aspects, the method produces recombinant myoglobin comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% heme-bearing recombinant bovine myoglobin.

[0177] In some aspects, the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthesis pathway or an exogenous component of the heme biosynthesis pathway.

[0178] In some aspects, the heme protein is selected from the group consisting of heme proteins of animal origin and heme proteins of plant origin.

[0179] In some aspects, the heme protein of animal origin is selected from the group consisting of heme proteins of bovine origin, heme proteins of porcine origin, heme proteins of ovine origin, heme proteins of equine origin and heme proteins of caprine origin.

[0180] In some aspects, the heme protein of animal origin 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.

[0181] In some aspects, the nucleic acid comprises a nucleic acid sequence having at least about 70% identity with the nucleic acid sequence of SEQ ID NO:1.

[0182] In some aspects, the nucleic acid comprises a nucleic acid sequence having 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% sequence identity with the nucleic acid sequence of SEQ ID NO:1.

[0183] In some aspects, the nucleic acid comprises a nucleic acid sequence having 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% sequence identity with the nucleic acid sequence of SEQ ID NO:1.

[0184] In some aspects, myoglobin comprises an amino acid sequence corresponding to SEQ ID NO:6. In some aspects, myoglobin comprises an amino acid sequence having 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% sequence identity to the amino acid sequence of SEQ ID NO:6. In some aspects, myoglobin comprises an amino acid sequence having 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% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0185] In some aspects, provided herein is a food composition comprising any heme protein disclosed herein or any bovine myoglobin disclosed herein.

[0186] In some aspects, provided herein is a meat analogue food composition comprising a lysate of any cultured Hansenula polymorpha provided herein.

[0187] In some aspects, provided herein is a meat analogue food composition comprising any heme protein disclosed herein or any bovine myoglobin disclosed herein.

[0188] Recombinant heme proteins can be isolated for use in preparing food compositions. In some aspects, recombinant heme proteins produced by modified Hansenula polymorpha can be used in their entirety, fractions, and modifications thereof, including dissolved, precipitated, partially or fully hydrolyzed, cross-linked, emulsified, texturized, cooked, extruded, high-shear mixed, Couette cell-produced, reacted (including nitrite-based curing) structured forms, to prepare meat and meat (i.e., meat analogue) foodstuffs, including ground meat and meat analogues, such as minced meat, meat strips, chunks, pieces, and steaks; reconstituted, 3D printed, 3D printed and shaped meat products, including hamburgers, meat slices, balls, rings, fingers, rings, fingers, strips, flat sheets; reconstituted and stuffed / filled meat (i.e., meat analogue) products, including sausages, ham-like products, spreadable foods; reconstituted and coated meat products, including chicken nuggets, meat patties, meat strips, popcorn-like pieces, rings, etc.

[0189] Extracts from Hansenula polymorpha.

[0190] In some aspects, provided herein are cell extracts from a culture of Hansenula polymorpha. Hansenula polymorpha is a methylotrophic yeast and is a good molecular source for the food and nutrition industries.

[0191] In some aspects, a Hansenula polymorpha extract is obtained from genetically modified Hansenula polymorpha that produces a recombinant heme protein. The recombinant heme protein can be a recombinant plant heme protein, a recombinant bacterial heme protein, a recombinant fungal heme protein, or a recombinant animal heme protein. In some aspects, the recombinant protein is selected from the group consisting of: hemoglobin, myoglobin, leghemoglobin, neuroglobin, cytoglobin, cytochrome P450s, cytochrome c oxidase, ligninase, catalase, peroxidase, cytochrome a, cytochrome b, and cytochrome c.

[0192] In some aspects, the Hansenula polymorpha extract 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 of heme.

[0193] In some aspects, the Hansenula polymorpha extract contains 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 or about 3.1% w / w of heme.

[0194] In some aspects, the Hansenula polymorpha extract contains from about 0.03% w / w to about 3.1% w / w, from about 0.1% w / w to about 3.1% w / w, from about 0.5% w / w to about 3.1% w / w, from about 1% w / w to about 3.1% w / w, from about 1.5% w / w to about 3.1% w / w, from about 2% w / w to about 3.1% w / w, from about 2.5% w / w to about 3.1% w / w, from about 0.1% w / w to about 3% w / w, from about 0.1% w / w to about 2.5% w / w, from about 0.1% w / w to about 2% w / w, from about 0.1% w / w to about 1.5% w / w, from about 0.1% w / w to about 1% w / w or from about 0.1% w / w to about 0.5% w / w of heme.

[0195] In some aspects, the Hansenula polymorpha extract contains at least 0.8% w / w of glutamic acid and at least 0.2% of glutathione.

[0196] In some aspects, the Hansenula polymorpha extract contains 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 of glutamic acid.

[0197] In some aspects, the Hansenula polymorpha extract contains 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 of glutamic acid.

[0198] In some aspects, the Hansenula polymorpha extract contains from about 0.8% w / w to about 4% w / w, from about 1% w / w to about 4% w / w, from about 1.5% w / w to about 4% w / w, from about 2% w / w to about 4% w / w, from about 2.5% w / w to about 4% w / w, from about 3% w / w to about 4% w / w, from about 3.5% w / w to about 4% w / w, from about 0.8% w / w to about 3.5% w / w, from about 0.8% w / w to about 3% w / w, from about 0.8% w / w to about 2.5% w / w, from about 0.8% w / w to about 2% w / w, from about 0.8% w / w to about 1.5% w / w or from about 0.8% w / w to about 1% w / w of glutamic acid.

[0199] In some aspects, the Hansenula polymorpha extract contains 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% of glutathione.

[0200] In some aspects, the Hansenula polymorpha extract contains 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% of glutathione.

[0201] In some aspects, the Hansenula polymorpha extract contains from about 0.2% to about 1%, from about 0.5% to about 1%, from about 0.7% to about 1%, or from about 0.2% to about 0.5% of glutathione.

[0202] In some aspects, the Hansenula polymorpha extract is rich in 5'-ribonucleotides such as 5'-GMP and / or 5'-IMP. In some aspects, the Hansenula polymorpha extract contains at least 0.007% of 5'-ribonucleotides. In some aspects, the Hansenula polymorpha extract contains at least 0.007% of 5'-GMP.

[0203] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-ribonucleotides.

[0204] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-ribonucleotides.

[0205] In some aspects, the Hansenula polymorpha extract contains from about 0.005% to about 0.05%, from about 0.007% to about 0.05%, from about 0.01% to about 0.05%, from about 0.02% to about 0.05%, from about 0.03% to about 0.05%, from about 0.04% to about 0.05%, from about 0.007% to about 0.04%, from about 0.007% to about 0.03%, from about 0.007% to about 0.02%, or from about 0.007% to about 0.01% of 5'-ribonucleotides.

[0206] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-GMP.

[0207] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-GMP.

[0208] In some aspects, the Hansenula polymorpha extract contains from about 0.005% to about 0.05%, from about 0.007% to about 0.05%, from about 0.01% to about 0.05%, from about 0.02% to about 0.05%, from about 0.03% to about 0.05%, from about 0.04% to about 0.05%, from about 0.007% to about 0.04%, from about 0.007% to about 0.03%, from about 0.007% to about 0.02%, or from about 0.007% to about 0.01% of 5'-GMP.

[0209] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-IMP.

[0210] In some aspects, the Hansenula polymorpha extract contains 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% of 5'-IMP.

[0211] In some aspects, the Hansenula polymorpha extract contains from about 0.005% to about 0.05%, from about 0.007% to about 0.05%, from about 0.01% to about 0.05%, from about 0.02% to about 0.05%, from about 0.03% to about 0.05%, from about 0.04% to about 0.05%, from about 0.007% to about 0.04%, from about 0.007% to about 0.03%, from about 0.007% to about 0.02% or from about 0.007% to about 0.01% of 5'IMP.

[0212] In some aspects, the Hansenula polymorpha extract contains at least: 0.03% w / w of heme, 0.8% w / w of glutamic acid and 0.2% w / w of glutathione.

[0213] In some aspects, the Hansenula polymorpha extract contains at least: 0.03% w / w of heme, 0.8% w / w of glutamic acid, 0.2% w / w of glutathione and 0.007% of 5'-ribonucleotide (e.g., 5'IMP and / or 5'GMP).

[0214] In some aspects, the Hansenula polymorpha extract contains at least: 0.03% w / w of heme, 0.8% w / w of glutamic acid, 0.2% w / w of glutathione and 0.007% of 5'GMP.

[0215] In some aspects, the Hansenula polymorpha extract contains at least 0.03% w / w of heme and at least 0.007% of 5'GMP.

[0216] In some aspects, the Hansenula polymorpha extract contains at least 0.8% w / w of glutamic acid, 0.2% of glutathione and 0.007% of 5'-ribonucleotide, and is substantially free of heme.

[0217] In some aspects, the Hansenula polymorpha extract contains about 0.8% w / w of glutamic acid and about 0.2% of glutathione.

[0218] In some aspects, the Hansenula polymorpha extract is rich in 5'-ribonucleotides such as 5'GMP and / or 5'IMP. In some aspects, the Hansenula polymorpha extract contains about 0.007% of 5'-ribonucleotide. In some aspects, the Hansenula polymorpha extract contains about 0.007% of 5'GMP.

[0219] In some aspects, the Hansenula polymorpha extract contains about 0.03% w / w of heme, about 0.8% w / w of glutamic acid and about 0.2% w / w of glutathione.

[0220] In some aspects, the Hansenula 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'-ribonucleotides (e.g., 5'IMP and / or 5'GMP).

