WO2023137192A1

WO2023137192A1 - Meat replica fungal food product

Info

Publication number: WO2023137192A1
Application number: PCT/US2023/010821
Authority: WO
Inventors: Brian Klopf; Erick WALTS; Eleanore Brophy ECKSTROM; Mark Andrew KOZUBAL; Richard Eugene MACUR; Yuval Charles AVNIEL
Original assignee: Fynder Group Inc
Current assignee: Fynder Group Inc
Priority date: 2022-01-15
Filing date: 2023-01-13
Publication date: 2023-07-20
Anticipated expiration: 2024-07-15

Abstract

Fungal food products are provided, in which an additive or modification allows a sensory perception of the fungal food product to replicate or closely mimic a sensory perception of a meat-based food product. This replication or mimicry is achieved by inclusion in the fungal food product of a heme-containing polypeptide that is mixed with fungal tissues present in the food product and/or synthesized by a genetically modified filamentous fungus of the food product. Methods of making such fungi and food products are also provided.

Description

MEAT REPLICA FUNGAL FOOD PRODUCT

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. Provisional Patent Application 63/299,935, filed 15 January 2022, the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to fungal food products, and particularly to fungal food products in which an additive or modification allows a sensory perception of the fungal food product to replicate or closely mimic a sensory perception of a meat-based food product.

BACKGROUND OF THE INVENTION

Food is any substance that is either eaten or drunk by any animal, including humans, for nutrition or pleasure. It is usually of plant, animal, or fungal origin, and can contain essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is ingested by an organism and assimilated by the organism’s cells in an effort to produce energy, maintain life, or stimulate growth.

Food typically has its origin in a photosynthetic organism, such as a plant. Some food is obtained directly from plants, but even animals that are used as food sources are raised by feeding them food which is typically derived from plants.

In most cases, the plant, animal, or fungal food source is fractionated into a variety of different portions, depending upon the purpose of the food. Often, certain portions of the plant, such as the seeds or fruits, are more highly prized by humans than others and these are selected for human consumption, while other less desirable portions, such as the stalks of grasses, are typically used for feeding animals.

Current plant-based meat substitutes have largely failed to cause a shift to a vegetarian diet. Meat substitute compositions are typically extruded soy/grain mixtures which largely fail to replicate the experience of cooking and eating meat. Common limitations of plant-based meat substitute products are a texture and mouth-feel that are more homogenous than that of equivalent meat products. Furthermore, as these products must largely be sold pre-cooked, with artificial flavors and aromas pre-incorporated, they fail to replicate the aromas, flavors, and other key features, such as texture and mouth-feel, associated with cooking or cooked meat. As a result, these products appeal largely to a limited consumer base that is already committed to vegetarianism/veganism, but have failed to appeal to the larger consumer segment accustomed to eating meat. It would be useful to have improved non-animal-derived meat substitutes which better replicate the aromas and flavors of meat, particularly during and/or after cooking.

SUMMARY OF THE INVENTION

In one aspect of the present disclosure, a food product comprises a) edible fungal mycelial matter comprising one or more fungal proteins; and b) at least one heme protein that is exogenous to the edible fungal mycelial matter.

In embodiments, the food product may comprise no meat or other animal -derived products.

In embodiments, before, during, and/or after a cooking process, a sensory perception of the food product is substantially similar to a sensory perception of a raw, cooking, or cooked meat product. The sensory perceptions may be selected from the group consisting of visual perceptions, auditory perceptions, olfactory perceptions, tactile perceptions, and gustatory perceptions. The sensory perceptions may be visual perceptions, and the visual perception of the food product before the cooking process may be substantially similar to a visual perception of a raw meat product. The sensory perceptions may be visual perceptions, and the visual perception of the food product during and/or after the cooking process may be substantially similar to a visual perception of a cooking or cooked meat product. The meat product may be a ground beef product. The cooking process may comprise exposing the food product to a temperature of at least about 150 °C for about 3 to about 5 minutes.

In embodiments, the at least one heme protein may be a globin, a cytochrome, or a methemalbumin.

In embodiments, at least one fungal protein may be a textured fungal protein.

In an aspect of the present invention, a food material comprises a heme polypeptide and a fungal mycelial biomass of a filamentous fungus belonging to an order selected from the group consisting of Ustilaginales Russulales Polyporales Agaricales , Pezizales. and Hypocr eales , wherein the fungal mycelial biomass comprises greater than about 40 wt. % protein content and less than about 8 wt. % RNA content.

In embodiments, the heme polypeptide may be produced by a yeast belonging to the order Saccharomycetales.

In embodiments, the filamentous fungus may belong to a family selected from the group consisting of Ustilaginaceae Hericiaceae, Polyporaceae. Grifolaceae. Lyophyllaceae , Strophariaceae , Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae , and Cordycipitaceae .

In embodiments, the filamentous fungus may belong to a species selected from the group consisting of Ustilago esculenta, Hericululm erinaceus, Polyporous squamosus, Grifola fondosa, Hypsizygus marmoreus, Hypsizygus ulmarius, Calocybe gambosa, Pholiota nameko, Ccdvatia gigantea Agaricus bisporus Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus ostreatus var. columbinus, Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa, Fusarium venenatum, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698), Disciotis venosa, and Cordyceps militaris.

In embodiments, the filamentous fungus may be a Fusarium species.

In embodiments, the filamentous fungus may be Fusarium venenatum.

In embodiments, the filamentous fungus may be Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698).

In embodiments, at least one of the following may be true: (a) the filamentous fungus comprises greater than about 45 wt. % protein content; and (b) the filamentous fungus comprises less than about 5 wt. % RNA content.

In embodiments, the filamentous fungus may comprise less than about 10 ppm of a mycotoxin selected from the group consisting of Alfatoxin Bl, Alfatoxin B2, Alfatoxin Gl, Alfatoxin G2, Fumonison Bl, Fumonison B2, Fumonison B3, Ochratoxin A, Nivalenol, Deoxynivalenol, Acetyl deoxynivalenol, Fusarenon X, T-2 Toxin, HT-2 Toxin, Neosolaniol, Diacetoxyscirpenol zearalenone, and any combinations thereof.

In embodiments, the filamentous fungus may comprise less than about 10 ppm total mycotoxin content. The filamentous fungus may comprise less than about 5 ppm total mycotoxin content.

In embodiments, the filamentous fungus may comprise greater than about 15 wt. % of branched chain amino acids.

In embodiments, the food material may be a meat analog food product.

In embodiments, the food material may be vegetarian. The food material may be vegan.

In embodiments, the food material may comprise meat, wherein the meat comprises a heme protein. The heme protein that is exogenous to the edible fungal mycelial matter may consist of, or may consist essentially of, the heme protein of the meat. The heme protein that is exogenous to the edible fungal mycelial matter may comprise a heme protein exogenous to the meat.

In embodiments, the heme polypeptide and one or more fungal proteins present in the filamentous fungus may collectively comprise all essential amino acids. The one or more fungal proteins present in the filamentous fungus may comprise all essential amino acids.

In embodiments, the filamentous fungus may be nonviable.

In an aspect of the present disclosure, a food composition comprises fungal mycelial biomass, in an amount from about 30 wt% to about 70 wt%; and a heme protein that is exogenous to the fungal mycelial biomass, in an amount from about 0.1 wt% to about 5 wt%.

In embodiments, the fungal mycelial biomass may be a cohesive fungal mycelial biomass. The fungal mycelial biomass may be produced by a liquid surface fermentation process, a solid- state fermentation process, or a membrane fermentation process.

In embodiments, the fungal mycelial biomass may be produced by a submerged fermentation process.

In embodiments, the heme protein may be dispersed in the fungal mycelial biomass. The food composition may be produced by contacting a liquid dispersion of the heme protein in water with the fungal mycelial biomass, and the heme protein may be dissolved, colloidally dispersed, or suspended in the water. A solubility of the heme protein in water may be at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 50 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, or at least about 250 g/L. At least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the heme protein may be dissolved in the water of the liquid dispersion. A mass of the liquid dispersion adsorbed by, adsorbed on a surface of, or coating the fungal mycelial biomass may be from about 23 wt% to about 51 wt% of a mass of the fungal mycelial biomass in the absence of the liquid dispersion.

In embodiments, the fungal mycelial biomass may not be spray-dried.

In embodiments, the fungal mycelial biomass may be spray-dried.

In embodiments, the fungal mycelial biomass may comprise at least one filamentous fungus belonging to an order selected from the group consisting of Ustilagincdes, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales. The filamentous fungus may belong to a family selected from the group consisting of Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae , Lycoperdaceae , Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae,Morchellaceae, Spar ssidaceae, Nectriaceae, and Cordycipitacea . The filamentous fungus may belong to the genus Fusarium. The filamentous fungus may belong to a species selected from the group consisting of Ustilago esculenta, Hericululm erinaceus, Polyporous squamosus, Grifola fondosa, Hypsizygus marmoreus, Hypsizygus ulmarius Calocybe gambosa Pholiotanameko Calvatia gigantea Agaricus bisporus Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus ostreatus var. columbinus, Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa, Fusarium venenatum, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), Disciotis venosa, and Cordyceps militaris.

In embodiments, the fungal mycelial biomass may comprise from about 35 wt% to about 60 wt% protein on a dry basis.

In embodiments, the fungal mycelial biomass may comprise from about 25 wt% to about 55 wt% carbohydrates on a dry basis.

In embodiments, the fungal mycelial biomass may comprise from about 20 wt% to about 40 wt% dietary fiber on a dry basis.

In embodiments, the fungal mycelial biomass may comprise from about 2.3 wt% to about 7.0 wt% lipids on a dry basis.

In embodiments, at least one of the following may be true: (i) the fungal mycelial biomass comprises from about 1.2 wt% to about 2.6 wt% c/.s,c/.s-polyunsaturated fatty acids on a dry basis; (ii) the fungal mycelial biomass comprises from about 0.1 wt% to about 0.6 wt% cis- monounsaturated fatty acids on a dry basis; (iii) the fungal mycelial biomass comprises from about 0.6 wt% to about 1.0 wt% saturated fatty acids on a dry basis; and (iv) the fungal mycelial biomass is substantially free of trans fatty acids.

In embodiments, the food composition may be a meat analog.

In embodiments, the food composition may be selected from the group consisting of an animal feed, a pet food, and an aquaculture feed.

In embodiments, the food composition may be selected from the group consisting of a hot dog analog, a burger analog, a ground meat analog, a sausage analog, a steak analog, a fdet analog, a roast analog, a meatbail analog, a meatloaf analog, and a bacon analog. In embodiments, the heme protein may be selected from the group consisting of androglobin, cytoglobin, globin E, globin X, globin Y, hemoglobin, myoglobin, leghemoglobin, erythrocruorin, beta hemoglobin, alpha hemoglobin, non-symbiotic hemoglobin, flavohemoglobin, protoglobin, cyanoglobin, Hell’s gate globin I, bacterial hemoglobin, ciliate myoglobin, histoglobin, neuroglobins, truncated 2/2 globin, HbN, HbO, Glb3, a heme peroxidase, a heme ligninase, a heme cytochrome, a heme oxidoreductase or catalase, and combinations thereof.

In embodiments, the food composition may further comprise one or more non-heme proteins exogenous to the fungal mycelial biomass. The one or more non-heme proteins may be derived from a vegetarian source. The vegetarian source may be a vegan source. The one or more non-heme proteins may be selected from the group consisting of seed proteins, legume proteins, tuber proteins, and combinations thereof. The one or more non-heme proteins may be selected from the group consisting of pea proteins, potato proteins, soy proteins, and combinations thereof. The one or more non-heme proteins may be present in an amount from about 6.5 wt% to about 33.5 wt% of the food composition.

In embodiments, the food composition may further comprise carbohydrates exogenous to the fungal mycelial biomass and selected from the group consisting of starch, dietary fiber, and combinations thereof. The carbohydrates may be present in an amount from about 0.1 wt% to about 10 wt% of the food composition.

In embodiments, the food composition may further comprise at least one binder or gelling agent. The at least one binder or gelling agent may be selected from the group consisting of methyl cellulose, hydrocolloids, carrageenans, calcium chloride, and combinations thereof. The at least one binder or gelling agent may be present in an amount from about 0.1 wt% to about 10 wt% of the food composition.

In embodiments, the food composition may further comprise at least one flavor, spice, or seasoning. The at least one flavor, spice, or seasoning may be selected from the group consisting of sodium chloride, a natural meat flavor additive, an artificial meat flavor additive, and combinations thereof. The at least one flavor, spice, or seasoning may be present in an amount from about 0.1 wt% to about 5 wt% of the food composition. In embodiments, the food composition may further comprise at least one food coloring. The at least one food coloring may be present in an amount from about 0.01 wt% to about 1 wt% of the food composition.

In embodiments, the food composition may further comprise at least one cooking fat or oil. The at least one cooking fat or oil may be selected from the group consisting of sunflower oil, coconut oil, and combinations thereof. The at least one cooking fat or oil may be present in an amount from about 1 wt% to about 15 wt% of the food composition.

In embodiments, at least one of the following may be true when the food composition is raw or uncooked: (i) a Hunter £ color value of the food composition is from about 44 to about 66;

(ii) a Hunter a color value of the food composition is from about 5 to about 19; and (iii) a Hunter b color value of the food composition is from about 15 to about 24. At least two of (i), (ii), and

(iii) may be true. All three of (i), (ii), and (iii) may be true.

In embodiments, at least one of the following may be true one hour after the food composition is cooked to an internal temperature of 160 °F: (i) a Hunter L color value of the food composition is from about 34 to about 53; (ii) a Hunter a value of the food composition is from about 7 to about 13; and (iii) a Hunter b value of the food composition is from about 14 to about 26. At least two of (i), (ii), and (iii) may be true. All three of (i), (ii), and (iii) may be true.

In embodiments, a method for making a food composition as disclosed herein comprises contacting a cohesive fungal mycelial biomass with a proteinaceous composition comprising at least one heme protein, whereby at least a portion of the proteinaceous composition is absorbed by, adsorbs onto a surface of, or coats the cohesive fungal mycelial biomass.

In embodiments, the proteinaceous composition may be a liquid dispersion of the heme protein. The liquid dispersion may comprise the heme protein and water, and the heme protein may be dissolved, colloidally dispersed, or suspended in the water. A solubility of the heme protein in water may be at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 50 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, or at least about 250 g/L. At least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the heme protein may be dissolved in the water of the liquid dispersion. A mass of the portion of the liquid dispersion adsorbed by, adsorbed on a surface of, or coating the fungal mycelial biomass may be from about 23 wt% to about 51 wt% of a mass of the fungal mycelial biomass in the absence of the liquid dispersion. In embodiments, the proteinaceous composition may be a powder or an aerosol.

BRIEF DESCRIPTION OF THE DRAWING

Figure l is a photograph of various burger analog food materials before and after cooking, according to embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, unless otherwise specified, the terms “analog” or “analog food product” and “replica” or “replica food product” are interchangeable and each refer to a food product comprising edible fungi that bears an aesthetic, culinary, nutritional, and/or sensory equivalence or resemblance to an identified non-fungal food product. By way of non-limiting example, a “meat analog food product,” as that term is used herein, refers to a food product comprising edible fungi that bears an aesthetic, culinary, nutritional, and/or sensory equivalence or resemblance to animal meat.