[0221] In some aspects, the Hansenula 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.

[0222] In some aspects, the Hansenula polymorpha extract contains about 0.03% w / w heme and about 0.007% 5'GMP.

[0223] In some aspects, the Hansenula polymorpha extract contains about 0.8% w / w glutamic acid, about 0.2% glutathione and about 0.007% 5'-ribonucleotides, and is substantially free of heme.

[0224] In some aspects, the method for obtaining the Hansenula polymorpha extract comprises the steps of: i) culturing and propagating Hansenula polymorpha cells, ii) optionally washing and harvesting the cells; and iii) lysing the cells. Cell lysis can be carried out by methods known in the art, such as autolysis, physical lysis, mechanical lysis, chemical lysis (mediated by acid or base solutions), enzymatic lysis, microbial lysis or combinations thereof.

[0225] In some aspects, after culturing and propagating Hansenula polymorpha cells, the cells are washed and harvested to produce Hansenula polymorpha biomass.

[0226] In some aspects, after culturing and propagating Hansenula polymorpha cells, the cells are collected with a culture medium or a portion of the culture medium to produce Hansenula polymorpha biomass.

[0227] In some aspects, after lysis, it is spray-dried in order to produce solid Hansenula polymorpha biomass. In some aspects, after lysis, it is spray-dried in order to produce solid Hansenula polymorpha extract.

[0228] In some aspects, to obtain a heme-rich extract, a low temperature is maintained due to the instability of the heme molecule and its rapid degradation at higher temperatures. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is below 70°C. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is from about 40°C to about 70°C. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is from about 45°C to about 60°C. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is from about 48°C to about 58°C. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is from about 49°C to about 57°C. In some aspects, the temperature of the method for obtaining a heme-rich Hansenula polymorpha extract is from about 50°C to about 55°C. If a heat pasteurization step is required, the temperature of the pasteurization step can be as high as about 90°C for about 1 second to about 100 seconds.

[0229] In some aspects, the temperature of the method for obtaining a Hansenula polymorpha extract is temperature independent, wherein the heme has been previously isolated and separated from the biomass.

[0230] In some aspects, the method of obtaining a Hansenula polymorpha extract is independent of temperature, and the resulting extract is substantially free of heme.

[0231] In some aspects, the H. polymorpha biomass contains at least 0.03% w / w heme per dry cell weight.

[0232] In some aspects, the Hansenula polymorpha extract contains at least 0.03% w / w heme per dry weight.

[0233] In some aspects, the H. polymorpha biomass contains about 0.03% w / w heme per dry cell weight.

[0234] In some aspects, the Hansenula polymorpha extract contains about 0.03% w / w heme per dry weight.

[0235] In some aspects, to obtain a H. polymorpha extract having high glutamate-derived compounds, the proteins of the H. polymorpha extract are hydrolyzed, and further induced to enzymatically convert glutamate into glutamate using glutaminase.

[0236] In some aspects, to obtain an extract of Hansenula polymorpha having high 5' ribonucleotides, it is preferred to hydrolyze nucleic acids with nucleases or chemicals. In some aspects, to produce 5' IMP by conversion of 5' AMP to 5' IMP, it is necessary to add an enzyme, such as 5' adenosine deaminase. In order to obtain the highest amount of 5' ribonucleotides, it is important to inactivate the endogenous acid, neutral or alkaline phosphatases of Hansenula polymorpha. Endogenous phosphatases can hydrolyze the phosphate groups in the nucleotides to produce nucleosides, which are no longer used for flavoring purposes. In some aspects, the extract of Hansenula polymorpha is enriched in 5' GMP and / or 5' IMP.

[0237] In some aspects, the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme and / or glutamate-derived compounds and enriched in 5' ribonucleotides, the temperature of the method is above 70°C. In some aspects, the temperature of the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme is from about 40°C to about 70°C. In some aspects, the temperature of the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme is from about 45°C to about 60°C. In some aspects, the temperature of the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme is from about 48°C to about 58°C. In some aspects, the temperature of the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme is from about 49°C to about 57°C. In some aspects, the temperature of the method of obtaining a hydrolyzed Hansenula polymorpha extract enriched in heme is from about 50°C to about 55°C. If a heat pasteurization step is required, the temperature of the pasteurization step can be as high as about 90°C for about 1 second to about 100 seconds.

[0238] In some aspects, the temperature of the method for obtaining a hydrolyzed Hansenula polymorpha extract is temperature independent, wherein the heme has been previously separated and isolated from the extract.

[0239] In some aspects, the method of obtaining a hydrolyzed Hansenula polymorpha extract is temperature independent, and the resulting hydrolyzed extract is substantially free of heme.

[0240] In some aspects, lysis of the Hansenula polymorpha cells is achieved at an alkaline pH of about 7 to about 12, about 7.5 to about 10.5, about 8 to about 10, or about 8.5 to about 9.0. At the pH disclosed herein, lysis of the cells is achieved, but endogenous acid phosphatases are also inactivated, and the viscosity is kept low enough to allow for adequate agitation during the process.

[0241] In some aspects, lysis of Hansenula polymorpha cells at alkaline pH is carried out by incubation with a lytic enzyme mixture containing a protease (preferably a protease active in the neutral to alkaline pH range), a glucanase (preferably a glucanase active in the neutral to alkaline pH range), a mannanase (preferably active in the neutral to alkaline pH range), and a chitinase (active in the neutral to alkaline pH range). Some well-known commercial lytic enzymes available are Kitalase, Zymolyase, Lyticase, Glusulase, (Sharma, M. et al., A review on microbial alkaline protease: an essential tool for various industrial approaches, Industrial Biotechnology, 15(2):69 - 78(2019); Sharma, K.M. et 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).

[0242] In some aspects, the proteolysis of the alkaline lysis mixture can be further carried out with other proteases, and the pH of the medium is adjusted to suit the activity of the selected protease. After lowering the pH to neutral or even acidic (pH from about 4 to about 7), the action of neutral or acidic proteolytic enzymes can be carried out. In some aspects, proteolysis can be achieved by a mixture of proteolytic enzymes. Commercially available proteolytic enzymes can be used, such as Protease Protease P, ProteAX, Papain, Bromelain, and Sumizyme BNP - L.

[0243] In some aspects, after proteolysis, the hydrolyzed Hansenula polymorpha extract contains mostly small peptides, such as peptides below 1.0 kDa.

[0244] In some aspects, after proteolysis, the conversion of glutamine to glutamate or its salts is used to increase the content of glutamate compounds, which are considered to impart umami. In some aspects, glutaminase (e.g., L-glutaminase from Escherichia coli, glutaminase from Bacillus amyloliquefaciens, glutaminase from Bacillus licheniformis, protein glutaminase (PG) from Chryseobacterium proteolyticum) can be used to achieve the conversion.

[0245] In some aspects, the proteolyzed Hansenula polymorpha extract is further treated with a deaminase containing at least 0.8% glutamate.

[0246] In some aspects, the proteolyzed Hansenula polymorpha extract is further treated with a deaminase containing at least 0.8% glutathione.

[0247] In some aspects, nucleic acid hydrolysis and the conversion of AMP to IMP can be carried out after or before proteolysis.

[0248] Flavor precursor mixture.

[0249] A flavor precursor mixture is a mixture of compounds characterized by providing flavor, taste, and aroma to a food product. The flavor and aroma are released by cooking the mixture or subjecting it to the Maillard reaction.

[0250] In some aspects, the flavor precursor mixture contains a Hansenula polymorpha extract.

[0251] In some aspects, the flavor precursor mixture contains a hydrolyzed Hansenula polymorpha extract.

[0252] In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 30 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 25 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 20 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 15 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 10 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 5 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 3 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 2 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 1 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain soluble molecules that are equal to or less than 0.5 kDa in size. In some aspects, the hydrolyzed Hansenula polymorpha extract is filtered to retain insoluble molecules.

[0253] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture 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 of heme.

[0254] In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains 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, or about 3.1% w / w of heme.

[0255] In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains at least 0.8% w / w of glutamic acid. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains at least 0.2% w / w of glutathione. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains at least 0.007% of 5'GMP. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains at least 0.03% w / w of heme and / or 0.8% w / w of glutamic acid and / or 0.2% w / w of glutathione and / or 0.007% of 5'GMP.

[0256] In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains about 0.8% w / w of glutamic acid. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains about 0.2% w / w of glutathione. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains about 0.007% of 5'GMP. In some aspects, the polymorphic Hansenula yeast biomass, extract, and / or hydrolyzed extract of the flavor precursor mixture contains about 0.03% w / w of heme, about 0.8% w / w of glutamic acid, about 0.2% w / w of glutathione, and / or about 0.007% of 5'GMP.

[0257] In some aspects, the content of Hansenula polymorpha biomass, extract and / or hydrolyzed extract in the mixture for obtaining the flavor precursor mixture is at least 0.01% of the mixture. In some aspects, the content of Hansenula polymorpha biomass, extract and / or hydrolyzed extract in the mixture for obtaining the flavor precursor mixture is 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%.

[0258] In some aspects, the content of Hansenula polymorpha biomass, extract and / or hydrolyzed extract in the mixture for obtaining the flavor precursor mixture is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 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 72%, about 73%, about 74%, about 75%, 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% of the mixture.

[0259] In some aspects, the content of Hansenula polymorpha biomass, extract and / or hydrolyzed extract in the mixture for obtaining the flavor precursor mixture is about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about 1% to about 5%, about 5% to about 90%, about 10% to about 90%, about 20% to about 90%, 20% to about 72%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90% or about 80% to about 90% of the mixture.

[0260] In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract flavor precursor mixture further comprises monosaccharides such as pentoses and hexoses. The monosaccharides can be reducing sugars selected from the group consisting of rhamnose, arabinose, ribose, D-xylose, glucose, glucosamine, galactose, fructose, steviol and synthetic sweeteners.

[0261] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises 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 of monosaccharides.

[0262] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises 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 of monosaccharides.

[0263] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises from about 0.1% w / w to about 35% w / w, from about 0.1% w / w to about 30% w / w, from about 0.1% w / w to about 25% w / w, from about 0.1% w / w to about 20% w / w, from about 0.1% w / w to about 15% w / w, from about 0.1% w / w to about 10% w / w, from about 0.1% w / w to about 5% w / w, from about 0.1% w / w to about 1% w / w, from about 1% w / w to about 35% w / w, from about 5% w / w to about 35% w / w, from about 10% w / w to about 35% w / w, from about 15% w / w to about 35% w / w, from about 20% w / w to about 35% w / w, from about 25% w / w to about 35% w / w, or from about 30% w / w to about 35% w / w of monosaccharides.

[0264] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture also contains disaccharides such as sucrose, lactose, and maltose.

[0265] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains amino acids such as alanine, arginine, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamine, and mixtures thereof. In some aspects, the flavor precursor mixture contains dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides. In some aspects, the flavor precursor mixture contains oligopeptides. In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains glutathione.

[0266] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains at least 0.4% w / w of histidine.

[0267] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains 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 of histidine.

[0268] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains 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 of histidine.

[0269] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains about 0.4% w / w to about 3% w / w, about 0.5% w / w to about 3% w / w, about 1% w / w to about 3% w / w, about 1.5% w / w to about 3% w / w, about 2% w / w to about 3% w / w, about 2.5% w / w to about 3% w / w, about 0.4% w / w to about 2.5% w / w, about 0.4% w / w to about 2% w / w, about 0.4% w / w to about 1.5% w / w, or about 0.4% w / w to about 1% w / w of histidine.

[0270] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture contains at least 15% w / w of amino acids.

[0271] In some aspects, the polymorphic biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises 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 of amino acids.

[0272] In some aspects, the polymorphic biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises 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 of amino acids.

[0273] In some aspects, the polymorphic biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises from about 0.1% w / w to about 15% w / w, from about 0.5% w / w to about 15% w / w, from about 1% w / w to about 15% w / w, from about 5% w / w to about 15% w / w, from about 10% w / w to about 15% w / w, from about 0.1% w / w to about 10% w / w, from about 0.1% w / w to about 5% w / w, from about 0.1% w / w to about 4% w / w, from about 0.1% w / w to about 3% w / w, or from about 0.1% w / w to about 1% w / w of amino acids.

[0274] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture comprises vitamins, such as vitamin A, vitamin C, vitamin D, vitamin E, vitamin B, vitamin K, and mixtures thereof. In some aspects, the vitamin is a vitamin B complex. In some aspects, the vitamin is vitamin B1.

[0275] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture further comprises extracts from Saccharomyces cerevisiae, Cyberlindnera jadinii, seaweed / kelp, and / or mushrooms.

[0276] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture further comprises fat. The fat can be vegetable oil or fatty acid. In some aspects, the fatty acid is a saturated fatty acid. In some aspects, the fatty acid is an unsaturated fatty acid. In some aspects, the fat is a mixture of unsaturated fatty acid and saturated fatty acid. In some aspects, the fat is an edible vegetable oil such as olive oil, canola oil, sunflower oil, soybean oil, safflower oil, chia oil, rapeseed oil, peanut oil, linseed oil, coconut oil, palm oil, and mixtures thereof.

[0277] In some aspects, the plant fat can be a saturated fatty acid such as lauric acid, myristic acid, palmitic acid, stearic acid.

[0278] In some aspects, the plant fat can be a monounsaturated fatty acid such as 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.

[0279] In some aspects, the plant fat can be free polyunsaturated fatty acids (PUFAs), such as hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, timnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic 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, and mixtures thereof.

[0280] In some aspects, the fat is a mixture of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs).

[0281] In some aspects, the fat is a mixture of vegetable oil and cultivated fat, and the cultivated fat is a culture of fat cells.

[0282] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture further comprises polypeptides, proteins, or protein fragments. In some aspects, the polypeptides, proteins, or protein fragments are from plants. In some aspects, the polypeptides, proteins, or protein fragments are from fungi. In some aspects, the polypeptides, proteins, or protein fragments are from animals. In some aspects, the polypeptides, proteins, or protein fragments are recombinant proteins. In some aspects, the polypeptides, proteins, or protein fragments are recombinant heme proteins. In some aspects, the polypeptides, proteins, or protein fragments are recombinant animal heme proteins.

[0283] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture further comprises a carrier substance. The carrier substance can be a polysaccharide, such as starch, modified starch, carrageenan, gum arabic, gum acacia, carboxymethyl cellulose, chitosan, and mixtures thereof. Other carriers can be Saccharomyces cerevisiae extract and salts.

[0284] Maillard reaction products.

[0285] In some aspects, provided herein is a method for producing a flavor composition from a flavor precursor mixture.

[0286] In some aspects, methods of producing a flavor composition include incubating a flavor precursor mixture under conditions of temperature and water content to obtain a flavor composition.

[0287] In some aspects, the flavor precursor mixture is added to a food matrix and the flavor is generated during cooking.

[0288] The Maillard reaction (MR) is a well-known process, where Maillard reaction products (MRP) are products derived from the MR. MRP are flavor compositions that can be added to food products. When the MRP is combined with another MRP obtained by using other types of amino acids or with a plant protein hydrolysate, the flavor can be improved.

[0289] In some aspects, a heat reaction is performed on a Hansenula polymorpha biomass, extract, and / or hydrolyzed extract flavor precursor mixture to obtain MRP derived from Hansenula polymorpha.

[0290] In some aspects, the temperature of the MR is in the range of about 85 °C to about 200 °C, more preferably about 90 °C to about 160 °C, and most preferably about 95 °C to about 140 °C. In some aspects, the flavor precursor mixture is heated for a period of about 10 minutes to about 60 minutes, about 60 minutes to about 1200 minutes, about 120 minutes to about 900 minutes, about 180 minutes to about 720 minutes, about 240 minutes to about 600 minutes, or about 300 minutes to about 540 minutes. Preferably, the pressure of the Maillard reaction is about 0.1 bar to about 10 bar, about 0.25 bar to about 7.5 bar, about 0.4 bar to about 5 bar, about 0.5 bar to about 3 bar, about 0.75 bar to about 2.5 bar, about 1 bar to about 2 bar. Preferably, the pH of the Maillard reaction is about 0.5 to about 11, about 1 to about 10.5, about 2 to about 10, about 3 to about 9, about 4 to about 8.5, or about 5 to about 8.

[0291] In some aspects, the temperature of the MR is in the range of 85 °C to 200 °C, more preferably 90 °C to 160 °C, and most preferably 95 °C to 140 °C. In some aspects, the flavor precursor mixture is heated for a period of 10 minutes to 60 minutes, 60 minutes to 1200 minutes, 120 minutes to 900 minutes, 180 minutes to 720 minutes, 240 minutes to 600 minutes, or 300 minutes to 540 minutes. Preferably, the pressure of the Maillard reaction is 0.1 bar to 10 bar, 0.25 bar to 7.5 bar, 0.4 bar to 5 bar, 0.5 bar to 3 bar, 0.75 bar to 2.5 bar, 1 bar to 2 bar. Preferably, the pH of the Maillard reaction is 0.5 to 11, 1 to 10.5, 2 to 10, 3 to 9, 4 to 8.5, or 5 to 8.

[0292] In some aspects, after the Maillard reaction, the obtained flavor composition (MRP) can be dried alone or mixed with a carrier (such as maltodextrin) by various methods known in the art, including spray drying, vacuum drying, freeze drying, etc. In some aspects, other flavorings can be added to the MRP. In some aspects of the present disclosure, it is desirable that the MRP derived from Hansenula polymorpha includes the addition of a spiro-1,2,4-trithiol compound, wherein the spiro compound is 3,5-bis(2'-methyltetrahydrofuranyl-3')spiro-1,2,4-trithiol. In some aspects, the amount of the spiro-1,2,4-trithiol compound is from about 1% to about 50% of the spiro compound / 100 g of the MRP composition. In some aspects, the amount of the spiro-1,2,4-trithiol compound is from about 5% to about 40%, from about 10% to about 35%, from about 15% to about 30%.

[0293] In some aspects, the MRP derived from Hansenula polymorpha includes the addition of MSG from other sources (e.g., MSG purified from Corynebacterium glutamicum or other natural sources). In some aspects, the MRP derived from Hansenula polymorpha includes the addition of IMP and / or GMP from other sources. In some aspects, the MRP derived from Hansenula polymorpha includes the addition of nutritional or flavor peptides from other sources from mushroom extracts and / or other natural sources.

[0294] Food product.

[0295] In some aspects, provided herein is a food product containing Hansenula polymorpha cells, cell components or cell fractions.

[0296] In some aspects, provided herein is a food product containing an extract of Hansenula polymorpha.

[0297] In some aspects, provided herein is a food product containing a hydrolyzed extract of Hansenula polymorpha.

[0298] In some aspects, provided herein is a food product containing a flavor precursor mixture comprising Hansenula polymorpha biomass, extract and / or hydrolyzed extract.

[0299] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract of a food product 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 of heme.