As used herein, unless otherwise specified, the term “animal protein” refers to any protein that is normally present in the body of an animal. By way of non-limiting example, an “animal protein” as that term is used herein may be an animal-derived protein (i.e., a protein molecule that is extracted, isolated, etc. from an animal), or may be a protein chemically and physically identical to a protein normally present in the body of an animal but obtained by other means (e.g. , by genetic engineering/recombination techniques of a non-animal cell).

As used herein, the term “biomass,” unless otherwise specified, refers to a mass of a living or formerly living organism. By way of non-limiting example, the phrase “filamentous fungal biomass” as used herein refers to a mass of a living or formerly living filamentous fungus. Filamentous fungal biomasses may include biomats and a mycoprotein paste as described in U.S. Patent 7,635,492 to Finnigan etal.

As used herein, unless otherwise specified, the term “cohesive” refers to any material that has sufficient structural integrity and tensile strength to be picked up and/or physically manipulated by hand as a solid object, without tearing or collapsing

As used herein, unless otherwise specified, the term “disperse” and its derived terms (“dispersed in,” “dispersion,” etc.) is used in either of two different senses, both of which refer to a type of physical contact between two different materials. Particularly, where a first material is referred to as being “dispersed in” a second material, and the second material is substantially entirely liquid or gas, the term is used in its usual chemical sense, i.e., a system in which discrete particles of the first material are dispersed (in the conventional sense) throughout a continuous phase of the second material, the system as a whole being referred to as a “dispersion.” By contrast, where a first material is referred to as being “dispersed in” a second material, and the second material includes an appreciable solid content (for example, where the second material is a living or formerly living biomass), the term refers to the condition achieved by a mixing step in which the first material can be visibly seen to be taken up, e.g., by absorption or adsorption, by the second material, rather than being repelled and remaining as distinct particles, droplets, etc. Particularly, when used in this second sense, “dispersing” one material in another (solid) material may often have the effect of causing the resulting “dispersion” to have a relatively uniform and/or homogeneous color or other visual appearance.

As used herein, unless otherwise specified, the term “filamentous fungus” refers to any multicellular fungus that is capable of forming an interconnected network of hyphae (vegetative hyphae or aerial hyphae, and most commonly both) known as “mycelium.” Examples of “filamentous fungi” as that term is used herein include, but are by no means limited to, fungi of the genera Acremonium, Alternaria, Aspergillus Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma, and Trichophyton, among many others. It is to be expressly understood that filamentous fungi, as that term is used herein, may be capable of forming other fungal structures, such as fruiting bodies, in addition to hyphae/mycelium.

As used herein, unless otherwise specified, the term “food product” refers to any product adapted, configured, and/or intended to be eaten or drunk by an animal, including but not limited to a human, for reasons of nutrition, pleasure, or both. Non-limiting examples of “food products” as that term is used herein include human foods, animal feeds, pet foods, and aquaculture feeds. Food products adapted, configured, and/or intended to be eaten or drunk by a human, specifically, are referred to herein as “human food products” or “culinary food products.”

As used herein, unless otherwise specified, the terms “fungal mycelial matter” and “fungal mycelial biomass” are interchangeable and each refer to any material that includes at least about 50 wt% fungal mycelium on a dry basis (z.e., disregarding the mass of any water). More specifically, as used herein, unless otherwise specified, the term “consisting essentially of fungal mycelium” refers to any material that includes at least about 95 wt% fungal mycelium on a dry basis, with any additional component of the material not materially affecting the characteristic of being a food. As used herein, unless otherwise specified, the term “meat” refers to flesh of any animal that is intended to be eaten by another animal as food. If not otherwise specified, “meat,” as that term is used herein, can include any part (skeletal muscle, fat and other tissues associated with skeletal muscle, offal, etc.) of any animal (mammals, fish, shellfish, insects, poultry, etc.) intended for consumption by another animal as food.

As used herein, unless otherwise specified, the term “vegan” refers to a food product that is substantially free of food components or ingredients, such as protein, derived from animals. Specific examples of non-vegan food ingredients or products include blood, eggs, isinglass, meat (and components thereof, e.g., animal proteins or fats), milk, rennet, and foods made using any one or more of these ingredients (e.g., pate, sausage, etc.). As disclosed herein, some vegan food products may be analogs of non-vegan food products.

As used herein, unless otherwise specified, the term “vegetarian” refers to a food product that is substantially free of meat and components thereof. “Vegetarian” food products, as that term is used herein, may (but need not) include food components or ingredients other than meat that are derived from animals (e.g., eggs, milk, etc.). Thus, as the terms are used herein, all “vegan” food products are “vegetarian,” but not all “vegetarian” food products are necessarily “vegan.”

As used herein, unless otherwise specified, the term “water holding capacity” refers to the ability of a material to “hold” (that is, tightly bind) water or a substantially aqueous fluid upon soaking, and is defined as the mass of a material after it has been soaked in an aqueous fluid and centrifuged to be separated from free water, divided by the mass of the material after soaking but before centrifugation. For example, a material that has a mass of 5 g after being soaked in water, and subsequently a mass of 2 g after being centrifuged to separate the free water, has a water holding capacity of 2 / 5 = 0.4, or 40%.

The following reference generally relates to fungal food products and systems and methods associated therewith, and is hereby incorporated by reference in their entireties: PCT Application Publication 2020/176758, entitled “Food Materials Comprising Filamentous Fungal Particles and Membrane Bioreactor Design,” published 3 September 2020 to Macur et al.

The subject matter of the present invention relates to the use of edible fungal matter in food products that provides numerous benefits as a primarily or completely non-meat protein source that can be efficiently produced and provides high amounts of protein, including complete protein. The edible fungal matter is improved by the inclusion of heme proteins to the product to improve one or more sensory attributes of the product. The heme protein can be produced independently of the fungal matter and added to it, and/or the heme protein can be produced by a filamentous fungus that has been engineered to produce the heme protein, and/or the heme protein can be produced by a filamentous fungus that has not been engineered, and/or the heme protein can be produced by another heme-producing organism (e.g. a plant, a yeast, a bacterium, an algae, a mammal, etc.).

One aspect of the present disclosure provides food products, often but not exclusively human food products, that include both edible fungal biomass (most typically fungal mycelial matter) and at least one heme protein exogenous to the edible fungal biomass. Typically, such food products can be made by mixing fungal mycelial matter together with a solid or liquid composition comprising the heme protein, such that the fungal mycelial matter and the heme protein do not readily separate into distinct phases (e.g., by causing the heme protein composition to be absorbed by, to adsorb onto a surface of, and/or to coat the fungal mycelial matter).

While incorporation of heme proteins into vegetable protein-based products to simulate meat products is known, there are no known attempts to incorporate exogenous heme proteins into fungal food products which present unique challenges compared to vegetable-based products because of the significant chemical, physical and structural differences between plant and fungal biomasses. Particularly, it has heretofore been difficult to ensure uniform and/or homogeneous dispersal and/or distribution of an exogenous heme-containing composition into and throughout a fungal biomass, and the results of attempts to disperse or distribute heme proteins into fungal materials has yielded unpredictable results. For example, depending on the physical form of the fungal biomass and the processing methods used to isolate and obtain the fungal biomass (e.g., spray-drying particles of filamentous fungal biomass derived from a submerged fermentation process), the biomass may evince hydrophobic properties that repel aqueous compositions with which the biomass comes into contact and cause the aqueous composition to bead on the surface of the biomass rather than be incorporated throughout the biomass. This represents a significant obstacle for the production of heme protein-containing fungal food products, which require sufficient distribution of the heme protein throughout the biomass to meet consumer needs for appearance, taste and flavor from the heme protein. The most typical delivery vehicle for exogenous heme proteins is as a liquid dispersion (e.g., solution, colloid, or suspension) of the heme protein(s) in water (one non-limiting example of such compositions being Hemami™, produced by Motif FoodWorks, Inc., Boston, MA). The methods, systems, and compositions of the present disclosure overcome many of the above-described challenges and difficulties that have been encountered in the art. Particularly, as further described throughout this disclosure, fungal biomasses used in food products according to the present disclosure have a surprising and advantageous affinity for and ability to take up (/.< ., to absorb, have absorbed thereon, or be coated by) aqueous dispersions, especially aqueous solutions of water-soluble heme proteins. This allows the heme proteins to be more readily and completely incorporated throughout the entirety of the food product, rather than causing separation of the food product into distinct “phases” of fungal matter and heme protein.

The fungal biomasses used in food products according to the present disclosure are filamentous fungal biomasses — that is, biomasses of one or more fungi that produce hyphae known collectively as mycelium. Particularly, in many embodiments, the filamentous fungal biomass in food products according to the present disclosure may be a fungal mycelial biomass as that term is defined herein (although other filamentous fungal biomasses, such as biomasses that contain a significant quantity of material derived from fruiting bodies of a filamentous fungus, are also contemplated and are within the scope of this disclosure). Most typically, the fungal mycelial biomasses used in food products according to the present disclosure are cohesive fungal mycelial biomasses (or cohesive mycelial biomasses), i.e., mycelial biomasses that have sufficient structural integrity and tensile strength to be picked up and physically manipulated by hand without tearing or collapsing. Cohesive fungal mycelial biomasses can also be characterized as having been grown from a nutritive source (which can be a liquid or solid media) into a space free from the nutritive source or other supporting materials or structures so that the mycelium form an interconnected network that is cohesive as that term is defined herein. Non-limiting examples of cohesive fungal mycelial biomasses that may suitably be used in food products of the present disclosure include fungal mycelial biomasses produced by a liquid surface fermentation process or membrane fermentation process as described in PCT Application Publication WO 2019/046480 (the entirety of which is incorporated herein by reference) and/or fungal mycelial biomasses produced by a solid-substrate fermentation process as described in, e.g, PCT Application Publication WO 2019/099474 (the entirety of which is incorporated herein by reference) and/or fungal mycelial biomasses produced by the processes as described in PCT Application Publication WO 2018/014004 (the entirety of which is incorporated herein by reference). As further described throughout this disclosure, the present inventors have found that cohesive fungal mycelial biomasses are particularly effective to take up aqueous dispersions of heme proteins, such that the heme proteins may be uniformly and/or homogeneously incorporated throughout a bulk, or about substantially all of a surface, of the cohesive fungal mycelial biomass. It is to be expressly understood, however, that in certain embodiments non-cohesive fungal mycelial biomasses, e.g., biomasses produced by a submerged fermentation process as described in PCT Application Publication 95/23843 (the entirety of which is incorporated herein by reference), can have sufficient ability to take up and retain aqueous dispersions of heme proteins to be suitable for use in food products according to the present disclosure, and such embodiments are within the scope of this disclosure as well.

The ingredients of food compositions of the present disclosure, and most especially the fungal mycelial biomass, may be selected to provide the fungal mycelial biomass (either alone or when mixed with other ingredients of the food product) with a desired water holding capacity, which may in many embodiments entail selecting a material with a water holding capacity sufficient to allow for absorption by, adsorption by or coating of the fungal mycelial biomass with a solute in, particle dispersed in or other material held by an aqueous composition. For example, where the food product is a burger analog food product, the fungal mycelial biomass may desirably have a water holding capacity of 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%, or at least about 55%, or alternatively any value in any range having a lower bound of any whole number of percentage points from 20% to 55% and an upper bound of any other number of percentage points from 20% to 55%. In particular embodiments, the fungal mycelial biomass may be a cohesive fungal mycelial biomass (e.g., a biomat produced by a surface fermentation process), which may have an advantageously higher water holding capacity compared to, e.g., a non-cohesive fungal mycelial biomass (e.g., a fungal “paste” derived from a submerged fermentation process).

The food products of the present disclosure may advantageously contain no meat or other animal-derived products and may therefore be suitable for people following vegetarian and/or vegan diets. Particularly, in some embodiments, the food products of the present disclosure may be meat analog food products that allow a person to perceive an aesthetic, culinary, nutritional, and/or sensory quality of meat, without consuming meat. Conversely, it may in many embodiments be desirable for the food product to contain a combination of edible fungal matter and a heme protein exogenous to the fungal matter with true animal meat, for example to provide an analog of a conventional meat product (e.g., a hot dog analog, a burger analog, a ground meat analog, a sausage analog, a steak analog, a filet analog, a roast analog, a meatball analog, a meatloaf analog, or a bacon analog) that provides sensory perceptions characteristic of the meat product but having improved nutritional value, lower cost, and/or lower environmental impact.

In many embodiments, the food products of the present disclosure may be characterized, when uncooked, by a sensory perception similar to a sensory perception of a raw meat product; may be characterized, when cooking, by a sensory perception similar to a sensory perception of a cooking meat product; and/or may be characterized, when cooked, by a sensory perception similar to a sensory perception of a cooked meat product. Such sensory perceptions may include any one or more of visual perceptions (e.g., a red meat analog food product of the disclosure may, due to the inclusion of the heme protein, be pink when uncooked and transition from pink to brown in the course of cooking, as a true red meat product does), auditory perceptions (e.g. , a meat analog food product of the disclosure may “sizzle” or “pop” while cooking in a manner similar to a true meat product), olfactory perceptions (e.g., a meat analog food product of the disclosure may, due to the inclusion of the heme protein, release one or more odor compounds characteristic of cooking meat during a cooking process), tactile perceptions (e.g., a meat analog food product of the disclosure may have a texture and/or mouthfeel akin to that of a meat product), and gustatory perceptions (e.g., a meat analog food product of the disclosure may have the umami and/or savory flavors characteristic of a meat product after being cooked). It may be particularly preferable for meat analog food products according to the present disclosure to achieve the sensory perceptions of a cooked meat product after being subjected to a cooking process identical or similar to the cooking process typical of the meat product of which it is an analog; by way of non-limiting example, where the cooking of a meat product entails exposing the meat product to a temperature of at least about 150 °C (e.g., a pan over medium or medium-high heat on a kitchen stove) for a period of several minutes (for many food products, such as burger patties, about 2 to about 7 minutes (total or per side), or about 3 to about 5 minutes (total or per side)), the same cooking process may be carried out to cause the meat analog product of the present disclosure to exhibit similar sensory perceptions as the cooking and/or cooked meat product.

Food products according to the present disclosure may include any one or more heme proteins, and in particular may include any one or more heme proteins that are “exogenous” to (i.e., not natively produced by) the filamentous fungus from which the edible mycelial matter of the food product is derived. Non-limiting examples of heme proteins suitable for use in the food products of the present disclosure include globins e.g., androglobin, cytoglobin, globin E, globin X, globin Y, hemoglobin, myoglobin, leghemoglobin, erythrocruorin, beta hemoglobin, alpha hemoglobin, non-symbiotic hemoglobin, flavohemoglobin, protoglobin, cyanoglobin, Hell’s gate globin I, bacterial hemoglobin, ciliate myoglobin, histoglobin, neuroglobins, truncated 2/2 globin, HbN, HbO, Glb3, a heme-containing peroxidase, a heme-containing ligninase, a heme-containing cytochrome, another heme-containing oxidoreductase or catalase, etc.), cytochromes, methemalbumins, and the like. The heme proteins are typically present in the food product in an amount from about 0.01 wt% to about 5 wt%, or in any range having a lower bound of about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, about 0.15 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.35 wt%, about 0.4 wt%, about 0.45 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2.0 wt%, about 2.1 wt%, about 2.2 wt%, about 2.3 wt%, about 2.4 wt%, about 2.5 wt%, about 2.6 wt%, about 2.7 wt%, about 2.8 wt%, about 2.9 wt%, about 3.0 wt%, about 3.1 wt%, about 3.2 wt%, about 3.3 wt%, about 3.4 wt%, about 3.5 wt%, about 3.6 wt%, about 3.7 wt%, about 3.8 wt%, about 3.9 wt%, about 4.0 wt%, about 4.1 wt%, about 4.2 wt%, about 4.3 wt%, about 4.4 wt%, about 4.5 wt%, about 4.6 wt%, about 4.7 wt%, about 4.8 wt%, or about 4.9 wt%, and an upper bound of about 5.0 wt%, about 4.9 wt%, about 4.8 wt%, about 4.7 wt%, about 4.6 wt%, about 4.5 wt%, about 4.4 wt%, about 4.3 wt%, about 4.2 wt%, about 4.1 wt%, about 4.0 wt%, about 3.9 wt%, about 3.8 wt%, about 3.7 wt%, about 3.6 wt%, about 3.5 wt%, about 3.4 wt%, about 3.3 wt%, about 3.2 wt%, about 3.1 wt%, about 3.0 wt%, about 2.9 wt%, about 2.8 wt%, about 2.7 wt%, about 2.6 wt%, about 2.5 wt%, about 2.4 wt%, about 2.3 wt%, about 2.2 wt%, about 2.1 wt%, about 2.0 wt%, about 1.9 wt%, about 1.8 wt%, about 1.7 wt%, about 1.6 wt%, about 1.5 wt%, about 1.4 wt%, about 1.3 wt%, about 1.2 wt%, about 1.1 wt%, about 1.0 wt%, about 0.9 wt%, about 0.8 wt%, about 0.7 wt%, about 0.6 wt%, about 0.5 wt%, about 0.45 wt%, about 0.4 wt%, about 0.35 wt%, about 0.3 wt%, about 0.25 wt%, about 0.2 wt%, or about 0.15 wt%. In particular embodiments, the heme protein can be present in an amount from about 0.1 wt% to about 1.5 wt%, from about 0.1 wt% to about 1.0 wt%, or from about 0.1 wt% to about 0.5 wt%. In embodiments, any one or more heme proteins present in the food product may be produced (either naturally or as a result of genetic engineering/recombinant techniques) by, and/or obtained from, an animal, a plant, a fungus other than the fungus from which the edible fungal mycelial matter is derived (e.g., a Saccharmycetales yeast), a protist, or a bacterium.

As further described above and throughout this disclosure, in many embodiments the edible fungal mycelial matter in food products according to the present disclosure may comprise or consist of cohesive fungal biomass, e.g., as produced by a liquid surface fermentation process, a solid-state fermentation process, or a membrane fermentation process, and may typically include a high proportion of mycelium, including vegetative and aerial hyphae. The use of a cohesive fungal biomass may be particularly advantageous in embodiments in which the heme protein is provided in a liquid dispersion, and especially an aqueous dispersion (i.e., where the one or more heme proteins are dissolved, colloidally dispersed, or suspended in a liquid medium such as water). As further described in Example 3, cohesive fungal biomasses used in embodiments of the fungal food products of the present disclosure have a surprising and advantageous affinity for and ability to take up aqueous dispersions, especially aqueous solutions of water-soluble heme proteins. To facilitate uptake of the liquid dispersion of heme protein by the fungal biomass (e.g., absorption by the fungal biomass, adsorption onto a surface of the fungal biomass by the liquid dispersion, and/or effective coating of the fungal biomass by the liquid dispersion), the heme protein may in embodiments have solubilities in the liquid dispersion medium (which will most typically be water) of at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 50 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, or at least about 250 g/L, and/or at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the heme protein in the food product may be dissolved in the liquid dispersion medium. Additionally or alternatively, the fungal mycelial matter of the food product may be able to absorb, or have adsorbed thereon, or be coated by a mass of a liquid dispersion of heme protein equal to 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%, or at least about 55% (or, alternatively, any amount in a range having a lower bound of any whole number of percentage points from 20% to 55% and an upper bound of any whole number of percentage points from 20% to 55%, e.g., from about 23% to about 51%) of its own mass in the absence of the liquid dispersion. Of course, it is to be expressly understood that in many embodiments the heme protein(s) may be provided in any one or more additional or alternative physical forms (e.g., a solid powder, an aerosol, etc.), and such embodiments are within the scope of the present disclosure.

This disclosure is based on methods and materials for modulating the taste and/or aroma profile of food products. As described herein, compositions containing one or more flavor precursors and one or more highly conjugated heterocyclic rings complexed to an iron (referred to herein as an iron complex) can be used to modulate the taste and/or aroma profile of food products. Such iron complexes include heme moieties or other highly conjugated heterocylic rings complexed to an iron ion (referred to as an iron complex). “Heme” refers to a prosthetic group bound to iron in the center of a porphyrin ring. Thus, an iron complex can be a heme moiety, or a porphyrin, porphyrinogen, corrin, corrinoid, chlorin, bacteriochorophyll, corphin, chlorophyllin, bacteriochlorin, or isobacteriochlorin moiety complexed to iron ion. The heme moiety that can be used to modulate the taste and/or aroma profile of food products can be a heme cofactor such as a heme protein; a heme moiety bound to a non-peptidic polymer or other macromolecule such as a liposome, a polyethylene glycol, a carbohydrate, a polysaccharide, a cyclodextrin, a polyethylenimine, a polyacrylate, or derivatives thereof; a siderophore (i.e., an iron chelating compound); or a heme moiety bound to a solid support (e.g., beads) composed of a chromatography resin, cellulose, graphite, charcoal, or diatomaceous earth.

In some embodiments, the iron complexes catalyze some reactions and produce flavor precursors without heating or cooking. In some embodiments, the iron complex destabilizes upon heating or cooking and releases the iron, e.g., the protein is denatured, so flavor precursors can be generated.

Suitable flavor precursors include sugars, sugar alcohols, sugar derivatives, oils (e.g., vegetable oils), free fatty acids, alpha-hydroxy acids, dicarboxylic acids, amino acids and derivatives thereof, nucleosides, nucleotides, vitamins, peptides, protein hydrolysates, extracts, phospholipids, lecithin, and organic molecules.

In some embodiments, one flavor precursor or combinations of two to one hundred flavor precursors, two to ninety, two to eighty, two to seventy, two to sixty, or two to fifty flavor precursors are used. For example, combinations of two to forty flavor precursors, two to thirty- five flavor precursors, two to ten flavor precursors, or two to six flavor precursors can be used with the one or more iron complexes (e g., heme co-factors such as a heme proteins). For example, the one or more flavor precursors can be glucose, ribose, cysteine, a cysteine derivative, thiamine, lysine, a lysine derivative, glutamic acid, a glutamic acid derivative, alanine, methionine, IMP, GMP, lactic acid, and mixtures thereof (e.g., glucose and cysteine; cysteine and ribose; cysteine, glucose or ribose, and thiamine; cysteine, glucose or ribose, IMP, and GMP; cysteine, glucose or ribose, and lactic acid). For example, the one or more flavor precursors can be alanine, arginine, asparagine, aspartate, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine, glucose, ribose, maltodextrin, thiamine, IMP, GMP, lactic acid, and creatine.

As used herein, the term “heme protein” can be used interchangeably with “heme polypeptide” or “heme-containing protein” or “heme-containing polypeptide” and includes any polypeptide that can covalently or noncovalently bind a heme moiety. In some embodiments, the heme polypeptide is a globin and can include a globin fold, which comprises a series of seven to nine alpha helices. Globin type proteins can be of any class (e.g., class I, class II, or class III), and in some embodiments, can transport or store oxygen. For example, a heme protein can be a non- symbiotic type of hemoglobin or a leghemoglobin. A heme polypeptide can be a monomer, i.e., a single polypeptide chain, or can be a dimer, a trimer, tetramer, and/or higher order oligomers. The life-time of the oxygenated Fe2⁺ state of a heme protein can be similar to that of myoglobin or can exceed it by 10%, 20%, 30% 50%, 100% or more under conditions in which the heme-protein food product is manufactured, stored, handled or prepared for consumption. The life-time of the unoxygenated Fe²⁺ state of a heme protein can be similar to that of myoglobin or can exceed it by 10%, 20%, 30% 50%, 100% or more under conditions in which the heme-protein food product is manufactured, stored, handled or prepared for consumption. Non-limiting examples of heme polypeptides can include an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobins, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e g., Glb3), a cytochrome, or a peroxidase.

Heme proteins that can be used in the compositions and food products described herein can be from mammals (e.g., farms animals such as cows, goats, sheep, pigs, ox, or rabbits), birds, plants, algae, fungi (e.g., yeast or filamentous fungi), ciliates, or bacteria. For example, a heme protein can be from a mammal such as a farm animal (e.g., a cow, goat, sheep, pig, ox, or rabbit) or a bird such as a turkey or chicken. Heme proteins can be from a plant such as Nicoticma tabacum or Nicotiana sylvestris (tobacco); Zea mays (com), Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicer arietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such as garden peas or sugar snap peas, Phaseolus vulgaris varieties of common beans such as green beans, black beans, navy beans, northern beans, or pinto beans, Vigna unguiculata varieties (cow peas), Vigna radiata (Mung beans), Lupinus albus (lupin), 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 (sugarbeet); Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa), Sesamum sp. (sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley). Heme proteins can be isolated from fungi such as Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, Aspergillus oryzae, Trichoderma reesei, Myceliopthera thermophile, Kluyvera lactis, or Fusarium oxysporum. Heme proteins can be isolated from bacteria such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or thermophilic bacteria such as Thermophilus. The sequences and structure of numerous heme proteins are known. See for example, Reedy, et al., Nucleic Acids Research, 2008, Vol. 36, Database issue D307-D313 and the Heme Protein Database available on the world wide web at http://hemeprotein.info/heme.php.

For example, a non-symbiotic hemoglobin can be from a plant selected from the group consisting of soybean, sprouted soybean, alfalfa, golden flax, black bean, black eyed pea, northern, garbanzo, moongbean, cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheat berries, spelt, barley, wild rice, or rice.

Heme proteins can be extracted from the source material (e.g., extracted from animal tissue, or plant, fungal, algal, or bacterial biomass, or from the culture supernatant for secreted proteins) or from a combination of source materials (e.g., multiple fungi species). Leghemoglobin is readily available as an unused by-product of commodity legume crops (e.g., soybean, alfalfa, or pea). The amount of leghemoglobin in the roots of these crops in the United States exceeds the myoglobin content of all the red meat consumed in the United States.

In some embodiments, extracts of heme proteins include one or more non-heme proteins from the source material (e.g., other animal, plant, fungal, algal, or bacterial proteins) or from a combination of source materials (e.g., different animal, plant, fungi, algae, or bacteria). In embodiments, food products according to the present disclosure may include one or more non- heme proteins exogenous to the fungal mycelial biomass (which may be provided as part of a heme protein extract or heme protein composition, or as a separate ingredient or component of the food product), such as, by way of non-limiting example, plant-derived non-heme proteins (as may be particularly desirable in, e.g., a vegetarian or vegan food product). Suitable non-heme proteins include, but are not limited to, seed proteins, legume proteins (e.g., pea proteins), tuber proteins (e.g., potato proteins), soy proteins, and the like. The non-heme proteins exogenous to the fungal mycelial biomass may, in particular embodiments, be present in the food product in an amount from about 6.5 wt% to about 65 wt% on a dry basis, or alternatively in any amount in any subrange having a lower bound of any half of a percentage point from 6.5 wt% to 65 wt% and an upper bound of any other half of a percentage point from 6.5 wt% to 65 wt% (e.g., a range from about 6.5 wt% to about 33.5 wt%).

In some embodiments, heme proteins are isolated and purified from other components of the source material (e.g., other animal, plant, fungal, algal, or bacterial proteins). As used herein, the term “isolated and purified” indicates that the preparation of heme protein is at least 60% pure, e.g., greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. Without being bound by theory, isolating and purifying proteins can allow the food products to be made with greater consistency and greater control over the properties of the food product as unwanted material is eliminated. Proteins can be separated on the basis of their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation. In some embodiments, the proteins can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Proteins also can be separated on the basis of their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents or solvent extraction. Proteins also can be separated by their affinity to another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography also can include using antibodies having specific binding affinity for the heme protein, nickel NTA for His-tagged recombinant proteins, lectins to bind to sugar moieties on a glycoprotein, or other molecules which specifically binds the protein.

Heme proteins also can be recombinantly produced using polypeptide expression techniques (e.g., heterologous expression techniques using bacterial cells, insect cells, fungal cells, plant cells such as tobacco, soybean, or Arabidopsis, or mammalian cells). In some cases, standard polypeptide synthesis techniques (e.g., liquid-phase polypeptide synthesis techniques or solidphase polypeptide synthesis techniques) can be used to produce heme proteins synthetically. In some cases, in vitro transcription-translation techniques can be used to produce heme proteins.

The protein used in the food product may be soluble in a solution. In some embodiments, the isolated and purified proteins are soluble in solution at greater than 5, 10, 15, 20, 25, 50, 100, 150, 200, or 250 g/L.

In some embodiments, the isolated and purified protein is substantially in its native fold and water soluble. In some embodiments, the isolated and purified protein is more than 50, 60, 70, 80, or 90% in its native fold. In some embodiments, the isolated and purified protein is more than 50, 60, 70, 80, or 90% water soluble.

Modulating Flavor and/or Aroma Profiles

As described herein, different combinations of flavor precursors can be used with one or more iron complexes (e.g., a ferrous chlorin, a chlorin-iron complex, or a heme-cofactor such as a heme protein or heme bound to a non-peptidic polymer such as polyethylene glycol or to a solid support) to produce different flavor and aroma profiles when the flavor precursors and iron complexes are heated together (e g., during cooking). The resultant flavor and/or aroma profile can be modulated by the type and concentration of the flavor precursors, the pH of the reaction, the length of cooking, the type and amount of iron complex (e.g., a heme-cofactor such as heme protein, heme bound to non-peptidic polymer or macromolecule, or heme bound to a solid support), the temperature of the reaction, and the amount of water activity in the product, among other factors. In embodiments in which a heme moiety is bound to a solid support such as cellulose or a chromatography resin, graphite, charcoal, or diatomaceous earth, the solid support (e g., beads) can be incubated with sugars and/or one or more other flavor precursors to generate flavors, and then the solid support with attached heme moiety can be re-used, i.e., incubated again with sugars and/or one or more other flavor precursors to generate flavors.