[0300] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract of a food product contains 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, or about 3.1% w / w of heme.

[0301] In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises at least 0.8% w / w of glutamic acid. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises at least 0.2% w / w of glutathione. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises at least 0.007% of 5'GMP. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises at least 0.03% w / w of heme, 0.8% w / w of glutamic acid, 0.2% w / w of glutathione and / or 0.007% of 5'GMP.

[0302] In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises about 0.8% w / w of glutamic acid. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises about 0.2% w / w of glutathione. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises about 0.007% of 5'GMP. In some aspects, the Hansenula polymorpha biomass, extract and / or hydrolyzed extract of a food product comprises about 0.03% w / w of heme, about 0.8% w / w of glutamic acid, about 0.2% w / w of glutathione and / or about 0.007% of 5'GMP.

[0303] In some aspects, the disclosure provided herein discloses a food product comprising MRP from Hansenula polymorpha.

[0304] In some aspects of the present invention, the food product is solid or liquid (such as a beverage). The food product of the present invention can be a condiment, a baked good, a protein supplement, a vitamin and mineral supplement, a meat product or a meat substitute product (meat analogue). The meat product or meat substitute can be a meat sausage replica, a meat paste replica, a ground meat replica or a muscle replica. The condiment can be a sauce. The meat substitute can be free of products of direct animal origin. The meat substitute can contain a small amount of products of direct animal origin. Products of direct animal origin are defined as the whole or part isolated from an animal, such as isolated protein, isolated tissue, isolated muscle, isolated cells. In the context of this specification, a recombinant protein produced in a non-animal host is not considered a product of direct animal origin. In the context of this specification, a recombinant nucleic acid encoding an animal protein is not considered a product of direct animal origin.

[0305] The sauce can be a liquid or semi-liquid preparation for consumption as a table appetizer. The composition of the sauce of the present invention can vary according to the desired consistency.

[0306] Tofu is a traditional food product made from soy protein. The process typically involves preparing soy milk by soaking and grinding soybeans, boiling the mixture, and filtering out the remaining particles. The soy residue is further coagulated and pressed into solid blocks. In this way, tofu can be considered a plant-based protein-rich food.

[0307] Tempeh is an Indonesian-based protein-rich food made from fermented soybean cakes and cooked soybeans (such as rice and millet), and is shared with a culture of Rhizopus oligosporus (Bakhsh, Allah et al., "Traditional plant-based meat alternatives, current and a future perspective: A review" J. Agric. Life Sci 55: 1-10 (2021)).

[0308] Seitan is a traditional food product made from wheat flour by activating gluten and washing away the starch. Alternatively, Seitan can be made from commercially available active wheat gluten and hydrated with the addition of water. Heat treatment can then be carried out to cook the product. The resulting product has a chewy texture and can be added as an ingredient to various dishes.

[0309] U.S. Publication No. 2005 / 003071 discloses a plant-based meat analogue processed by sequentially mixing methylcellulose into an ice / water mixture, then into water containing modified gluten and highly soluble plant protein and initiating gelation by gentle heating, and preparing an oil for the emulsion phase, and modified starch. U.S. Patent No. 7,070,827 discloses a method for preparing a plant protein meat analogue, which includes sequentially mixing methylcellulose into a water / ice mixture to form a cream, then mixing modified gluten, a plant protein product highly soluble in water and capable of forming a gel under gentle heat treatment, an oil for preparing an emulsion matrix, and modified food starch and flavoring ingredients to form a flavored emulsion matrix. U.S. Patent No. 10,039,306 discloses a meat replica matrix containing plant protein, sugar, sulfur compounds, and heme-containing protein. U.S. Patent No. 10,863,761 discloses a meat replica comprising a muscle replica, a fat tissue replica, and a connective tissue replica. The muscle replica consists of muscle replicas formed by asymmetric fibers, such as spun fibers or extruded fibers. The fat replica includes a gel in which fat droplets are suspended, where the fat can be vegetable oil, and the gel can be plant protein. The connective tissue replica contains approximately 50% by total weight of protein, approximately 50% by liquid weight, and has low fat and polysaccharide components, where the protein is prolamin.

[0310] In some aspects, a composition derived from Hansenula polymorpha can be used as a dietary supplement to supplement the deficiencies of certain minerals (such as iron), amino acids, vitamins, and nucleotides in the human or animal diet.

[0311] A dietary supplement refers to a product for ingestion that, among other requirements, contains a "dietary ingredient" for supplementing the diet. Dietary supplements containing a composition derived from Hansenula polymorpha can have various forms, such as pills, tablets, capsules, gummies, soft capsules, liquids, and powders.

[0312] In some aspects, a dietary supplement containing a composition derived from Hansenula polymorpha can be used as a nutraceutical composition for treating anemia and other iron deficiency-related diseases. The dietary supplement can contain dead cells of Hansenula polymorpha, where the cells can be intact or incomplete. The dietary supplement can contain lysed cells of Hansenula polymorpha. The dietary supplement can contain a hydrolyzed extract of Hansenula polymorpha. The dietary supplement can contain a solution obtained by filtering the hydrolyzed extract of Hansenula polymorpha. The dietary supplement can contain an insoluble fraction obtained by filtering the hydrolyzed extract of Hansenula polymorpha.

[0313] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract of the dietary supplement 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 of heme.

[0314] In some aspects, the Hansenula polymorpha biomass, extract, and / or hydrolyzed extract of the dietary supplement contains 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, or about 3.1% w / w of heme.

[0315] In some aspects, the Hansenula polymorpha - derived composition can be applied to the surface of the food. In some aspects, the Hansenula polymorpha - derived composition can be dissolved or suspended and injected or mixed into the food. In some aspects, the spray - dried Hansenula polymorpha - derived composition can be mixed with the food.

[0316] Examples

[0317] Example 1. Optimization of the medium for promoting the growth of Hansenula polymorpha and heme biosynthesis.

[0318] 1.1 Carbon and nitrogen source selection: To determine a cost - effective and commercially viable medium for using the Hansenula polymorpha yeast strain for growth and heme biosynthesis, single - factor screening and optimization were performed using different carbon and nitrogen sources. These experiments were carried out in 500 mL conical flasks with a working volume of 100 mL in batch mode. The carbon sources tested were glucose, fructose, pure glycerol, and crude glycerol. The nitrogen sources evaluated were synthetic medium (SYN6), corn steep liquor, and corn gluten hydrolysate. After optimization, the medium components and operating parameters were verified in a 150 L bioreactor operating in fed - batch fermentation mode.

[0319] 1.2 Fermentation conditions: Inoculate a YPD agar plate (2% glucose) with frozen cells from a glycerol cell bank culture and incubate at 30 °C - 37 °C for 45 - 65 hours. Inoculate the seed culture from a fresh YPD agar plate into YPD medium (2% w / v glucose) and grow overnight (20 - 25 hours, 180 rpm, d = 2.5 cm, 30 °C - 37 °C).

[0320] In a bioreactor, the cultivation was started in batch mode with a carbon (glycerol) source concentration of 2% w / v. An additional carbon (glycerol) source was continuously added in fed-batch mode within 35 - 40 hours at a feeding rate of 2 - 6 g / (l*h). The substrate feeding, aeration, and impeller speed (RPM) were adjusted in such a way that the dissolved oxygen was maintained between 20% - 40% DO2 at the maximum stirring speed. During fermentation, the pH was set and maintained at 4.5 - 5.0 (corrected medium: 12.5% (v / v) ammonia solution and 28% (v / v) phosphoric acid solution). The antifoaming agent (10% (w / v) PEG 6000) was added appropriately by an AF controller. After 40 hours of cultivation, 0.5% v / v methanol was added as an inducer of oxidative stress, and the cultivation was further extended for 24 hours before harvesting.

[0321] 1.3 Cell lysis and heme extraction: The cells were washed and resuspended 3 - 4 times using continuous centrifugation to remove insoluble or extracellular deposits or medium components on the cell surface. After the final wash, the cells were resuspended in 20 mM NaOH PBS buffer for cell disruption. For cell lysis, a Dyno mill, high-pressure homogenizer, and / or ultrasonic wave were used. After cell disruption, the suspension was centrifuged to remove any insoluble particles, and the supernatant could be stored at -20 °C for further use. Heme was extracted with 4 volumes of acidic acetone and incubated at -20 °C for 20 minutes, further centrifuged, and the sample was diluted 10x with acidic acetonitrile for quantification using HPLC.

[0322] 1.4 Heme quantification: An HPLC method using gradient flow with acetonitrile (A) and 0.1% acetic acid in water (B) as the mobile phase was developed. Stationary phase: Luna 5uM, C18(2), 100A, 250*4.6 mm column. Flow rate: 0.5 mL / min. Column pressure: 50 - 60 Kgf / cm2. Detector: SPD-M20A, diode array detector. Wavelength: 406 nm. Hemin stock solution: 41 mg of hemin (2.5 mM) was dissolved in 25 mL of PBS containing 20 mM NaOH. Hemin working stock solution: The hemin stock solution was diluted 50-fold (50 uM) using acidic acetonitrile (80% acetonitrile containing 20% 1.6 M HCl), resulting in a pink or red suspension (pH < 5). HPLC standards: Using the 50 μM working stock solution, more samples were prepared by diluting to the desired concentration using acidic acetonitrile.