Flavor and aroma profiles are created by different chemical compounds formed by chemical reactions between the heme co-factor (e.g., heme protein) and flavor precursors. Gas chromatography-mass spectrometry (GCMS) can be used to separate and identify the different chemical compounds within a test sample. For example, volatile chemicals can be isolated from the head space after heating a heme protein and one or more flavor precursors. In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein is heated in the presence of ground chicken, to increase specific volatile flavor and odorant components typically elevated in beef. For example, propanal, butanal, 2-ethyl-furan, heptanal, octanal, trans-2-(2-pentenyl)furan, (Z)-2-heptenal, I-2-octenal, pyrrole, 2,4-dodecadienal, 1 -octanal, (Z)-2-decenal, or 2-undecenal can be increased in the presence of the heme protein, which can impart a more beefy flavor to the chicken.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein is heated in the presence of cysteine and glucose or other combinations of flavor precursors to provide a different profile of volatile odorants than when any subset of the three components are used individually. Volatile flavor components that are increased under these conditions include but are not limited to furan, acetone, thiazole, furfural, benzaldehyde, 2-pyridinecarboxaldehyde, 5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2- thiophenecarboxaldehyde, 3-thiophenemethanol and decanol. Under these conditions, cysteine and glucose alone or in the presence of iron salts such as ferrous glucanate produced a sulfurous, odor, but addition of heme proteins reduced the sulfurous odor and replaced it with flavors including but not limited to chicken broth, burnt mushroom, molasses, and bread.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein is heated in the presence of cysteine and ribose to provide a different profile of volatile odorants. Heating in the presence of ribose created some additional compounds as compared to when a heme protein and glucose were heated together.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein can be heated in the presence of thiamine and a sugar to affect the formation of 5-Thiazoleethanol, 4-methyl-furan, 3,3'-dithiobis[2-methyl-furan, and/or 4- Methylthiazole. These compounds are known to be present in meat and have beefy, meaty taste notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein can be heated in the presence of a nucleotide such as inosine monophosphate and/or guanosine monophosphate to control the formation of flavor compounds such I(E)-4-octene, 2-ethyl-furan, 2-pentanone, 2, 3 -butanedi one, 2-methyl-thiazole, methylpyrazine, tridele, (E)-2-octenal, 2-thiopenecarboxaldehyde, and/or 3-thiopenecarboxaldehyde. These compounds are known to be present in meat and have a beefy, meaty, buttery, and or savory flavor notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein can be heated in the presence of lysine, a sugar such as ribose, and cysteine to control the formation of flavor compounds such as dimethyl trisulfide, nonanal, 2- pentyl thiophene, 2-nonenal furfural, 1 -octanol, 2-nonenal, thiazole, 2-acetylthiazole, phenylacetaldehyde, and/or 2-acetylthiazole. These compounds are known to be present in meat and some have a beefy, meaty, and or savory flavor.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein can be heated in the presence of lactic acid, a sugar such as ribose, and cysteine to control the formation of the flavor compounds nonanal, thiazole, 2-acetylthiazole, and/or 8-methyl 1 -undecene. These compounds are known to be present in meat and have beefy, savory, browned, bready, and malty notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein can be heated in the presence of amino acids, sugars such as glucose, ribose, and maltodextrin, lactic acid, thiamine, IMP, GMP, creatine, and salts such as potassium chloride and sodium chloride, to control the formation of flavor compounds such as 1,3- bis(l,l-dimethylethyl)-benzene, 2-methyl 3-furanthiol, and/or bis(2-methyl-4,5-dihydro-3-furyl) disulfide. These compounds are known to be present in meat and have beefy notes.

In some embodiments, a particular type of heme protein is chosen to control the formation of flavor compounds. The addition of different types of heme-proteins (LegH, Barley, B. myoglobin, or A. aeolicus) in flavor reaction mixtures containing one or more flavor precursor compounds results in many of the same key meat flavors, including but not limited to pentanone, 3 -methyl butanal, 2-methyl butanal, 2-heptenal, 1 -octene, nonanal, 2-propenal, 2-decenal, 2- nonanone, 2-octanone, 2-tridecen-l-ol, 2-octanone, 2-octenal, 4-methyl-2-heptanone, octanal, 2- undecenal, butyrolactone, l-octen-3-one, 3 -methylheptyl acetate, and 2-pentyl-thiophene. These differences in flavor compounds can change the overall taste profile.

In some embodiments, an iron complex (e.g., a ferrous chlorin or a heme-cofactor such as a heme protein) described herein and one or more flavor precursors can be reacted (e.g., in vitro) with heating to generate a particular flavor and/or aroma profile of interest and the resultant flavor additive composition can be added to the food product of interest, which can then be eaten as-is or can be additionally modified, e.g., by additional cooking.

In some embodiments, any undesirable flavors can be minimized by deodorizing with activated charcoal or by removing enzymes such as lipoxygenases (LOX), which can be present in trace amounts when using preparations of fungal proteins, and which can convert unsaturated triacylglycerides (such as linoleic acid or linolenic acid) into smaller and more volatile molecules. LOX are naturally present in legumes such as peas, soybeans, and peanuts, as well as rice, potatoes, and olives. When legume flours are fractionated into separate protein fractions, LOX can act as undesirable “time-bombs” that can cause undesirable flavors on aging or storage. Compositions containing plant or fungal proteins (e.g., from ground plant seeds) can be subjected to purification to remove LOX using, for example, an affinity resin that binds to LOX and removes it from the protein sample. The affinity resin can be linoleic acid, linolenic acid, stearic acid, oleic acid, propyl gallate, or epigallocatechin gallate attached to a solid support such as a bead or resin. See, e.g., WO2013138793. In addition, depending on the protein component of the food product, certain combinations of antioxidants and/or LOX inhibitors can be used as effective agents to minimize off-flavor or off-odor generation especially in the presence of fats and oils. Such compounds can include, for example, one or more of P-carotene, a-tocopherol, caffeic acid, propyl gallate, or epigallocatechin gallate.

The ingredients of food compositions of the present disclosure, and most especially the fungal mycelial biomass and (if any) flavors, spices, and seasonings provided, may be selected to provide a desired flavor profile to the food product when uncooked and/or when cooked. For example, where the food product is a beef burger analog product, the source and amount of the fungal mycelial biomass, and in some embodiments the flavor(s), spice(s), and/or seasonings, incorporated into the food product may be selected to provide the food product, when cooked, with a flavor resembling that of a cooked beef hamburger. Non-limiting examples of flavor profiles that may be achieved by the selection of an appropriate fungal mycelial biomass and amount thereof (e.g., a cohesive fungal mycelial biomass, in an amount of about 45 wt% of the food product) and appropriate flavor(s), spice(s), and/or seasonings (e.g., sodium chloride, natural beef flavor, artificial beef flavor, etc.) include an umami flavor profile, a salty flavor profile, a beef-like flavor profile, a caramelized flavor profile, a “grill” (e.g. , charcoal or wood smoke) flavor profile, an iron or blood flavor profile, any combination of these flavor profiles, and the like. In some embodiments, the one or more heme proteins exogenous to the fungal mycelial biomass may also be selected with a desired flavor profile in mind; by way of non-limiting example, where the food product is a beefburger analog food product, it may be desirable for the one or more heme proteins exogenous to the fungal mycelial biomass to include beef myoglobin, as this may provide the food product with a desirable “beefy” or iron flavor profile.

In some embodiments, specific flavor compounds can be isolated and purified from the flavor additive composition. These isolated and purified compounds can be used as an ingredient to create flavors useful to the food and fragrance industry.

A flavor additive composition can be in the form, of but not limited to, soup or stew bases, bouillon, e.g., powder or cubes, flavor packets, or seasoning packets or shakers. Such flavor additive compositions can be used to modulate the flavor and/or aroma profile for a variety of food products, and can be added to a food product before, during, or after cooking of the food product. Food Products

Food products containing fungal biomass and one or more heme proteins, optionally with one or more flavor precursors, can be used as a base for formulating a variety of additional food products, including meat substitutes, soup bases, stew bases, snack foods, bouillon powders, bouillon cubes, flavor packets, or frozen food products. Meat substitutes can be formulated, for example, as hot dogs, burgers, ground meat, sausages, steaks, filets, roasts, breasts, thighs, wings, meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes.

In addition, food products described herein can be used to modulate the taste and/or aroma profile of other food products (e.g., meat replicas, meat substitutes, tofu, mock duck or other gluten based vegetable product, textured vegetable protein such as textured soy protein or textured fungal protein, pork, fish, lamb, or poultry products such as chicken or turkey products) and can be applied to the other food product before or during cooking. As used herein, the term “textured protein” refers to any defatted protein product that has been heated to denature the proteins into a fibrous, insoluble, porous network; by extension, as used herein, unless otherwise specified, the term “textured vegetable protein” refers to a textured protein product in which the proteins are derived from vegetables, “textured fungal protein” refers to a textured protein product in which the proteins are derived from fungi, etc. Using the food products described herein can provide a particular meaty taste and smell, for example, the taste and smell of beef or bacon, to a non-meat product or to a poultry product. Food products described herein can be packaged in various ways, including being sealed within individual packets or shakers, such that the composition can be sprinkled or spread on top of a food product before or during cooking.

Food products described herein can include additional ingredients including food-grade oils such as canola, com, sunflower, soybean, olive or coconut oil, seasoning agents such as edible salts (e.g., sodium or potassium chloride) or herbs (e.g., rosemary, thyme, basil, sage, or mint), flavoring agents, proteins (e.g., soy protein isolate, wheat gluten, pea vicilin, and/or pea legumin), protein concentrates (e.g., soy protein concentrate), emulsifiers (e.g., lecithin), gelling agents (e.g., k-carrageenan or gelatin), fibers (e.g., bamboo filer or inulin), or minerals (e.g., iodine, zinc, and/or calcium). In some embodiments, food products according to the present disclosure may further include one or more cooking fats or oils, such as, by way of non-limiting example, sunflower oil, coconut oil, and the like. Cooking fats and/or oils may, in some embodiments, be provided to augment the total lipid content of the food product, to improve a cooking performance of the food product e.g., to allow the food product to cook more evenly and/or without sticking to a cooking vessel), to simulate a sensory characteristic of a conventional food product during cooking (e.g., a cooking oil may flow out of a food product according to the present disclosure during cooking in the same way that fat may render from a meat product, such as a burger patty or strip of bacon, during cooking), and so on. The one or more cooking fats/oils may, in particular embodiments, be present in the food product in an amount from about 1 wt% to about 15 wt% of the food product on a dry basis, or alternatively in any amount in any subrange having a lower bound of any whole number of tenths of a percentage point from 1 wt% to 159 wt% and an upper bound of any other whole number of tenths of a percentage point from 1 wt% to 15 wt%. In some embodiments, food products according to the present disclosure may include one or more binders or gelling agents, such as, by way of non-limiting example, methyl cellulose, hydrocolloids, carrageenans, calcium chloride, and the like. The one or more binders or gelling agents may, in particular embodiments, be present in the food product in an amount from about 0.1 wt% to about 10 wt% of the food product on a dry basis, or in any subrange within about 0.1 wt% to about 10 wt% of the food product. In embodiments, food products according to the present disclosure may further include one or more flavors, spices, or seasonings, such as, by way of non-limiting example, sodium chloride (table salt), a natural meat flavor additive, an artificial meat flavor additive (e.g., a commercially available beef flavor additive, chicken flavor additive, pork flavor additive, etc ), and the like. The one or more flavors, spices, or seasonings may, in particular embodiments, be present in the food product in an amount from about 0.1 wt% to about 5 wt% of the food product on a dry basis, or in any subrange within about 0.1 wt% to about 5 wt% of the food product.

Food products described herein also can include a natural coloring agent such as turmeric or beet juice, or an artificial coloring agent such as azo dyes, triphenylmethanes, xanthenes, quinines, indigoids, titanium dioxide, red #3, red #40, blue #1, or yellow #5. Food colorings may, in some embodiments, be added to food products according to the present disclosure to more closely simulate or approximate a conventional food product of which the food product of this disclosure is an analog; by way of non-limiting example, a red or pink food coloring may be added to a red meat analog food product (such as a raw hamburger or raw ground beef analog food product) to more closely approximate the red or pink color of raw red meat. In some embodiments, the food coloring may be selected, adapted, and/or configured to change color (e.g., from red to brown) during a cooking process to simulate the color change of a conventional food product upon cooking. The one or more food colorings may, in particular embodiments, be present in the food product in an amount from about 0.01 wt% to about 1 wt% of the food product on a dry basis, or alternatively in any amount in any subrange having a lower bound of any whole number of hundredths of a percentage point from 0.01 wt% to 1 wt% and an upper bound of any other whole number of hundredths of a percentage point from 0.01 wt% to 1 wt%.

In embodiments, food products according to the present disclosure may further include one or more carbohydrates exogenous to the fungal mycelial biomass, such as, by way of non-limiting example, starches and dietary fiber compounds. The carbohydrates exogenous to the fungal mycelial biomass may, in particular embodiments, be present in the food product in an amount from about 0.1 wt% to about 10 wt% of the food product on a dry basis, or in any subrange within about 0.1 wt% to about 10 wt% of the food product.

Food products described herein also can include meat shelf life extenders such as carbon monoxide, nitrites, sodium metabisulfite, Bombal, vitamin E, rosemary extract, green tea extract, catechins and other anti-oxidants.

Food products described herein can be free of animal products (e.g., animal heme proteins or other animal products). In some embodiments, the food products can be soy-free, wheat-free, yeast-free, MSG- free, and/or free of protein hydrolysis products, and can taste meaty, highly savory, and without off odors or flavors.

Polypeptides

This disclosure provides for compositions and methods for the expression of a polypeptide in a host cell (e.g., yeast or bacteria). A polypeptide can refer to subunits or domains of a polypeptide. A polypeptide of the disclosure can be a heme polypeptide. The term heme polypeptide can refer to all proteins or protein subunits that are capable of covalently or noncovalently binding a heme moiety. Heme polypeptides can transport or store oxygen. In some instances, the polypeptide of the disclosure can be a globin. Polypeptides can comprise the globin fold, which can comprise a series of eight alpha helices. A polypeptide can comprise an alpha globin and/or a beta globin. A polypeptide can comprise a characteristic higher structure (e.g., the “myoglobin fold”) generally associated with globins. A polypeptide can be an oligomer. Polypeptides can be monomers, dimers, trimers, tetramers, and/or higher order oligomers. In some instances, a polypeptide can be an iron-containing polypeptide.

A polypeptide of the disclosure can include, but is not limited to, androglobin, cytoglobin, globin E, globin X, globin Y, hemoglobin, myoglobin, leghemoglobins, erythrocruorins, beta hemoglobins, alpha hemoglobins, non-symbiotic hemoglobins, flavohemoglobins, protoglobins, cyanoglobins, cytoglobin, Hell’s gate globin I, bacterial hemoglobins, ciliate myoglobins, histoglobins, neuroglobins, chlorocruorin, erythrocruorin, protoglobin, truncated 2/2 globin, HbN, HbO, Glb3, and cytochromes, ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructose bisphosphate aldolase, phosphofructokinases, triose phosphate isomerases, phosphoglycerate kinases, phosphoglycerate mutases, enolases, pyruvate kinases, proteases, lipases, amylases, glycoproteins, lectins, mucins, glyceraldehyde-3-phosphate dehydrogenases, pyruvate decarboxylases, actins, translation elongation factors, histones, ribulose-1,5- bisphosphate carboxylase oxygenase (rubisco), ribulose-l,5-bisphosphate carboxylase oxygenase activase (rubisco activase), albumins, glycinins, conglycinins, globulins, vicilins, conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin, phaseolin (protein), proteinoplast, secalin, extensins, triticeae gluten, collagens, zein, kafirin, avenin, dehydrins, hydrophilins, late embyogenesis abundant proteins, natively unfolded proteins, any seed storage protein, oleosins, caloleosins, steroleosins orother oil body proteins, vegetative storage protein A, vegetative storage protein B, moong seed storage 8S globulin, globulin, pea globulins, and pea albumins. In some instances, a polypeptide can be introduced into a host cell. For example, a polypeptide can be expressed, secreted, and/or purified from a fungus, and in particular from a filamentous fungus.