[0323] 1.5 Results: Figure 1AShows the heme production (per gram of cell dry weight) obtained from Hansenula polymorpha cultured on different carbon sources with and without methanol induction. Due to its reducibility, glucose is the most preferred carbon source for the growth and development of any microorganism, resulting in the highest cell dry weight (CDW) of 43.12 g / L and a heme production of 0.39 mg / g CDW (0.039% w / w). However, the highest heme production was observed with pure glycerol induced with methanol after 24 hours, which was 0.53 mg / g CDW (0.053% w / w). Further experiments were conducted using glycerol as the preferred carbon source.

[0324] Figure 1B Depicts heme production in relation to the nitrogen source. When the synthetic medium (SYN6) was replaced with a complex medium containing corn protein hydrolysate powder, the heme production increased by 4-fold (15 mg / L compared to 60 mg / L) (0.053% w / w compared to 0.225% w / w). Using 2% w / v CPH powder, a heme production of 2.25 mg per gram of cell dry weight was observed.

[0325] Figure 1C Shows the heme quantification in a 150 L volume process scale-up. The optimal medium parameters and process conditions were observed in flask laboratory-scale experiments, and the maximum heme production was 30 mg per gram of cell dry weight (3% w / w).

[0326] 1.6 Nutritional profile overview. Two different batches of samples were analyzed using standard ISO, AOAC, ELISA, spectrophotometry, gas chromatography (GC), high-performance liquid chromatography (HPLC), and mass spectrometry (MS) techniques to evaluate microbial parameters, heavy metals, carbohydrates, total protein, dietary fiber, glycomics analysis, vitamins, minerals, amino acids, carcinogens, aromatic volatiles, etc. This affects the nutritional value of the said product.

[0327] Table 1

[0328]

[0329]

[0330]

[0331] Example 2. Genetically modified Hansenula polymorpha expressing recombinant myoglobin.

[0332] 2.1 - Myoglobin gene selection, codon optimization, and synthesis.

[0333] The myoglobin (MG) gene from Bos taurus was codon-optimized (SEQ ID NO:2) for expression in Hansenula polymorpha and synthesized by ThermoFisher Scientific. Gene optimization not only increases the chance of efficient expression in the target host but also facilitates cloning by removing unwanted restriction sites.

[0334] 2.2 - Host transformation and selection

[0335] The Hansenula polymorpha strains used were RB11 (ura3) and ALU3 (ade1, leu2, ura3), and were obtained from Artes Biotechnology, GmbH.

[0336] pFPMT121 and its derivatives were used as circular plasmids for the transformation of RB11 and ALU3 strains. B14 ( Figure 5 ), a derivative of pFPMT121, is a plasmid without an antibiotic resistance gene and was used to transform the Hansenula polymorpha RB11 strain.

[0337] To construct the expression plasmid B14 - BtMG ( Figure 6 ), the plasmid DNA provided by ThermoFisher Scientific / Gene art was digested with the restriction enzymes EcoRI and BamHI ( Figure 7 ), generating a 480 bp gene (myoglobin) fragment with an optimized nucleotide sequence encoding bovine myoglobin (BtMG). After the restriction reaction electrophoresis, the fragment was purified from the agarose gel. The vector plasmid B14 was also digested with EcoRI and BamHI overnight, and the linearized plasmid was gel - purified. The annealing of EcoRI overlaps produced the fusion of the heterologous gene with the promoter element (FMDp), and the ligation of the BamHI site produced the fusion of the heterologous gene with the terminator element (MOXt).

[0338] The Hansenula polymorpha RB11 and ALU3 strains were transformed based on the yeast electroporation protocol. Thirty - six RB11 and seventy - two ALU3 transformants were screened using minimal (selective) and complex (non - selective) media to achieve stable integration of the plasmid. The stain - free imaging technique utilizes polyacrylamide gels containing a proprietary trihalo compound that causes proteins to fluoresce directly in the gel upon brief photo - activation, enabling immediate visualization of proteins at any point during electrophoresis and western blotting. This trihalo compound covalently binds to tryptophan residues, and when exposed to UV light, the fluorescence of tryptophan residues is enhanced, enabling the detection of proteins at levels as low as 10 - 25 ng.

[0339] The myoglobin expression level of RB11 (10%-16%) is at least 10x higher than that of the ALU3 (3%-6%) strain. The sample pool RB11 / b14-BtMB#35 gave a very high expression of approximately 16.3% of the total protein loaded onto the lane ( Figure 6 ). After successful construction of the recombinant plasmid and transformation of Hansenula polymorpha, the plasmids and genomes of the transformants were sequenced. The obtained polynucleotide sequence was translated into an open reading frame using the Expasy tool (ExPASy - translation tool), and the obtained full-length 3'-5' ORF was compared with the bovine myoglobin sequence using the CLUSTAL - multiple sequence alignment (MSA) tool. The myoglobin gene sequence integrated into the chromosome of Hansenula polymorpha was observed to have 100% similarity with the bovine myoglobin gene ( Figure 7 ).

[0340] 2.3 - Production of recombinant myoglobin in a bench - top bioreactor.

[0341] The production strain Hansenula polymorpha RB11 / b14 - BtMB#35 was screened from 36 pools and cultured in a 3L bioreactor for protein production and further characterization. The strain was cultured in a fed - batch fermentation mode with an initial feed of 2% glycerol, and derepression was carried out by providing glycerol feed at a rate of 2–6 g / L per hour until 48 hours. When the culture reached the stationary phase, protein synthesis was induced by adding 1% methanol and incubated for 24 hours.

[0342] The cell biomass was harvested and lysed using a bead mill to obtain a lysate containing myoglobin. The obtained protein was subjected to SDS PAGE and quantified by densitometry signals.

[0343] Sequence analysis confirmed the expression of a 154 - amino - acid polypeptide chain similar to bovine myoglobin, and it was observed by densitometry analysis that the Hansenula polymorpha RB11 / BtMG strain produced 1.6 g / L (about 10 - 15% of the total protein) of myoglobin, with a conversion yield of 20 mg / g cell dry weight. In addition, information on the heme loading is crucial and negligible for determining the functionality of the protein. Therefore, a heme assay kit (a heme assay kit sufficient for 250 colorimetric tests, Sigma - Aldrich (sigmaaldrich.com)) was used to optimize the heme quantification assay. Colorimetric assays using commercial myoglobin (left) (animal - derived) and Moolec myoglobin (right) gave 200 μM (8.6 mg / mL) and 30 μM (1.6 mg / mL), respectively.

[0344] 2.4 - Production of recombinant myoglobin in a semi - pilot - scale (10L) bioreactor.

[0345] Production was started batchwise in a Sartorius C-DCU 15-L stainless steel fermenter (C-DCU 15-L) with 2% glycerol as the sole carbon source; when the concentration of glycerol in the production medium was below 10 g / L, a further fed-batch fermentation mode was started at 12 h. The linear feeding of glycerol was carried out during the 12 h to 50 h of cultivation at a feeding rate of 2–6 g / L / h. The substrate feeding rate and the dissolved oxygen (DO) concentration in the reactor were adjusted so as to maintain a DO measurement value of 20% - 40% throughout the cultivation time at the maximum stirring speed. During fermentation, the pH was set and maintained at 4.8 (correction medium: 20% (v / v) ammonia solution and 20% (v / v) phosphoric acid solution). (10% (w / v) Structol J 673) Antifoaming agent was added appropriately by an AF controller. After 50 h of cultivation, methanol induction was carried out by batchwise addition of methanol according to the following scheme: 0 h and 5 h: addition of 1.0% (v / v) methanol sol each; 8 h, 11 h, 14 h and 17 h: addition of 0.5% (v / v) methanol-sol each ( Figure 8 ).

[0346] 2.5 - Isolation and purification of myoglobin from the accumulated cell biomass

[0347] The resulting microbial cell biomass was harvested by a basket centrifuge, and the cell pellet was resuspended in soft water and further centrifuged. This process was repeated at least 3 times, and samples of known volume were collected at each step for qualitative and quantitative characterization of microbial cells, metabolites and other characteristic compounds. After the final washing step, the cells were resuspended in lysis buffer for cell disruption.

[0348] The microbial cells were lysed by passing the suspension from step 1 through a Dyno mill 3 times. During milling, the suspension was heated to 30 °C. After cell disruption, the suspension was centrifuged at 6362 RCF for 60 minutes. Thereafter, the supernatant was collected and frozen at -20 °C.

[0349] In the present disclosure, the expressed myoglobin is intracellular, and thus the microbial biomass accumulated after cultivation was separated and purified by the following steps.

[0350] 2.5.1 - Cell washing:

[0351] Microbial cell biomass produced by fed-batch culture was harvested by a bench-top centrifuge (630RS), and the cell pellet was resuspended in soft water and centrifuged further at 6362 RCF. This process was repeated at least 3 times, and samples of known volume were collected at each step for qualitative and quantitative characterization of microbial cells, metabolites, and other characteristic compounds. After the final washing step, the cells were resuspended in lysis buffer for cell disruption.

[0352] 2.5.2 - Quantification of microbial growth:

[0353] Weigh 5 mL of each sample and fill it with approximately 45 mL of deionized water. Centrifuge the suspension at 3333 g for 10 minutes and then at 5000 g for 5 minutes in a second round. Transfer the pellet together into a pre-weighed glass bottle. Then, incubate it overnight at 93 °C and for 5 hours at 105 °C the next morning. Finally, calculate the dry weight in g / kg.

[0354] 2.5.3 - Cell lysis:

[0355] Microbial cells were lysed by passing the suspension from step 1 through a Dyno mill 3 times. During milling, the suspension was heated to 30 °C. After cell disruption, the suspension was centrifuged at 6362 RCF for 60 minutes. Thereafter, the supernatant was collected and frozen at -20 °C.