A polypeptide may be expressed, but may not be properly secreted and/or folded using the methods of the disclosure. A polypeptide may be expressed, but may be not be correctly localized in the cell using the methods of the disclosure. A polypeptide may be expressed, but may not retain levels of activity comparable to a wild-type polypeptide. A polypeptide may retain at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% activity level of a wild-type polypeptide. A polypeptide may retain at most about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% activity level of a wild-type polypeptide. A polypeptide comprising at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% amino acid sequence identity to a polypeptide may be expressed, but may not be properly secreted and/or folded using the methods of the disclosure. A polypeptide comprising at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% amino acid sequence identity to a polypeptide may be expressed, but may not be properly secreted and/or folded using the methods of the disclosure. A polypeptide comprising at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% amino acid sequence identity to a polypeptide may be expressed, but may not be retain activity compared to a wild-type polypeptide. A polypeptide comprising at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% amino acid sequence identity to a polypeptide may be expressed, but may contain less heme cofactor compared to a wild-type polypeptide.

In some instances, a sequence of a polypeptide to be expressed in a host cell can be a sequence comprising at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% amino acid sequence identity to an endogenous polypeptide sequence, e.g., an endogenous heme polypeptide, of the host cell. In some instances, a sequence of a polypeptide to be expressed in a host cell can be a sequence comprising at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% amino acid sequence identity to an endogenous polypeptide sequence of the host cell. For example, a polypeptide can be a polypeptide sequence found in an animal, a mammal, a vertebrate, an invertebrate, a plant, a fungus, a bacterium, a yeast, an alga, an archaea, a genetically modified organism such as a genetically modified fungus. A polypeptide sequence can be chemically synthesized, and/or synthesized by in vitro synthesis.

A polypeptide sequence can be a sequence of a polypeptide, e.g., a heme polypeptide, found in plants. Non-limiting examples of plants can include grains such as, e.g., com, maize, oats, rice, wheat, barley, rye, triticale, teff, oilseeds including cottonseed, sunflower seed, safflower seed, crambe, camelina, mustard, rapeseed, leafy greens such as, e.g., lettuce, spinach, kale, collard greens, turnip greens, chard, mustard greens, dandelion greens, broccoli, cabbage, sugar cane, trees, root crops such as cassava, sweet potato, potato, carrots, beets, turnips, plants from the legume family, such as, e.g., clover, peas such as cowpeas, English peas, yellow peas, green peas, beans such as, e.g., soybeans, fava beans, lima beans, kidney beans, garbanzo beans, mung beans, pinto beans, lentils, lupins, mesquite, carob, soy, and peanuts, coconut, vetch (vicia), stylo (stylosanthes), indigofera, acacia, leucaena, cyamopsis, and sesbania. Plants not ordinarily consumed by humans, including biomass crops, including, for example, switchgrass, miscanthus, tobacco, Arundo donax, energy cane, sorghum, other grasses, alfalfa, corn stover, kelp, or other seaweeds. Polypeptides that can be found in any organism in the plant kingdom may be used in the present disclosure. In some instances, the plant can be soy. In some instances, the plant can be barley.

In some instances, a polypeptide sequence can be a sequence, e g., a heme polypeptide sequence, found in metazoa. For example, a polypeptide sequence of the disclosure can be a polypeptide sequence found in mammals such as cow, pig, rat, dog, or horse. In some instances, the polypeptide sequence comes from cow. In some instances, the polypeptide sequence comes from pig. In some instances, a polypeptide sequence can be a sequence found in protists. For example, a polypeptide sequence of the disclosure can be a polypeptide sequence found in protists such as algae. In some instances, a polypeptide sequence can be a sequence found in archaea. For example, a polypeptide sequence of the disclosure can be a polypeptide sequence found in archaea such as halobacteria or pyrococcus. In some instances, a polypeptide sequence can be a sequence found in eubacteria. For example, a polypeptide sequence of the disclosure can be a polypeptide sequence found in eubacteria such as Bacdlus, Clostridia, or Escherichia.

As used herein, the term “heme protein” includes any polypeptide that can covalently or noncovalently bind to a heme moiety. In some embodiments, the heme polypeptide is a globin and can include a globin fold, which comprises a series of seven to nine alpha helices. Globin type proteins can be of any class (e.g., class I, class II, or class III), and in some embodiments, can transport or store oxygen. For example, a heme polypeptide can be a non-symbiotic type of hemoglobin or a leghemoglobin. A heme polypeptide can be a monomer, i.e., a single polypeptide chain, or can be a dimer, a trimer, tetramer, and/or higher order oligomers. The life-time of the oxygenated Fe⁺ state of a heme polypeptide can be similar to that of myoglobin or can exceed it by 10%, 20%, 30%, 40%, 50%, 100% or more.

Non-limiting examples of heme polypeptides can include an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a histoglobin, a neuroglobin, a chlorocruorin, a truncated hemoglobin (e g., HbN, HbO, a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3)), a cytochrome, or a peroxidase.

Heme polypeptides can be from mammals (e.g., farms animals such as cows, goats, sheep, pigs, ox, or rabbits), birds, plants, algae, fungi (e.g., yeast or filamentous fungi), ciliates, or bacteria. For example, a heme polypeptide can be from a mammal such as a farm animal (e.g., a cow, goat, sheep, pig, ox, or rabbit) or a bird such as a turkey or chicken. Heme polypeptides can be from a plant such as Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn), Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicer arietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such as garden peas or sugar snap peas, Phaseolus vulgaris varieties of common beans such as green beans, black beans, navy beans, northern beans, or pinto beans, Vigna unguiculata varieties (cow peas), Vigna radiate (Mung beans), Lupinus albus (lupin), 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 (sugarbeet); Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp. (sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley). Heme polypeptides can be isolated from fungi such as Saccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusarium graminearum, or Fusarium oxysporum. Heme polypeptides can be isolated from bacteria such as Escherichia coli, Bacillus subtilis, Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, or thermophilic bacteria such as Thermophilus.

The sequences and structure of numerous heme polypeptides are known. See for example, Reedy, et al., Nucleic Acids Research, 2008, Vol. 36, Database issue D307-D313 and the Heme Protein Database available on the world wide web at http://hemeprotein.info/heme.php.

A non-symbiotic hemoglobin can be from a plant selected from the group consisting of soybean, sprouted soybean, alfalfa, golden flax, black bean, black eyed pea, northern, garbanzo, moong bean, cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheat berries, spelt, barley, wild rice, or rice.

It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., fungi) is obtained, using appropriate codon bias tables for that species.

Heme polypeptides can be extracted from the source material (e.g., extracted from animal tissue, or plant, fungal, algal, or bacterial biomass, or from the culture supernatant for secreted proteins) or from a combination of source materials (e.g., multiple fungi species). Leghemoglobin is readily available as an unused by-product of commodity legume crops (e.g., soybean, alfalfa, or pea). The amount of leghemoglobin in the roots of these crops in the United States exceeds the myoglobin content of all the red meat consumed in the United States.

In some embodiments, extracts of heme polypeptides include one or more non-heme polypeptides from the source material (e.g., other animal, plant, fungal, algal, or bacterial proteins) or from a combination of source materials (e.g., different animal, plant, fungi, algae, or bacteria).

A polypeptide of the disclosure (e.g., a globin, a heme polypeptide, or an iron-containing protein), can be referred to as a “purified” polypeptide. A polypeptide of the disclosure can be purified from other components of the source material (e g., other animal, plant, fungal, algal, or bacterial proteins). A purified polypeptide can refer to a polypeptide that has been enriched in a composition, has been manipulated in some fashion to remove unwanted debris (e.g., cell debris, genomic DNA, and/or other polypeptides), and/or is removed from the host cell in which it was synthesized (e.g., transcribed/translated) (e.g., cell lysis). A “purified” polypeptide can be a polypeptide extracted from its host cell. In some embodiments, a “purified” polypeptide is at least 1% pure, e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. Proteins can be separated on the basis of their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation. In some embodiments, the proteins can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Proteins also can be separated on the basis of their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents or solvent extraction. Proteins also can be separated by their affinity to another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography can also include using antibodies having specific binding affinity for the heme polypeptide, antibody to the protein, nickel NTA for His-tagged recombinant proteins, lectins to bind to sugar moieties on a glycoprotein, or other molecules which specifically binds the protein. Hemoglobin

Hemoglobin (Hb) can be the major constituent of an erythrocyte which can carry oxygen from the lungs throughout the body. When contained in red blood cells, human Hb can exist as a tetramer structure composed of two oxygen linked ap dimers, each having a molecular weight of about 32 kD. Each a and subunit of each dimer can have a protein chain and a heme molecule. Hemoglobin, or “Hb” can refer to (a) an iron respiratory pigment found in vertebrate red blood cells that comprises a globin composed of four subunits (a tetramer) each of which is linked to a heme molecule, that functions in oxygen transport to the tissues after conversion to oxygenated form in the gills or lungs, and that assists in carbon dioxide transport back to the gills or lungs after surrender of its oxygen. A hemoglobin can refer to a recombinantly produced hemoglobin; aP- dimers of hemoglobin, inter- or intramolecularly crosslinked hemoglobin, as well as modified versions of the hemoglobins provided in the disclosure, which can include but are not limited to modifications increasing or decreasing the oxygen affinity of hemoglobin (e.g., such as substituting an alanine, valine, leucine, or phenylalanine for histidine. All hemoglobins can be capable of binding heme. A hemoglobin can be a variant hemoglobin. Variant hemoglobins can comprise amino acid mutations, substitutions, additions, and/or deletions. Hemoglobin variants can include hemoglobin Kansas, hemoglobin S, hemoglobin C, hemoglobin E, hemoglobin D- Punjab, hemoglobin O-Arab, hemoglobin G-Philadelphia, hemoglobin Hasharon, hemoglobin Lepore, and hemoglobin M.

Leghemoglobin

In some instances, the sequence (amino acid and/or nucleic acid) of a leghemoglobin can be a plant leghemoglobin sequence. Various legumes species and their varieties, for example, Soybean, Fava bean, Lima bean, Cowpeas, English peas, Yellow peas, Lupine, Kidney bean, Garbanzo beans, Peanut, Alfalfa, Vetch hay, Clover, Lespedeza and Pinto bean, comprise nitrogen-fixing root nodules in which leghemoglobin can have a key role in controlling oxygen concentrations. Leghemoglobins from different species can be homologs and have similar color properties. Some plant species can express several leghemoglobin isoforms (for example soybean has four leghemoglobin isoforms). Minor variations in precise amino acid sequence can modify overall charge of the protein at a particular pH and can modify precise structural conformation of an iron containing heme group in leghemoglobin. In some instances, an alanine, valine, leucine, or phenylalanine can be substituted for histidine. Differences in structural conformation of the hemegroup of different leghemoglobins can influence oxidation and reduction rates of the heme iron. These differences may contribute to color and flavor generation properties of different leghemoglobins.

Variants

A polypeptide of the disclosure can be a variant (e.g., comprise a mutation such as an amino acid substitution, e.g., a non-conservative or conservative amino acid substitution, an amino acid deletion, an amino acid insertion, ornon-native sequence). In some instances, a variant polypeptide can include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 mutations. In some instances, a variant polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more mutations. In some instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50% of the sequence of a polypeptide of the disclosure can be mutated. In some instances, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50% of the sequence of a polypeptide of the disclosure can be mutated. In some instances, a polypeptide of the disclosure can comprise at least about 10, 20, 30, 40, 50, 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% amino acid sequence identity to a naturally occurring polypeptide of the disclosure. In some instances, a polypeptide of the disclosure can comprise at most about 10, 20, 30, 40, 50, 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% amino acid sequence identity to a naturally occurring polypeptide of the disclosure.

In some instances, the polypeptide of the disclosure comprises a non-native sequence (e.g., a tag or a label). A tag can be covalently bound to the polypeptide sequence of the polypeptide. The tag can be bound to the N-terminus, or the C-terminus, or to an intervening amino acid. The tag can be inserted in the polypeptide sequence (e.g., in a solvent accessible surface loop). Examples of tags can include, but are not limited to, affinity tags (e.g., myc, maltose binding protein, or 6 his, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system), and fluorescent tags (e.g., green fluorescent protein).

A tag can be a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, tags suitable for use in the present disclosure can include biotin, digoxigenin, or haptens as well as proteins which can be made detectable, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), dyes (e.g., alexa, cy3 cy5), chemical conjugates (e.g., quantum dots), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

A tag can be detected. For example, where the tag is radioactive, means for detection can include a scintillation counter or photographic film, as in autoradiography. Where the tag is a fluorescent tag, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic tags may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent tags may be detected simply by observing the color associated with the tag.

In some instances, a tag can be a signal peptide. A signal peptide can be a peptide sequence usually present at the N-terminal end of newly synthesized secretory or membrane polypeptides which directs the polypeptide across or into a cell membrane of the cell (the plasma membrane in prokaryotes or the endoplasmic reticulum membrane in eukaryotes). It can be subsequently removed (e.g., by a protease). In particular, the signal peptide may be capable of directing the polypeptide into a cell’s secretory pathway. In some instances, the signal peptide is a secretory pathway signal peptide. In some such embodiments, the signal peptide can be referred to as a signal peptide or a secretion signal peptide.

Protoporphyrins

A polypeptide can bind to a tetrapyrrole (e.g., protoporphyrin). A polypeptide can bind to a protoporphyrin with its protoporphyrin binding portion (e.g., domain). A polypeptide can bind to a protoporphyrin as the polypeptide is being translated/folded. A polypeptide can bind to a protoporphyrin after the polypeptide is translated/folded. A polypeptide can remain bound to a protoporphyrin after it has been subcellularly localized (e.g., localized to a subcellular compartment, secreted).

Protoporphyrins can comprise side chains including methyl groups, propionic acid groups, and vinyl groups. Suitable protoporphyrin structures can include, but are not limited to, diiododeuteroporphyrin, mesoporphyrin, metalloporphyrins, and protoporphyrin IX. In some instances, a polypeptide can bind to more than one protoporphyrin. A polypeptide can bind to one, two, three, four, five, six, seven, eight, nine, ten or more protoporphyrins.

A protoporphyrin can be a protoporphyrin IX. Protoporphyrin IX (PpIX), Pheophorbide, a naturally occurring photosensitizer, can be the immediate precursor of heme in the heme biosynthetic pathway. Protoporphyrin IX can be referred to as heme. Heme can comprise a protoporphyrin ring and an iron atom, wherein the iron atom is coordinated by the members of the ring (e.g., the iron atom is inside the ring). In some instances, the protoporphyrin can be heme A, heme B, heme C, heme D, heme I, heme M, heme O or Heme S. In some instances, a protoporphyrin can coordinate an atom other than iron (i.e., metalloporphyrin). Other atoms can include for example, zinc, gadolinium, magnesium, manganese, cobalt, nickel, tin, and copper.

Vectors and Genetically Modified Organisms

Exogenous Nucleic Acids

The disclosure can provide for an exogenous nucleic acid encoding a polypeptide of the disclosure (e.g., a heme polypeptide, a globin). An exogenous nucleic acid can encode any of the heme polypeptides described herein. An exogenous nucleic acid can be RNA or DNA, and can be single stranded, double stranded, and/or codon optimized. An exogenous nucleic acid sequence encoding a polypeptide of the disclosure can be transcribed and/or translated. The term “polynucleotide” can be used interchangeably herein with “exogenous nucleic acid.”