[0356] 2.5.4 - Protein recovery and purification:

[0357] The cell lysate obtained from 2.9 - was subjected to microfiltration and ultrafiltration through different retention membranes with sizes in the range of 5 kDa to 800 kDa.

[0358] 2.5.5 - Qualitative myoglobin analysis:

[0359] Samples collected during methanol induction and downstream processes were subjected to qualitative analysis of myoglobin using SDS PAGE. Sequence analysis confirmed the expression of a 154 - amino acid polypeptide chain similar to bovine myoglobin, and it was observed by densitometric analysis that the Hansenula polymorpha RB11 / BtMG strain produced 1.6 g / L (approximately 10 - 15% of the total protein) of myoglobin with a conversion yield of 20 mg / g of cell dry weight.

[0360] The protein band pattern in SDS PAGE analysis ( Figure 8 ) showed the appearance of a characteristic 17 kDa band after methanol induction (lane T7), which remained present until the end of induction (lane T end). Similarly, qualitative analysis of myoglobin was performed on samples obtained at the end of each step during the downstream process.

[0361] According to Western blot analysis (Figure 9 ) A characteristic band of myoglobin at approximately 17 kDa was observed from the final sample.

[0362] 2.5.6 - Quantification of heme:

[0363] In addition to the information regarding the quantification of recombinant myoglobin produced by engineered Hansenula polymorpha strains, the heme loading is crucial for determining the functionality of the protein. Thus, the heme quantification assay was optimized using a heme assay kit (heme assay kit sufficient for 250 colorimetric tests, Sigma - Aldrich (sigmaaldrich.com)).

[0364] The procedure for the heme assay was as follows: According to the manufacturer's protocol, 50 μl of purified myoglobin was mixed with 200 μl of the reagent and incubated at room temperature for 5 minutes. After incubation, the sample absorbance was recorded at 400 nm using a spectrophotometer. Meanwhile, a linear standard curve could be prepared by different dilutions using the heme calibrator. The protocol stated that the provided heme calibrator corresponded to 62.5 μM heme, with a linear detection range of 0.6 - 125 μM.

[0365] The optical density reading of 0.148 corresponded to 300.95 μM of heme, corresponding to 501.58 μM of myoglobin, with a 60% heme loading.

[0366] Table 2

[0367]

[0368] In addition, the myoglobin concentration and heme loading of the samples obtained from the 5 kDa filter module were quantified, which resulted in myoglobin with a purity of 60% - 70% at 30 - 40 g / L and a 50% - 60% heme loading onto apomyoglobin.

[0369] 2.6 - Production of recombinant myoglobin in Pichia pastoris:

[0370] To provide further details to support the efficiency of Hansenula polymorpha as a suitable host for the production of heme - containing proteins, the myoglobin (MG) gene from Bos taurus was codon - optimized for expression in Pichia pastoris. The myoglobin gene was expressed under the AOX1 promoter and terminator sequences.

[0371] The positive transformants obtained after myoglobin gene transformation and confirmation were cultured in yeast extract, peptone, and dextrose (YPD) medium for expression studies.

[0372] Inoculate single colonies of positive transformants from the plates screened by PCR identification into 10 ml of YPD liquid culture.

[0373] After 24 hours, the culture medium was milky white. Take 10 ml of the culture medium and inoculate it into 1 L of YPD medium (inoculation amount 1%), and incubate at 28 °C and 230 rpm.

[0374] Preparation by methanol induction: Dissolve hemin with 0.2 M NaOH, the concentration of hemin is 100 mM, then dilute it 10 times with methanol, filter sterilize it, and obtain methanol containing 10 mM hemin.

[0375] When the OD600 value of the bacteria reaches 0.6 - 0.7 after dilution 50 times (about 24 hours), pour the culture broth into two sterilized and cooled 500 ml test tubes, centrifuge on the ultra-clean workbench, centrifuge at 6000 rpm at 4 °C for 5 minutes. Pour out the supernatant, resuspend the yeast with 900 ml of YP medium, and pour the resuspended system back into a 5 L conical flask. Add 100 ml of sterilized 1 M phosphate buffer (the working concentration of phosphate is 0.1 M), and add 10 ml of methanol containing 10 mM hemin to the system to start induction (the working concentration of methanol is 1%, and the working concentration of hemin is 0.1 mM).

[0376] Add 10 ml of methanol containing 10 mM hemin to the system every 24 hours.

[0377] Take samples every day for TCA concentration electrophoresis. Record the time of adding methanol for the first time as zero time. After about 48 hours (different proteins may have different induction times), pour the culture broth into two 500 ml centrifuge tubes, centrifuge at 6000 rpm, and centrifuge at 4 °C for 10 minutes. Collect the supernatant and freeze it at -20 °C for standby.

[0378] As described above, in the absence of added external hemin, no hemin loading onto myoglobin was observed; however, after supplementing with exogenous hemin, up to 22% of myoglobin was loaded with hemin.

[0379] Example 3 - Hansenula polymorpha cell lysis.

[0380] Thaw the polymorphic Hansenula yeast frozen biomass to room temperature and then dilute it in tap water to a concentration of approximately 30% dry matter (moisture analyzer, Mettler Toledo HB43). Two conditions were prepared, control biomass (pH 5.2) and biomass adjusted to pH 8.5 in combination with 2.5% alkaline protease (Novozymes, 2.4L pure (2.4AU-A / g)) (33% NaOH added). The two biomass solutions were incubated with continuous shaking at 50 °C for 4 hours. During this process, the pH of the alkaline protease sample was maintained at approximately 8.5 by adding small amounts of 33% v / v NaOH. Samples were collected hourly and stored at -10 °C for subsequent analysis.

[0381] Example 4. Enzymatic hydrolysis.

[0382] After 4 hours of incubation, the control biomass sample was completed. First, the pH of the biomass treated with alkaline protease was set to 5.5 using 5% hydrochloric acid, after which the first batch of enzymes was added (Step 1), RP-1G (0.16%, Amano enzyme, a 5'-phosphodiesterase that hydrolyzes RNA to 5'-nucleotides) and deaminase (0.067%, Amano enzyme, that converts the nucleotide 5'AMP to 5'IMP). The nucleotides 5'GMP and 5'IMP are known flavor enhancers. The enzymes were incubated with shaking at 50 °C for 5 hours.

[0383] Next, the solution was adjusted to pH 7.0 with 33% NaOH, after which the second enzyme mixture was added (Step 2). It is known that Protana UBoost (0.17%, Novozymes, a glutaminase that produces glutamic acid from glutamine), ProtanaPrime (0.17%, Novozymes, a mixture of exopeptidases capable of releasing free amino acids), and ProteAXH (0.17%, Amano enzyme, exhibiting protease and peptidase activities) contribute to umami enhancement and were incubated with shaking at 50 °C for 12 hours. Samples were collected after each enzyme digestion step.

[0384] To determine that the lysis and hydrolysis processes did not degrade heme, samples were taken at each step and the heme concentration was determined according to the method used in Example 1 (1.4), as shown in Table 3.

[0385] Table 3

[0386] Sample Heme (% w / w) Crude Biomass 2.9 Cell Lysate 2.2 Step 1 3.1 Step 2 2.4

[0387] Example 5 - Microfiltration and ultrafiltration

[0388] The hydrolysis products were first separated by microfiltration. An 800 kDa membrane was installed in the filtration system of the SANI membrane (Vibro-Lab3500), and then the digested solution was pumped through the membrane module while continuously vibrating. The retentate (insoluble fraction) consisted of mostly polysaccharides (β-glucans, a group of glucose polymers that form the yeast cell wall), and the permeate represented the intercellular part of the yeast cells (a clear dark brown solution). Subsequently, the 800 kDa permeate (soluble fraction) was used for ultrafiltration using a 1 kDa membrane, which produced a product of soluble small peptides and nucleotides collected in the permeate.

[0389] Example 6 - Quantification of GMP

[0390] The spray-dried fraction of the yeast hydrolysate obtained after filtration (obtained after Example 5) was used to determine the concentration of flavor-enhancing GMP using an isocratic or gradient HPLC elution method, with 0.5% orthophosphoric acid (A) and 100% methanol (B) as the mobile phase, and a Gemini phenyl C6 reverse-phase 5 μm, 110A, 50*4.6 column as the stationary phase. The flow rate was maintained at 0.5 mL / min, the column pressure: 50 - 60 Kgf / cm2. Detector: SPD-M20A, diode array detector. Wavelength: 254 nm.

[0391] Result: The average concentration of GMP in the yeast hydrolysate of Hansenula polymorpha per gram was observed to be 0.007% w / w.

[0392] Example 7 - Quantification of glutathione

[0393] The yeast hydrolysate obtained after filtration was used to determine the concentration of glutathione (an umami flavor component) by UPLC-QTOF using a C18 column, which was subjected to positive electrospray ionization. The gradient elution method included 0.1% formic acid in water (A) and 0.1% formic acid / acetonitrile solution (B) as the mobile phase, and an Acquity UPLC BEH C18, 50*2.1 mm chromatographic 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.

[0394] Result: The average concentration of oxidized glutathione in the spray-dried yeast product per gram was observed to be 11.62% w / w, and the average concentration of reduced glutathione was 0.29% w / w.

[0395] Example 8 - Flavorings derived from Hansenula polymorpha.