The term “exogenous” as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non- naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a cDNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (cDNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally- occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a regulatory element (e.g., a promoter sequence and/or a signal sequence) and a sequence encoding a heme polypeptide (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host (e.g., fungus). For example, an entire chromosome isolated from a cell of plant x is an exogenous nucleic acid with respect to a cell of plant y once that chromosome is introduced into a cell of plant y.

In contrast, the term “endogenous” as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell “endogenously expressing” a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host “endogenously producing” or that “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.

The degeneracy of the genetic code can permit variations of the nucleotide sequence, while still producing a polypeptide having the identical amino acid sequence as the polypeptide encoded by the native polynucleotide sequence. Variations in the polynucleotide sequence can be customized for any organism of interest. In some instances, a polynucleotide encoding a polypeptide can be codon optimized for expression in a fungus (e.g., a filamentous fungus such as Fusarium venenatum).

The frequency of individual synonymous codons for cognate amino acids varies widely from genome to genome among eukaryotes and prokaryotes. These differences in codon choice patterns can contribute to the overall expression levels of individual genes by modulating peptide elongation rates.

Methods of Expression The disclosure can provide for methods for expression of a polypeptide (e.g., globin) in a host cell (e.g., a yeast cell or a bacterial cell).

In some instances, expression of a polypeptide can include introducing a vector comprising a polynucleotide sequence encoding the polypeptide into the host cell and inducing expression of the polypeptide.

In some embodiments, the methods of the disclosure provide for a host cell that comprises a stably integrated sequence of interest (i.e., polypeptide-encoding nucleic acid). However, in alternative embodiments, the methods of the present disclosure provide for maintenance of a selfreplicating extra-chromosomal transformation vector.

Methods of introducing the polynucleotide into cells for expression of the polynucleotide sequence can include, but are not limited to electroporation, transformation, transduction, high velocity bombardment with DNA-coated microprojectiles, infection with modified viral (e g., phage) nucleic acids; chemically-mediated transformation, or competence. In some embodiments, polynucleotides encoding a polypeptide of the disclosure can be transcribed in vitro, and the resulting RNA can be introduced into the host cell.

Following introduction of a polynucleotide comprising the coding sequence for a polypeptide of the disclosure, the host cell can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, and/or amplifying expression of a polypeptide-encoding polynucleotide. The culture conditions, such as temperature, pH and the like, can be those previously used for the host cell selected for expression. The progeny of cells into which such polynucleotide constructs have been introduced can be considered to comprise the poly pepti de-encoding poly nucl eoti de .

In some embodiments, the polypeptide or variant thereof can be expressed as a fusion protein by the host fungal cell. Although cleavage of the fusion polypeptide to release the desired protein can often be useful, it is not necessary. Polypeptides and variants thereof expressed and secreted as fusion proteins can retain their function.

Expression of a polypeptide of the disclosure can comprise transient expression and/or constitutive expression (e.g., developing of a stable cell line).

Expression of Recombinant Polypeptides

Expression of a polypeptide can comprise inducing the host cell to transcribe and/or translate the polypeptide encoded in the polynucleotide introduced to the host cell. Induction can occur after the host cell has been cultured for at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more hours. Induction can occur after the host cell culture has an optical density (OD) of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 ormore. Induction can occur after the host cell culture has an optical density (OD) of at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 5, 10, or 20 or more. Induction may be caused by addition of chemicals such as IPTG, arabinose or in response to a limiting nutrient such as Nitrogen, phosphorus, glucose or oxygen. In some instances, the polypeptide is linked to a promoter, such as aprE, liaG, lepA, cry3Aa, or gsiB that leads to constitutive expression of the polypeptide.

In some instances, chemical agents can be added to the media. In some instances, the chemical agents can aid the stability, heme content, and/or protein folding capability of the expressed polypeptide. A chemical agent can comprise a small molecule such as a metal. Examples of suitable metals for addition to media can include iron fluorides (iron difluoride, iron trifluoride), iron dichloride, iron trichloride, iron dibromide, iron tribromide, iron diiodide, iron triiodide, iron oxide, diiron trioxide, tri-iron tetraoxide, iron sulfide, iron persulfide, iron selenide, iron telluride, di-iron nitride, iron pentacarbonyl, diiron nonacarbonyl, triiron dodecacarbonyl, iron dichloride dihydrate, iron trifluoride trihydrate, iron dibromide hexahydrate, iron dichloride tetrahydrate, iron nitrate hexahydrate, iron trichloride hexahydrate, iron difluoride tetrahydrate, iron sulphate heptahydrate, iron trinitrate nonahydrate, diiron tri sulfate nonahydrate, iron chromate, iron citrate, iron gluconate, magnesium iron hexahydride, iron lactate, iron phosphate, iron pentacarbonyl, ammonium iron sulfate, ammonium ferric citrate, ferric oxalate, and triiron diphosphate octahydrate.

A chemical agent can be added to the media at a final concentration of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 5.0, 10.0 or more millimolar. A chemical agent can be added to the media at a final concentration of at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 5.0, 10.0 or more millimolar.

In some instances, a chemical agent can be a heme derivative. A hemederivative can increase the heme content of the expressed polypeptide (e.g., increase the number of globin molecules that comprise a heme). Suitable heme derivatives can include delta-aminolevulinic acid, derivatives of heme A, derivatives of heme B, derivatives of heme C, derivatives of heme O, heme precursors, derivatives of heme I, derivatives of heme m, derivatives of heme D, and derivatives of heme S. A hemederivative can be added to the media at a final concentration of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 5.0, 10.0 or more millimolar. A heme derivative can be added to the media at a final concentration of at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 5.0, 10.0 or more millimolar. In some instances, no heme derivative is added to the media.

After inducing the polypeptide, the host cell can be cultured for a period of time favoring maximal expression levels of the polypeptide. For example, a polypeptide can be expressed for at least about 0.1 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 days, 2 days, 3 days, 4 days,

5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 or more weeks. A polypeptide can be expressed in a host cell for at most about at least about 0.1 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 days, 2 days, 3 days, 4 days, 5 days,

6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 or more weeks.

A polypeptide can be expressed at a variety of temperatures. A polypeptide can be expressed at a temperature of at least about 4, 10, 16, 18, 21, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 42, 43, 44, 45, 46, 47, 48, 49, or 50° C. A polypeptide can be expressed at a temperature of at most about 4, 10, 16, 18, 21, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 42, 43, 44, 45, 46, 47, 48, 49, or 50° C.

Accessory proteins such as thiol -di sulfide oxidoreductases or chaperones may be beneficial to help fold the polypeptide into its active conformation. Thiol-disulfide oxidoreductases and protein disulfide isomerases can catalyze the formation of the correct disulfide bonds in the protein. Chaperones can help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavorable interactions and prolyl-peptidyl cis-trans isomerases assist in formation of the proper conformation of the peptide chain adjacent to proline residues. In some embodiments, the host cells can be transformed with an expression vector encoding at least one thiol-disulfide oxidoreductase or chaperone. In some embodiments, the fraction of properly folded polypeptide can be increased by the addition of chemicals to the growth medium that reduce/oxidize disulfide bonds, and/or alter the general redox potential, and/or chemicals that alter solvent properties thus affecting protein conformation and aggregation. In some embodiments, a reagent that reduces disulfide bonds, such as 2 -mercaptoethanol, can increase the fraction of correctly folded protein. In some embodiments and depending on the medium used, other disulfide reducing or oxidizing agents (e.g., DTT, TCEP, reduced and oxidized glutathione, cysteine, cystine, cysteamine, thioglycolate, S2O3 ², S2O4², S2O5 ², SO3 ², S2O7 ², Cu+, etc.), either used alone or in combination, can find use in the present disclosure. It can be contemplated that other adjuvants that alter solvent properties, (e.g., urea, DMSO, TWEEN®-80, etc ), either added to the growth medium alone or preferably in combination with disulfide reducing/oxidizing agents, such as PME, can also increase the fraction of correctly folded secretory protein and find use in various embodiments of the present disclosure. In some embodiments, the pME can be used at concentrations ranging from 0.5 to 4 mM, or from about 0.1 mM to 10 mM.

The polypeptide can be recovered from the culture (e g., by centrifugation, purification, etc.), as described below and herein.

Methods of Secretion in Host Cells

In some instances, an expressed polypeptide can be secreted from a host cell (e.g., a yeast cell or a bacterial cell). Secretion of a polypeptide can comprise releasing the polypeptide from a cell or subcellular compartment in a cell (e.g., nucleus, cell wall, plasma membrane). Secretion can occur through plasma membranes, which can surround cells and/or subcellular compartments. In some instances, secretion can refer to releasing a polypeptide to the cell envelope. In some instances, secretion can refer to releasing a polypeptide to the extracellular space (e.g., into the culture media).

A host cell of the disclosure can comprise secretory pathways, which can comprise a number of proteins that function together to secrete a protein. In some instances, the host cell can comprise a twin-arginine translocation (TAT) secretory pathway. In some instances, an organism can comprise a SEC secretory pathway. The TAT secretory pathway can comprise secretion of polypeptides (e.g., globins) in a folded state. The TAT secretory pathway can transport proteins across a plasma membrane (e g., lipid layer, i.e., lipid bilayer). The disclosure provides for secretion factors and methods that can be used in host cells to ameliorate the bottleneck to protein secretion and the production of proteins in secreted form, in particular when the polypeptides are recombinantly introduced and expressed by the host cell. Enhancement of Endogenous Polypeptides

In some embodiments, the disclosure provides for enhanced production of an endogenous polypeptide (e.g., an endogenous heme polypeptide) in a fungal organism that comprises the fungal biomass. Enhanced production of an endogenous polypeptide can be accomplished by modulating the pathway that produces the endogenous polypeptide. Modulation can refer to modulation of transcription, translation, subcellular localization, localization to different tissues, timing of expression, folding, affinity for binding partners, and the like. Modulation can occur at the DNA level (e g., knock-out the gene, knock-in an enhancer/promoter element). Modulation can occur at the RNA level (e.g., silence the gene via RNA interference). Modulation can occur at the protein level (e.g., modulation by allosteric inhibitors, small molecule binders).

In some instances, modulation can refer to altering the activity and/or levels of the endogenous polypeptide by at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more higher or lower relative to the wild-type levels of the endogenous polypeptide. In some instances, modulation can refer to altering the activity and/or levels of the endogenous polypeptide by at most about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more higher or lower relative to the wild-type levels of the endogenous polypeptide. In some instances, modulation can refer to altering the activity and/or levels of the endogenous polypeptide by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wildtype levels of the endogenous polypeptide. In some instances, modulation can refer to altering the activity and/or levels of the endogenous polypeptide by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wild-type levels of the endogenous polypeptide.

In some instances, polypeptides in the heme biosynthesis pathway that can produce the heme cofactor can be modulated. The modulation of polypeptides in the heme biosynthetic pathway can be at the DNA, RNA, or protein level. In some instances, the modulation of other polypeptides in the pathway can refer to increasing the levels and/or activity of an activator of the pathway. In some instances, the modulation of other polypeptides in the pathway can refer to decreasing the levels and/or activity of a suppressor of the pathway. In some instances, modulation can refer to altering the activity and/or levels of polypeptides in the heme biosynthesis pathway (including the heme cofactor that associates with a heme polypeptide of the disclosure) by at least about 1 fold, 2-fold, 3-fold, 4-fold, 5-fold, 10- fold, 20-fold, 50-fold, or 100-fold or more higher or lower relative to the wild-type levels of the polypeptide in the pathway. In some instances, modulation can refer to altering the activity and/or levels of polypeptides in the heme biosynthesis pathway (including the heme cofactor) by at most about 1 fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more higher or lower relative to the wild-type levels of the polypeptide in the pathway. In some instances, modulation can refer to altering the activity and/or levels of polypeptides in the heme biosynthesis pathway (including the heme cofactor) by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wild-type levels of the polypeptide in the pathway. In some instances, modulation can refer to altering the activity and/or levels of polypeptides in the heme biosynthesis pathway (including the hemecofactor) by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the wild-type levels of the polypeptide in the pathway.

Methods of Purification

An expressed and/or secreted polypeptide of the disclosure may be recovered (e.g., from the culture medium or from the host cells). For example, when the expressed heme polypeptide is secreted from the host cells, the polypeptide can be purified and/or extracted from the culture medium. In some embodiments, the host cells expressing the polypeptide can be removed from the media before purification of the polypeptide (e.g., by centrifugation).

When the expressed recombinant desired polypeptide is not secreted from a host cell, the host cell can be disrupted and the polypeptide released into an aqueous “extract” which can be the first stage of purification. The expression host cells can be collected from the media before the cell disruption. The cell disruption may be performed by using any suitable means, such as by lysozyme or beta-glucanase digestion, grinding, sonication, homogenization, milling, forcing the cells through high pressure, or high pressure homogenization (i.e. a combination of pressure, shearing, heat, and cavitation).

A recovered polypeptide may be purified. Purification may be accomplished by means of a salt (e.g., ammonium sulfate) or low pH (typically less than 3) wash/fractionation or chromatographic procedures (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography, size exclusion chromatography etc.). During purification, the cumulative abundance by mass of protein components other than the specified protein, which can be a single monomeric or multimeric protein species, can be reduced by a factor of 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more or 1000 or more relative to the source material from which the specified protein was isolated.

In some instances, a polypeptide can be recovered from a bioreactor. A cohesive fungal mycelial biomass or feedstock of the bioreactor can generally comprise cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired protein product, which can be removed from the biomass or feedstock or from a waste stream. Suitable processes for such removal can include conventional solid-liquid separation techniques (e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes), to produce a cell-free filtrate. In some embodiments, it can be acceptable to further concentrate the feedstock or the cell-free filtrate prior to the purification and/or crystallization process using techniques such as ultrafiltration, evaporation and/or precipitation. In some instances, the polypeptide is further purified to reduce the cumulative abundance by mass of protein components other than the specified protein, which can be a single monomeric or multimeric protein species, by a factor of 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more or 1000 or more relative to the source material from which the specified protein was isolated. Purification may be accomplished by means of a salt (e.g., ammonium sulfate) or low pH (typically less than 3) wash/fractionation or chromatographic procedures (e g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, and/or hydrophobic charge induction chromatography etc).

Characterization of a Polypeptide

A purified polypeptide can be characterized for purity, heme content, oligmerization state, stability, degradation, binding affinity and the like. For some applications the polypeptides (e g., globins) produced using the present disclosure can be very highly pure (e.g., having a purity of more than 99%). A purified polypeptide can be characterized for odor, taste and color.

The purified polypeptide can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure. The purified polypeptide can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure. The purified polypeptide can comprise at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% impurities. The purified polypeptide can comprise at most about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% impurities. The purified polypeptide can comprise at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per million impurities. The purified polypeptide can comprise at most about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per million impurities. The purified polypeptide can comprise at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per billion impurities. The purified polypeptide can comprise at most about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per billion impurities.

In some instances, the purified globin can be tested for activity, oligomerization state, proper protein folding, stability, secondary structure and/or heme content. Activity, oligomerization state, protein folding, and/or stability can be determined by a number of methods including spectroscopy, ELISA, binding assays, analytical ultracentrifugation, circular dichroism, x-ray crystallography, surface plasmon resonance, mass spectrometry, or NMR.

A polypeptide of this disclosure may have similar properties to myoglobin isolated from animal tissues. In one embodiment a group of people can be asked to rate a myoglobin isolated from an animal tissue, according to properties that describe the myoglobin. These ratings can be used as an indication of the properties of the animal tissue derived myoglobin. The polypeptide of the present invention can then be compared to the animal derived globin to determine how similar the polypeptide of this disclosure is to the animal tissue derived myoglobin. So, in some embodiments, the polypeptide is rated similar to animal tissue derived myoglobin according to human evaluation. In some embodiments the polypeptide is indistinguishable from animal tissue derived myoglobin to a human.

In some embodiments the polypeptides of this disclosure are compared to animal tissue derived myoglobin based upon olfactometer readings. In various embodiments the olfactometer can be used to assess odor concentration and odor thresholds, odor suprathresholds with comparison to a reference gas, hedonic scale scores to determine the degree of appreciation, or relative intensity of odors. In some embodiments the olfactometer allows the training and automatic evaluation of expert panels. In some embodiments the food product is a product that causes similar or identical olfactometer readings. In some embodiments the similarity is sufficient to be beyond the detection threshold of human perception.

Gas chromatography — mass spectrometry (GCMS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to separate and identify different substances within a test sample. GCMS can, in some embodiments, be used to evaluate the properties of polypeptides of this disclosure. For example, volatile chemicals can be isolated from the head space around animal tissue derived myoglobin. These chemicals can be identified using GCMS. A profile of the volatile chemicals in the headspace around animal tissue derived myoglobin is thereby created. In some instances each peak of the GCMS can be further evaluated. For instance, a human could rate the experience of smelling the chemical responsible for a certain peak. This information could be used to further refine the profile. GCMS could then be used to evaluate the properties of a polypeptide of the disclosure. The GCMS profile could be used to refine the polypeptide.

Heme content can refer to the percentage of polypeptide molecules that comprise the correct amount of heme moi eties. For example, if a polypeptide of the disclosure binds one heme moiety, then heme content can refer to the number of polypeptides that are bound to the hememoiety. Heme content of a polypeptide can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, or 100%. Heme content of a polypeptide can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, or 100%. In some instances, heme content can be expressed as a molar ratio of polypeptide concentration to hemeconcentration. The molar ratio heme content can be at least about 1 :1, 1:2, 1 :3, 1:4, 1 :5, 1:6, 1 :7, 1:8, 1 :9, 1: 10, 1 :20, 1 :30, or 1:40 or less. The molar ratio heme content can be at most about 1 :1, 1:2, 1 :3, 1:4, 1 :5, 1:6, 1 :7, 1:8, 1 :9, 1: 10, 1 :20, 1:30, or 1 :40 or less. The heme content can be 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, or 40-fold or more lower than the heme content of a full-occupied polypeptide (e.g., the polypeptide is 100% occupied with heme). The heme content can be 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, or 40- fold or more higher than the heme content of a fully-unoccupied polypeptide (e g., the polypeptide is 0% occupied with heme). Heme content can be determined by a number of methods including spectroscopy (Raman, UV-Vis), electron paramagnetic resonance (EPR), protein denaturation assays, heme stealing assays, and heme reduction assays. Methods for Using a Polypeptide in a Meat Replica Food Product

The disclosure provides for methods for the use of a polypeptide of the disclosure in a meat replica food product. The food products can compete with, supplement or replace animal-based foods. For instance, the food products can be meat replicas that include at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, or at least about 70 wt%, or alternatively any amount in any range having a lower bound of any whole number of percentage points between 30 wt% and 70 wt% and an upper bound of any other whole number of percentage points between 30 wt% and 70 wt%, of filamentous fungal biomass. In embodiments, the filamentous fungal biomass is a filamentous fungal mycelial biomass. In other embodiments, the filamentous fungal mycelial biomass is a cohesive filamentous fungal mycelial biomass, which can be produced by liquid surface fermentation, by solid state fermentation, by membrane fermentation, or other like methods. In other embodiments, the filamentous fungal mycelial biomass is a filamentous fungal mycelial biomass produced by submerged fermentation.

The food products can be made to mimic the cut or appearance of meat as it is currently sold. For instance, a food product may be visually similar to or indistinguishable from ground beef or a particular cut of beef. Alternatively, the food products can be made with a unique look or appearance. For instance, the food product could contain patterns or lettering that is based upon the structure of the food product. In some instances, the food products can look like traditional meat products after they are prepared. For example, a food product may be produced which is larger than a traditional cut of beef but which, after the food product is sliced and cooked appears the same as a traditional cooked meat. In some embodiments the food product may resemble a traditional meat shape in two dimensions, but not in a third. For example, the food product may resemble a cut of meat in two dimensions (for example when viewed from the top), but may be much longer (or thicker) than the traditional cut. A meat replica (e.g., substitute) can have similar physical characteristics as traditional meat (taste, texture, force, nutrients). In some embodiments, a meat replica can comprise a similar cook loss characteristic as meat. In some embodiments a meat replica can comprise a similar fat and protein content as ground beef has the same reduction in size when cooked as real ground beef. Methods of producing a meat replica are described in PCT/US2012/046560, which is hereby incorporated by reference in its entirety.

In some instances, a meat replica can comprise a polypeptide of the disclosure. A polypeptide of the disclosure can be used as a colorant or indicator of cooking of the meat replica. In some instances, the disclosure provides for a method for expressing a polypeptide (e.g., globin), in a host cell, secreting the polypeptide from the host cell, purifying the secreted polypeptide, and mixing the purified polypeptide with fats and lipids to produce a meat substitute.

In some instances, the disclosure provides for a method for enhancing the expression of an endogenous polypeptide (e.g., globin) in a host cell, purifying the polypeptide from the cell, and mixing the purified polypeptide with fats and lipids to produce a meat substitute.

In some instances, the disclosure provides for a method for expressing a polypeptide (e. g, globin), in a host cell (e.g., a fungus), purifying the secreted polypeptide, and mixing the purified polypeptide with fungal biomass, and optionally, other components, such as fats and lipids, to produce a meat substitute.

Compositions

Meat Replica Food Product Compositions

In some instances, a composition of the disclosure can comprise a meat replica and a host cell (e.g., yeast cell or part of a yeast cell). In some instances, a composition can further comprise a polypeptide of the disclosure. In some instances, a composition comprises a polypeptide and a meat replica (i.e., meat substitute) comprised of fungal biomass.

A meat replica food product can refer to meat-like product (e.g., a meat substitute) that is not made of meat. A meat replica food product can refer to a meat substitute that is made from non-animal products (e.g., fungal biomass). A meat replica food product can be meat replicas made entirely, mostly, or primarily from fungal sources. The food products may also be made from a combination of fungus-based sources and animal-based sources. The food products can be made to mimic the cut or appearance of meat as it is currently sold. For instance, a food product may be visually similar to or indistinguishable from ground beef or a particular cut of beef. In some instances, the food products look like traditional meat products after they are prepared. The meat replica can be substantially or entirely composed of ingredients derived from non-animal sources, yet recapitulates key features associated with the cooking and consumption of an equivalent meat product derived from animals.

A composition can comprise a meat replica and a polypeptide of the disclosure. A meat replica can comprise at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (w/w) of one or more polypeptides of the disclosure. In some instances, a meat replica can comprise at most about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of one or more polypeptides of the disclosure. A meat replica can comprise at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% weight/volume of one or more polypeptides of the disclosure. In some instances, a meat replica can comprise at most about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% weight/volume of one or more polypeptides of the disclosure.

A composition can comprise a meat replica and a host cell (e.g., yeast cell). A host cell of the composition can be the host cell from which the polypeptide was expressed and/or secreted. A composition can comprise a meat replica and at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2,

3, 4, 5, 6, 7, 8, 9, or 10% (w/w) or more host cells. A composition can comprise a meat replica and at most about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% host cells. A composition can comprise a meat replica and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per million host cell. A composition can a meat replica and comprise at most about 1, 2, 3,

4, 5, 6, 7, 8, 9, or 10 parts per million host cell. A composition can comprise a meat replica and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per billion host cell. A composition can comprise a meat replica and at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per billion host cell. A composition can comprise a meat replica and be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a host cell. A composition comprises a meat replica and can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a host cell.

A composition can comprise a part of a meat replica and a component of a host cell (e g., a part of a fungal cell). A component of a host cell can include a cell wall, a subcellular compartment (e.g., Golgi complex, endoplasmic reticulum, nucleus), a flagella, nucleic acid, protein, genomic DNA, or a plasma membrane. A component of a host cell can be a part of a fungal cell from which the polypeptide was expressed and/or secreted. A composition can comprise a meat replica and at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% or more of part of a host cell. A composition can comprise a meat replica and at most about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of a component of a host cell. A composition can comprise a meat replica and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per million of part of a host cell. A composition can comprise a meat replica and at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per million of a component of a host cell. A composition can comprise a meat replica and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per billion of a component of a host cell. A composition can comprise a meat replica and at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per billion of a component of a host cell. A composition can comprise a meat replica and be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a component of a host cell. A composition can comprise a meat replica and be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a component of a host cell.

A composition can comprise a meat replica and a component of a host cell (e.g., fungus, e.g., a filamentous fungus). A part of a host cell can include a cell wall, a subcellular compartment (e.g., Golgi complex, endoplasmic reticulum, nucleus), a shoot, a stem, a leave, a seed, a bean, a xylem, a rosette, a root, nucleic acid, protein, genomic DNA, and a plasma membrane. A component of a host cell can be a part of a fungus from which the polypeptide was expressed and/or secreted. A composition can comprise a meat replica and at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% or more of a component of a host cell. A composition can comprise a meat replica and at most about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of a component of a host cell. A composition can comprise a meat replica and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per million of a component of a host cell. A composition can comprise a meat replica and at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per million of a component of a host cell. A composition can comprise a meat replica and at least about

1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more parts per billion of a component of a host cell. A composition can comprise a meat replica and at most about 11, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts per billion of a component of a host cell. A composition can comprise a meat replica and can be at least about 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a component of a host cell. A composition can comprise a meat replica and be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% free of a component of a host cell.

In some embodiments, the disclosure can provide for a food product that can be substantially or entirely composed of ingredients derived from non-animal sources, yet recapitulates key features associated with the cooking and consumption of an equivalent meat product derived from animals. The equivalent meat product can be a white meat or a dark meat. The equivalent meat product can be derived from any animal. Non-limiting examples of animals used to derive the equivalent meat product include farmed animals such as, e.g., cattle, sheep, pig, chicken, turkey, goose, duck, horse, dog or game animals (whether wild or farmed) such as, e g., rabbit, deer, bison, buffalo, boar, snake, pheasant, quail, bear, elk, antelope, pigeon, dove, grouse, fox, wild pig, goat, kangaroo, emu, alligator, crocodile, turtle, groundhog, marmot, possum, partridge, squirrel, raccoon, whale, seal, ostrich, capybara, nutria, guinea pig, rat, mice, vole, any variety of insect or other arthropod, seafood such as, e. g, fish, crab, lobster, oyster, muscle, scallop, abalone, squid, octopus, sea urchin, tunicate and others. Many meat products are typically derived from skeletal muscle of an animal but it is understood that meat can also come from other muscles or organs of the animal. In some embodiments, the equivalent meat product is a cut of meat derived from skeletal muscle. In some embodiments, the equivalent meat product is an organ such as, e g., a kidney, heart, liver, gallbladder, intestine, stomach, bone marrow, brain, thymus, lung, tongue. Accordingly, in some embodiments the compositions of the present are food products similar to skeletal muscle or organs.

In some instances, the disclosure provides meat substitute products comprising one or more of a first composition comprising a muscle tissue replica, a second composition comprising an adipose tissue replica, and/or a third composition comprising a connective tissue replica, wherein the one or more compositions are combined in a manner that recapitulates the physical organization of meat. In other aspects, the present disclosure provides compositions for a muscle tissue replica (herein referred to as “muscle replica”), an adipose tissue replica (herein referred to as “fat replica”), and a connective tissue replica (herein referred to as “connective tissue replica”). In some embodiments, the compositions and meat substitute products are principally or entirely composed of ingredients derived from non-animal sources. In alternative embodiments, the muscle, fat, and/or connective tissue replica, or the meat substitute products comprising one or more of said replicas, are partially derived from animal sources but supplemented with ingredients derived from non-animal sources.

In some embodiments, meat products can be substantially derived from animal sources but which are supplemented with one or more of a muscle tissue replica, a fat replica, and/or a connective tissue replica, wherein the replicas can be derived substantially or entirely from non- animal sources. A non-limiting example of such a meat product is an ultra-lean ground beef product supplemented with a non-animal derived fat replica which can improve texture and mouthfeel while preserving the health benefits of a food product low in animal fat. Such alternative embodiments result in products with properties that more closely recapitulate key features associated with preparing and consuming meat but which are less costly and associated with a lesser environmental impact, less animal welfare impact, or improved health benefits for the consumer.

The physical organization of the meat substitute product can be manipulated by controlling the localization, organization, assembly, or orientation of the muscle, fat, and/or connective tissue replicas described herein. In some embodiments the product is designed in such a way that the replicas described herein are associated with one another as in meat. In some embodiments the food product is designed so that after cooking the replicas described herein are associated with one another as in cooked meat. In some embodiments, one or more of the muscle, fat, and/or connective tissue replicas are combined in a manner that recapitulate the physical organization of different cuts or preparations of meat. In an example embodiment, the replicas are combined in a manner that approximates the physical organization of natural ground meat. In other embodiments, the replicas are combined in a manner that approximates different cuts of beef, such as, e.g., rib eye, filet mignon, London broil, among others.

Indicators of Cooking Meat

In some instances, a polypeptide of the disclosure can be used in a composition of the disclosure as an indicator for cooking meat. The release of odorants upon cooking is an important aspect of meat consumption. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking beef In some embodiments, the food product when cooked generates an aroma recognizable by humans as typical of cooking pork. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking bacon. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking chicken. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking lamb. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking fish. In some embodiments, the food product is a meat replica entirely composed of non-animal products that when cooked generates an aroma recognizable by humans as typical of cooking turkey. In some embodiments the food product is a meat replica principally or entirely composed of ingredients derived from non-animal sources, with an odorant that is released upon cooking. In some embodiments the food product is a meat replica principally or entirely composed of ingredients derived from non-animal sources, with an odorant that is produced by chemical reactions that take place upon cooking. In some embodiments the food product is a meat replica principally or entirely composed of ingredients derived from non-animal sources, comprising a polypeptide of the disclosure and mixtures of proteins, peptides, amino acids, nucleotides, sugars and polysaccharides and fats in combinations and spatial arrangements that enable these compounds to undergo chemical reactions during cooking to produce odorants and flavor-producing compounds. In some embodiments the food product is a meat replica principally or entirely composed of ingredients derived from non-animal sources (e.g., a polypeptide of the disclosure), with a volatile or labile odorant that is released upon cooking. In some embodiments the food product is a method for preparing a meat replica where meat replicas principally or entirely composed of ingredients derived from non-animal sources are heated to release a volatile or labile odorant.