[0396] 8.1 - Flavor 1. Mix the Hansenula polymorpha yeast extract (20%-24% ds) (370 g) as a protein source, amino acids (cysteine, methionine, glutamine, glycine) 16 g, vitamin B1 (3-5 g), reducing carbohydrate source (d-xylose, ribose, arabinose, rhamnose, glucose) 40 g, water 80 g, and 33% caustic alkali (8-10 g) together using a high-shear mixer (mixture 1) and pour it into a Parr high-pressure stirred reactor. The slurry is heated to 120 °C within 30 minutes. After reaching 120 °C, keep the slurry at this temperature for 60 minutes (an increase in pressure is observed: 3 bar). After the reaction, the batch is cooled to 50 °C within 30 minutes. Mix the resulting liquid with a certain amount of carrier, and then spray dry or vacuum dry or keep it as a liquid.

[0397] 8.2 - Flavor 2. Mix the Hansenula polymorpha yeast extract 20%-24% ds (200 g), amino acids (lysine, arginine, histidine, cysteine 25 g), flavor enhancers IMP / GMP 5 g, acids (lactic acid, succinic acid, tartaric acid) 15 g, reducing carbohydrate source (glucose, arabinose) 120 g, and 33% caustic alkali 37 g together using a high-shear mixer (mixture 2) and pour it into a Parr high-pressure stirred reactor. The slurry is heated to 120 °C within 30 minutes. After reaching 120 °C, keep the slurry at this temperature for 30 minutes. After the reaction, the batch is cooled to 50 °C within 30 minutes. Use the resulting liquid as it is or mix it with a certain amount of carrier, and then spray dry or vacuum dry.

[0398] 8.3 Flavor 3. Lyse the cell culture of genetically modified Hansenula polymorpha expressing myoglobin at 65 °C with papain (0.1%) for 2 hours, and then lyse it at 50 °C with 0.05% protease A Amano 2SD (Amano Enzyme Inc.) for 24 hours. Mix 580 g of genetically modified Hansenula polymorpha yeast extract with 15.8 g of yeast extract, 60 g of gum arabic, 120 g of water, 14 g of amino acids (Gly, Ala, Cys, and Met), 0.35 g of vitamin B1, 3.6 g of reducing sugar (rhamnose, arabinose, ribose, D-xylose), and 7.2 g of disodium phosphate as a buffer (mixture 3). Heat-treat mixture 3 at 120 °C for 30 minutes at a pressure of 1-2.5 bar and at pH = 6.5 to produce a Maillard reaction product (flavor 3).

[0399] 8.4 Flavoring 4. The Maillard reaction product of flavoring 3 (750 g) is mixed with maltodextrin (255 g), gum arabic (45 g) and water (380 g) and spray-dried. The spray-dried mixture (280 g) is further mixed with other ingredients: 75 g of furfuryl alcohol, 90 g of yeast extract KU012, 100 g of yeast extract KA65, 160 g of a mixture of organic acids (lactic acid, malic acid, citric acid, tartaric acid, succinic acid and acetic acid) and amino acids (glycine, alanine, cysteine, glutamine, methionine) and salt (200 g). The resulting flavoring at 0.4% plus 0.2% salt is tasted in warm water to obtain a unique meaty flavor with a metallic bloody aftertaste.

[0400] Example 9 - Application to food products.

[0401] 9.1 - Sauce. Sauce 10%, flavoring 1 5%, salt 19%, water 56%, beef essence 0.5%, spice extract (chili powder, chili, garlic, onion) 0.1%, flavor enhancer IMP / GMP / msg (msg is monosodium glutamate) 3.5%, tomato ketchup 1%, molasses 3%, flavoring 2 1%, xanthan gum 0.3%. The product is mixed and heated to 80 °C and heat-packed in containers. The product has a good round Korean beef flavor.

[0402] 9.2 - Food matrix. Several food matrices have been developed based on well-known techniques. Hansenula polymorpha cells, extracts, hydrolysates and flavorings are incorporated into these food matrices. An overview of the food products and main ingredients is shown in Tables 4 - 6.

[0403] Table 4.

[0404]

[0405] *Binder: wheat gluten, protein and hydrocolloid enzymes, starch

[0406] Table 5

[0407]

[0408] *Such as: wheat gluten, protein and hydrocolloid enzymes, starch

[0409] Table 6

[0410]

[0411] ***

[0412] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0413] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

[0414] Table 7 - Sequences

[0415]

[0416]

[0417]

[0418]

Claims

1. A Hansenula polymorpha extract comprising at least about 0.03% w / w of heme.

2. The Hansenula polymorpha extract according to claim 1, wherein the Hansenula polymorpha comprises from about 0.03% to about 3.1% w / w of heme.

3. The Hansenula polymorpha extract according to claim 1 or claim 2, wherein the extract further comprises at least about 0.8% w / w of glutamic acid.

4. The Hansenula polymorpha extract according to any one of claims 1 to 3, wherein the extract further comprises at least about 0.007% of 5'-ribonucleotide.

5. The Hansenula polymorpha extract according to claim 4, wherein the 5'-ribonucleotide is 5'-GMP.

6. The Hansenula polymorpha extract according to any one of claims 1 to 5, wherein the extract comprises at least about 0.8% w / w of glutamic acid and at least about 0.007% of 5'-ribonucleotide.

7. The Hansenula polymorpha extract according to claim 6, wherein the 5'-ribonucleotide is 5'-GMP.

8. The Hansenula polymorpha extract according to any one of claims 1 to 7, wherein the extract further comprises at least about 0.2% w / w of glutathione.

9. The Hansenula polymorpha extract according to any one of claims 1 to 8, wherein the extract further comprises at least about 0.4% w / w of histidine.

10. The Hansenula polymorpha extract according to any one of claims 1 to 9, wherein the Hansenula polymorpha is genetically modified to produce a recombinant heme protein, and wherein the heme protein is produced at least about 0.1% by weight of the total protein.

11. The Hansenula polymorpha extract according to claim 10, wherein 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.

12. The Hansenula polymorpha extract according to claim 10, wherein the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

13. A hydrolyzed Hansenula polymorpha extract containing at least about 0.03% w / w of heme.

14. The hydrolyzed Hansenula polymorpha extract according to claim 13, wherein the hydrolyzed Hansenula polymorpha extract comprises from about 0.03% to about 3.1% w / w of heme.

15. The hydrolyzed Hansenula polymorpha extract according to claim 13 or claim 14, wherein the hydrolyzed Hansenula polymorpha extract further comprises at least about 0.8% w / w of glutamic acid.

16. The hydrolyzed Hansenula polymorpha extract according to any one of claims 13 to 15, wherein the hydrolyzed Hansenula polymorpha extract further comprises at least about 0.007% w / w of 5'-ribonucleotide.

17. The hydrolyzed Hansenula polymorpha extract according to claim 16, wherein the 5'-ribonucleotide is 5'-GMP.

18. The hydrolyzed Hansenula polymorpha extract according to any one of claims 13 to 17, wherein the hydrolyzed Hansenula polymorpha extract further comprises at least about 0.8% w / w of glutamic acid and at least about 0.007% of 5'-ribonucleotide.

19. The hydrolyzed Hansenula polymorpha extract according to claim 18, wherein the 5'-ribonucleotide is 5'-GMP.

20. The hydrolyzed Hansenula polymorpha extract according to any one of claims 13 to 19, wherein the hydrolyzed Hansenula polymorpha extract further comprises at least about 0.4% w / w of histidine.

21. The hydrolyzed Hansenula polymorpha extract according to any one of claims 13 to 20, wherein the hydrolyzed Hansenula polymorpha extract further comprises at least about 0.2% w / w of glutathione.

22. The hydrolyzed Hansenula polymorpha extract according to any one of claims 13 to 21, wherein the Hansenula polymorpha is genetically modified to produce a recombinant heme protein, and wherein the heme protein is produced at least about 0.1% by weight of the total protein.

23. The hydrolyzed Hansenula polymorpha extract according to claim 22, wherein 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.

24. The hydrolyzed Hansenula polymorpha extract according to claim 22, wherein the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

25. A flavor precursor mixture comprising Hansenula polymorpha biomass, protein, amino acids, carbohydrates, and vitamins.

26. The flavor precursor mixture according to claim 25, wherein the Hansenula polymorpha comprises at least about 0.03% w / w of heme.

27. The flavor precursor mixture according to claim 25 or claim 26, wherein the Hansenula polymorpha biomass is an extract.

28. The flavor precursor mixture according to claim 27, wherein the Hansenula polymorpha biomass is a hydrolyzed extract.

29. The flavor precursor mixture according to any one of claims 25 to 28, wherein the Hansenula polymorpha further comprises at least about 0.8% w / w of glutamic acid.

30. The flavor precursor mixture according to any one of claims 25 to 29, wherein the Hansenula polymorpha further comprises at least about 0.007% of 5'-ribonucleotide.

31. The flavor precursor mixture according to any one of claims 25 to 30, wherein the Hansenula polymorpha comprises at least about 0.8% w / w of glutamic acid and at least about 0.007% of 5'-ribonucleotide.

32. The flavor precursor mixture according to claim 30, wherein the 5'-ribonucleotide is 5'-GMP.

33. The flavor precursor mixture according to claim 31, wherein the 5'-ribonucleotide is 5'-GMP.

34. The flavor precursor mixture according to any one of claims 25 to 33, wherein the Hansenula polymorpha further comprises at least about 0.2% w / w glutathione.

35. The flavor precursor mixture according to any one of claims 25 to 34, wherein the Hansenula polymorpha further comprises at least about 0.4% w / w histidine.

36. The flavor precursor mixture according to any one of claims 25 to 35, wherein the Hansenula polymorpha is genetically modified to produce a recombinant heme protein, and wherein the heme protein is produced at least about 0.1% by weight of the total protein.

37. The flavor precursor mixture according to claim 36, wherein 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.

38. The flavor precursor mixture according to claim 36, wherein the recombinant heme protein is an animal-derived heme protein or a plant-derived protein.

39. The flavor precursor mixture according to any one of claims 25 to 38, wherein the flavor precursor mixture comprises at least about 1% w / w of Hansenula polymorpha biomass, at least about 5% w / w of monosaccharides, and at least about 15% w / w of amino acids.

40. A food product comprising Hansenula polymorpha biomass.

41. The food product according to claim 40, wherein the Hansenula polymorpha biomass is lysed biomass.

42. The food product according to claim 41, wherein the lysed biomass of Hansenula polymorpha is hydrolyzed biomass.

43. The food product according to claim 41 or claim 42, wherein the food product comprises from about 0.001% to about 80% w / w of Hansenula polymorpha biomass.

44. The food product according to any one of claims 40 to 43, wherein the Hansenula polymorpha comprises at least about 0.03% w / w of heme.

45. The food product according to any one of claims 40 to 43, wherein the Hansenula polymorpha comprises from about 0.03% to about 3.1% w / w of heme.

46. The food product according to any one of claims 40 to 45, wherein the Hansenula polymorpha further comprises at least about 0.8% w / w of glutamic acid.

47. The food product according to any one of claims 40 to 46, wherein the Hansenula polymorpha further comprises at least about 0.007% w / w of 5'-ribonucleotide.

48. The food product according to any one of claims 41 to 46, wherein the Hansenula polymorpha further comprises at least about 0.8% w / w of glutamic acid and at least about 0.007% w / w of 5'-ribonucleotide.

49. The food product according to claim 47, wherein the 5'-ribonucleotide is 5'-GMP.

50. The food product according to claim 48, wherein the 5'-ribonucleotide is 5'-GMP.

51. A food product according to any one of claims 40 to 50, wherein the Hansenula polymorpha further comprises at least about 0.4% w / w histidine.

52. A food product according to any one of claims 40 to 51, wherein the Hansenula polymorpha further comprises at least about 0.2% w / w glutathione.

53. A food product according to any one of claims 40 to 52, wherein the food product does not contain products of animal origin.

54. A food product according to any one of claims 40 to 53, wherein the product is selected from the group consisting of: sauces, marinades, seasonings, dressings, brines, broths, soups, traditional protein foods (such as tofu, tempeh, seitan), fermented vegetables and legumes, meat analogs, meat products, extruded meat products, cultured meat products, and dietary supplements.

55. A food product comprising a flavor precursor mixture according to any one of claims 25 to 39.

56. A method for obtaining a Hansenula polymorpha extract, comprising: a. culturing and propagating Hansenula polymorpha cells, b. lysing the cells, c. adjusting the pH if necessary, and d. drying and / or concentrating by removing water to obtain a solid Hansenula polymorpha extract.

57. The method according to claim 56, wherein after the lysis of the cells, the method further comprises inactivating acid phosphatase.

58. The method according to claim 57, wherein inactivating the acid phosphatase comprises raising the pH from about 5 to about 14.

59. The method according to claim 56, wherein the method further comprises hydrolyzing endogenous proteins and nucleic acids up to about 30 kDa.

60. The method according to claim 56, wherein the method further comprises maintaining the temperature below about 60 °C.

61. The method according to claim 56, wherein the method further comprises hydrolysis of proteins and / or nucleic acids up to about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa, about 2 kDa, about 1 kDa, and / or 05 kDa.

62. A method for preparing a flavor derived from Hansenula polymorpha, comprising: e. obtaining Hansenula polymorpha biomass, f. adding a carbohydrate, fat, and amino acid source to the Hansenula polymorpha biomass and mixing; and g. subjecting the biomass to heat treatment induction at about 100 °C to about 130 °C for about 30 minutes to about 180 minutes to obtain the flavor derived from Hansenula polymorpha.

63. The method according to claim 62, wherein the carbohydrate is selected from the group consisting of: monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, oligosaccharides, and polysaccharides.

64. The method according to claim 62 or 63, wherein the fat is selected from the group consisting of: animal fats, insect fats, fungal fats, plant fats, plant fats, synthetic fats, and microbial fats.

65. The method according to any one of claims 62 to 64, wherein the amino acid source is selected from the group consisting of: natural proteins, partially hydrolyzed proteins, and free amino acids.

66. The method according to any one of claims 62 to 65, wherein the method further comprises the step of inducing lysis of the Hansenula polymorpha cells to obtain an extract.

67. The method according to claim 66, wherein the method further comprises the step of inducing proteolysis of the obtained extract.

68. The method according to claim 66, wherein the method further comprises the step of inducing nucleic acid hydrolysis of the extract.

69. The method according to any one of claims 62 to 68, wherein the method further comprises the step of inducing the conversion of amino acids to glutamic acid.

70. A dietary supplement comprising Hansenula polymorpha biomass containing at least about 0.03% w / w of heme.

71. The dietary supplement according to claim 70, wherein the Hansenula polymorpha biomass contains from about 0.03% to about 3.1% w / w of heme.

72. The dietary supplement according to claim 70 or claim 71, wherein the biomass is lysed biomass.

73. The dietary supplement according to claim 72, wherein the lysed biomass is further hydrolyzed.

74. The dietary supplement according to claim 73, wherein a soluble fraction is obtained after filtering the hydrolyzed biomass.

75. The dietary supplement according to claim 74, wherein the soluble fraction contains particles smaller than about 100 kDa.

76. The dietary supplement according to claim 74, wherein the soluble fraction contains particles smaller than about 50 kDa.

77. The dietary supplement according to claim 74, wherein the soluble fraction contains particles smaller than about 5 kDa.

78. The dietary supplement according to claim 74, wherein the soluble fraction contains particles smaller than about 1 kDa.

79. The dietary supplement according to claim 74, wherein the soluble fraction contains particles smaller than about 0.5 kDa.

80. A method for producing a heme protein having a heme loading greater than about 50%, wherein the method comprises the following steps: a. providing a culture of transgenic Hansenula polymorpha comprising a nucleic acid encoding a heme protein operably linked to a promoter; b. culturing the transgenic Hansenula polymorpha without adding exogenous heme to the culture; and c. isolating and purifying the heme protein.

81. The method according to claim 80, wherein the promoter is a methanol-inducible promoter.

82. The method according to claim 80 or 81, wherein the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthetic pathway or an exogenous component of the heme biosynthetic pathway.

83. The method according to any one of claims 80 to 82, wherein the heme protein is selected from the group consisting of heme proteins of animal origin and heme proteins of plant origin.

84. The method according to claim 83, wherein the animal-derived heme protein is selected from the group consisting of: bovine-derived heme protein, porcine-derived heme protein, ovine-derived heme protein, equine-derived heme protein, and caprine-derived heme protein.

85. The method according to claim 83 or claim 84, wherein the animal-derived 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.

86. The method according to any one of claims 80 to 85, wherein the nucleic acid comprises a nucleic acid sequence having at least about 70% identity to the nucleic acid sequence of SEQ ID NO:

1.

87. A method for producing an animal-derived heme protein having a heme loading greater than about 50%, wherein the method comprises: a. introducing a nucleic acid construct into a Hansenula polymorpha yeast strain, wherein the nucleic acid construct comprises a promoter operably linked to a nucleic acid encoding an animal-derived heme protein; b. culturing the transgenic Hansenula polymorpha cells without adding exogenous heme molecules to the culture; and c. isolating the animal heme protein from the culture.

88. The method according to claim 87, wherein the promoter is a methanol-inducible promoter.

89. The method according to claim 87 or claim 88, wherein the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthetic pathway or an exogenous component of the heme biosynthetic pathway.

90. The method according to any one of claims 87 to 89, wherein the animal-derived heme protein is selected from the group consisting of: bovine-derived heme protein, porcine-derived heme protein, ovine-derived heme protein, equine-derived heme protein, and caprine-derived heme protein.

91. The method according to any one of claims 87 to 90, wherein the animal-derived 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.

92. The method according to any one of claims 87 to 91, wherein the nucleic acid comprises a nucleic acid sequence having at least about 70% identity to the nucleic acid sequence of SEQ ID NO:

1.

93. A method for producing recombinant bovine myoglobin comprising greater than about 50% of heme-loaded recombinant bovine myoglobin, wherein the method comprises: a. introducing a nucleic acid encoding recombinant bovine myoglobin operably linked to a promoter into Hansenula polymorpha yeast cells; and b. culturing the Hansenula polymorpha containing the recombinant bovine myoglobin nucleic acid without adding heme to the culture to promote the expression of the recombinant myoglobin, thereby expressing the recombinant myoglobin.

94. The method according to claim 93, wherein the promoter is a methanol-inducible promoter.

95. The method according to claim 93 or 94, wherein the transgenic Hansenula polymorpha does not contain an exogenous transcriptional activator of the heme biosynthesis pathway or an exogenous component of the heme biosynthesis pathway.

96. The method according to any one of claims 93 to 95, wherein the method further comprises extracting and purifying the recombinant bovine myoglobin.

97. The method according to any one of claims 93 to 96, wherein the nucleic acid comprises a nucleic acid sequence having at least about 70% identity to the nucleic acid sequence of SEQ ID NO:

1.

98. A food composition comprising a heme protein produced by claim 80 or claim 87 or a bovine myoglobin produced by claim 93.

99. A meat analogue food composition comprising a lysate of cultured Hansenula polymorpha produced by claim 80, claim 87 or claim 93.

100. A meat analogue food composition comprising a heme protein produced by claim 80 or 87 or a bovine myoglobin produced by claim 93.

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