Odorants released during cooking of meat are generated by reactions that can involve as reactants fats, protein, amino acids, peptides, nucleotides, organic acids, sulfur compounds, sugars and other carbohydrates. In some instances, a reactant can be a polypeptide of the disclosure (e.g, a globin, a secreted globin). In some embodiments the odorants that combine during the cooking of meat are identified and located near one another in the food product, such that upon cooking of the food product the odorants combine. In some embodiments, the characteristic flavor and fragrance components are produced during the cooking process by chemical reactions involving amino acids, fats and sugars found in plants as well as meat. In some embodiments, the characteristic flavor and fragrance components are mostly produced during the cooking process by chemical reactions involving one or more amino acids, fats, peptides, nucleotides, organic acids, sulfur compounds, sugars and other carbohydrates found in plants as well as meat.

Some reactions that generate odorants released during cooking of meat can be catalyzed by iron, in particular the iron of heme, which may be comprised (e.g., bound) by a polypeptide of the disclosure. Thus in some embodiments, some of the characteristic flavor and fragrance components are produced during the cooking process by chemical reactions catalyzed by iron. In some embodiments, some of the characteristic flavor and fragrance components are produced during the cooking process by chemical reactions catalyzed by heme. In some embodiments, some of the characteristic flavor and fragrance components are produced during the cooking process by chemical reactions catalyzed by the heme iron in leghemoglobin. In some embodiments, some of the characteristic flavor and fragrance components are produced during the cooking process by chemical reactions catalyzed by the heme iron in a heme protein (e.g., hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell’s gate globin I, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins, androglobin, cytoglobin, globin E, globin X, globin Y, myoglobin, leghemoglobins, erythrocruorins, beta hemoglobins, alpha hemoglobins, non-symbiotic hemoglobins, protoglobins, cyanoglobins, Hell’s gate globin I, bacterial hemoglobins, ciliate myoglobins, histoglobins and neuroglobins, etc).

Color Indicators

The color of meat is an important part the experience of cooking and eating meat. For instance, cuts of beef are of a characteristic red color in a raw state and gradually transition to a brown color during cooking. As another example, white meats such as chicken or pork have a characteristic pink color in their raw state and gradually transition to a white or brownish color during cooking. The amount of the color transition is used to indicate the cooking progression of beef and titrate the cooking time and temperature to produce the desired state of done-ness. In some aspects, the disclosure provides a non-meat based meat substitute product that provides a visual indicator of cooking progression. In some embodiments, the visual indicator is a color indicator that undergoes a color transition during cooking. In particular embodiments, the color indicator recapitulates the color transition of a cut of meat as the meat progresses from a raw to a cooked state. In some embodiments, the color indicator colors the meat substitute product a red color before cooking to indicate a raw state and causes the meat substitute product to transition to a brown color during cooking progression. In some embodiments, the color indicator colors the meat substitute product a pink color before cooking to indicate a raw state and causes the meat substitute product to transition to a white or brown color during cooking progression.

The ingredients of food compositions of the present disclosure, and most especially the food colorings provided (if any), may be selected to provide a desired color to the food product before cooking, during cooking, after cooking, or a combination thereof. For example, where the food product is a burger analog food product (most commonly a beefburger analog food product, but possibly also a chicken burger analog food product, a turkey burger analog food product, etc ), colorings and other ingredients may be selected to provide the uncooked food product with a color resembling that of uncooked ground beef, chicken, turkey, etc. meat (e.g. colors defined by a Hunter L value from about 44 to about 66 (or any value in any subrange therebetween), a Hunter a value from about 5 to about 19 (or any value in any subrange therebetween), and/or a Hunter b value from about 15 to about 24 (or any value in any subrange therebetween)). Additionally or alternatively, colorings and other ingredients may be selected to provide a burger analog food product according to the present disclosure, after it has been cooked (and, in some embodiments, allowed to rest, as many meat products are before being consumed), with a color resembling that of a cooked beef, chicken, turkey, etc. burger patty (e. ., colors defined by a Hunter L value from about 34 to about 53 (or any value in any subrange therebetween), a Hunter a value from about 7 to about 13 (or any value in any subrange therebetween), and/or a Hunter b value from about 14 to about 26 (or any value in any subrange therebetween)). The cooking process to which a food product according to the present disclosure is subjected to achieve a desired color (or change in color) may, in many embodiments, be similar to the cooking process to which an analogous conventional food product; by way of non-limiting example, in the case of a burger analog food product, the cooking process may entail being exposed to a cooking surface, such as a pan, heated to about 150 °C/300 °F, for about 2 to about 7 minutes (total or per side) or for about 3 to about 5 minutes (total or per side), or otherwise being cooked to an internal temperature of about 160 °F (and, optionally, being allowed to rest at room temperature after heating).

The main determinant of the color of meat is the concentration of iron carrying proteins in the meat. In the skeletal muscle component of meat products, one of the main iron-carrying proteins is myoglobin. So, in some embodiments, the composition is a meat replica food product which comprises an iron-carrying protein. In some embodiments, the composition comprises about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, or more than about 2% of an iron- carrying protein by dry weight or total weight. In some embodiments, the composition comprises at least about 10% by dry weight or total weight of a polypeptide of the disclosure. In some embodiments, the composition comprises at most about 10% by dry weight or total weight of a polypeptide of the disclosure, for example any range between about 0.05% and about 10%. In some cases, the iron carrying protein has been isolated and purified from a source. In other cases, the iron carrying protein has not been isolated and purified. In some cases, the source of the iron- carrying protein is an animal source, or a non-animal source such as a plant, fungus, or genetically modified organisms such as, e.g., bacteria or yeast. In some cases, the iron-carrying protein is myoglobin. In some embodiments the composition comprises a food product that is a fungus-based meat replica that has animal myoglobin added. So, for example a replica of young beef can have about 0.4-1% myoglobin. In some cases, the iron-carrying protein is leghemoglobin. In some embodiments the composition comprises a food product that is a fungus-based meat replica that has leghemoglobin added. So, for example a replica of young beef can have about 0.4-1% leghemoglobin. In some cases, the iron-carrying protein is a cytochrome. In some embodiments the composition comprises a food product that is a fungus-based meat replica that has a cytochrome added. So, for example a replica of young beef can have about 0.4-1% of a cytochrome. In some aspects the food product is a fungus-based meat replica containing hemoglobin. In some instances, the iron-carrying protein is a polypeptide of the disclosure (e.g., a globin).

Additional iron containing proteins exist in nature. In some embodiments the composition (e.g., food product) comprises an iron containing protein that is not myoglobin. In some embodiments the composition (e.g., food product) does not contain myoglobin. In some embodiments the compositions (e.g., food product) does not contain hemoglobin. In some embodiments the food product is a meat replica that comprises an iron containing protein other than myoglobin or hemoglobin (e.g., the globins described herein, e.g., hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell’s gate globin I, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins).

In some embodiments the composition comprises a food product that is a meat replica principally or entirely composed of ingredients derived from non-animal sources, including a muscle tissue replica, an adipose tissue replica, a connective tissue replica, and leghemoglobin. In some embodiments the composition comprises a food product that is a meat replica principally or entirely composed of ingredients derived from non-animal sources, containing a heme protein. In some embodiments the composition comprises a food product that is a meat replica principally or entirely composed of ingredients derived from non-animal sources, containing a leghemoglobin. In some embodiments the composition comprises a food product that is a meat replica principally or entirely composed of ingredients derived from non-animal sources, containing a member of the globin protein family. In some embodiments the composition comprises a food product that is a meat replica principally or entirely composed of ingredients derived from non-animal sources, with a high iron content from a heme protein. In some embodiments the iron content is similar to meat. In some embodiments the food product has the distinctive red color of meat, such color provided by leghemoglobin.

Leghemoglobin is, in some embodiments, used as an indicator that the food product is finished cooking. In some embodiments of the disclosure there is a method for cooking a food product comprising detecting leghemoglobin which has migrated from the interior of the food product to the surface when the product is cooked. In some embodiments of the disclosure there is a method for cooking a food product comprising detecting the change in color of from red to brown when the product is cooked.

The oxidation state of the iron ion in leghemoglobin can be important for its color. Leghemoglobin with the heme iron in the +2 oxidation state can appear vivid red in color, while leghemoglobin with the heme iron in the +3 oxidation state can appear brownish red. Thus, in using leghemoglobin as a source of red color in a meat replica for example, it can be desirable to reduce the heme iron from the +3 state to the +2 state. Heme iron in leghemoglobin can be switched from oxidized (+3) state to reduced (+2) state with reducing reagents.

A heme protein can, in some embodiments, be used as an indicator that the food product is finished cooking. In some embodiments, there is a method for cooking a food product comprising detecting leghemoglobin which has migrated from the interior of the food product to the surface when the product is cooked. In some embodiments, there is a method for cooking a food product comprising detecting the change in color of from red to brown when the product is cooked.

A heme protein (e.g., Hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell’s gate globin I, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins), can be, in some embodiments, used as an indicator that the food product is finished cooking. So, in some embodiments, the disclosure provides for a method for cooking a food product comprising detecting leghemoglobin which has migrated from the interior of the food product to the surface when the product is cooked. The disclosure can provide for a method for cooking a food product comprising detecting the change in color of from red to brown when the product is cooked. In embodiments, a reducing agent may be added to the food product to cause a more readily apparent change in the color of the food product during cooking.

Food Products Comprising Purified Polypeptide Polypeptides, for example leghemoglobin and hemoglobin, can be combined with other fungus-based meat replica components. In some embodiments the polypeptides are captured in a gel that contains other components, for example lipids and or proteins. In some aspects multiple gels are combined with non-gel based heme proteins. In some embodiments the combination of the polypeptides and the other compounds of the food product are done to insure that the heme proteins are able to diffuse through the food product. In some embodiments the food product comprises a heme-protein solution, for instance a leghemoglobin solution. In some embodiments the food product is soaked in a heme protein solution, for instance a leghemoglobin solution for 1, 5, 10, 15, 20 or 30 hours. In some embodiments the food product is soaked in a heme solution, for instance a leghemoglobin solution for 1, 5, 10, 15, 30, or 45 minutes. In some embodiments, a fungal mycelial biomass is contacted with an aqueous heme protein solution for from about 10 to about 120 minutes, or alternatively for any period in any subrange having a lower bound of any whole number of minutes from 10 minutes to 120 minutes and an upper bound of any whole number of minutes from 10 minutes to 120 minutes (e.g., from about 15 minutes to about 90 minutes, or from about 20 minutes to about 60 minutes, or from about 25 minutes to about 45 minutes).

Muscle Replicas

A large number of meat products comprise a high proportion of skeletal muscle. Accordingly, the present disclosure provides a fungal-based composition which replicates or approximates key features of animal skeletal muscle. In another aspect, the present disclosure provides a meat substitute product that comprises a composition derived from fungal sources which replicates or approximates animal skeletal muscle. Such a composition is termed herein as “muscle replica”. In some embodiments, the muscle replica and/or meat substitute product comprising the muscle replica are partially derived from animal sources. In some embodiments, the muscle replica and/or meat substitute product comprising the muscle replica are entirely derived from non-animal sources.

Many meat products comprise a high proportion of striated skeletal muscle in which individual muscle fibers are organized mainly in an isotropic fashion. Accordingly, in some embodiments the muscle replica comprises fibers that are to some extent organized isotropically. In some embodiments the fibers comprise a protein component. In some embodiments, the fibers comprise about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%), about 90%, about 95%, about 99% or more of a protein component. Animal skeletal muscle typically contains around 1% myoglobin, but can be as much as 7% of muscle mass in some whale muscles. In some embodiments the muscle replica comprises heme proteins of this disclosure.

In some embodiments, the protein component comprises one or more isolated, purified proteins. For example, the one or more isolated, purified protein can comprise the 8S globulin from Moong bean seeds, or the albumin or globulin fraction of pea seeds. These proteins provide examples of proteins with favorable properties for constructing meat replicas because of their ability to form gels with textures similar to animal muscle or fat tissue. Examples and embodiments of the one or more isolated, purified proteins are described herein. The list of potential candidates here is essentially open and may include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses, or genetically engineered microorganisms, or synthesized in vitro. The proteins may be artificially designed to emulate physical properties of animal muscle tissue. The proteins may be artificially designed to emulate physical properties of animal muscle tissue. In some embodiments, one or more isolated, purified proteins accounts for about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the protein component by weight, or any subrange between 0. 1% and 99%.

Skeletal muscle of animals such as beef cattle typically contain substantial quantities of glycogen, which can comprise on the order of 1%> of the mass of the muscle tissue at the time of slaughter. After slaughter, a fraction of this glycogen continues to be metabolized yielding products including lactic acid, which contributes to lowering the pH of the muscle tissue, a desirable quality in meat. Glycogen is a branched polymer of glucose linked together by alpha (1— 4) glycosidic bonds in linear chains, with branch points comprising alpha (1— >6) glycosidic bonds. Starches from plants and fungi, particularly amylopectins are also branched polymers of glucose linked together by alpha (1^-4) glycosidic bonds in linear chains, with branch points comprising alpha (1— >6) glycosidic bonds and can therefore be used as an analog of glycogen in constructing meat replicas. Thus in some embodiments, the muscle or meat replica includes a starch or pectin.

Additional components of animal muscle tissue include sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides and sulfur compounds. Thus in some embodiments, the muscle replica can include sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides and sulfur compounds. In some embodiments the concentration of sodium, potassium, calcium, magnesium, other metal ions, lactic acid, other organic acids, free amino acids, peptides, nucleotides and/or sulfur compounds in the muscle replica or food product are within 10%> of the concentrations found in a muscle or meat being replicated.

In another aspect, the disclosure provides methods for making a muscle replica. In some embodiments, the composition comprising fungal biomass is formed into asymmetric fibers prior to incorporation into the food product. In some embodiments these fibers replicate muscle fibers. In other embodiments the muscle replica composition can be extruded to align mycelial fibers. Alternatively, the muscle replica composition can treated by other processes such as by freezing to align fibers during ice crystal formation.

In some embodiments extrusion can be conducted using an MPF19 twin-screw extruder (APV Baker, Grand Rapids, Mich.) with a cooling die. The cooling die can cool the extrudate prior to return of the extrudate to atmospheric pressure, thus substantially inhibiting expansion or puffing of the final product. In the MPF19 apparatus, dry feed and liquid can be added separately and mixed in the barrel. Extrusion parameters can be, for example: screw speed of 200 rpm, product temperature at the die of 150 C, feed rate of 23 g/min, and water-flow rate of 11 g/min, although it is to be expressly understood that these and other extrusion parameters may vary greatly based on the nature and composition of the biomass. Product temperature can be measured during extrusion by a thermocouple at the end of the extrusion barrel. Observations can be made on color, opacity, structure, and texture for each collected sample. Collected samples can be optionally dried at room temperature overnight, then ground to a fine powder (<60 mesh) using a Braun food grinder. The pH of samples can be measured in duplicate using 10% (w/v) slurries of powdered sample in distilled water.

Fat Replica

Animal fat is important for the experience of eating cooked meat. Accordingly, the present disclosure provides a composition derived from non-animal sources which recapitulates key features of animal fat. In another aspect, the present disclosure provides a meat substitute product that comprises a composition derived from non-animal sources which recapitulates animal fat. Such a composition is termed herein as a “fat replica”. In some embodiments, the fat replica and/or meat substitute product comprising the fat replica are partially derived from animal sources.

In some embodiments the meat substitute product has a fat component. In some embodiments the fat content of the food product is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% fat, or any subrange between 1% and 60%. In some embodiments, the fat replica comprises a gel with droplets of fat suspended therein. In some embodiments, the gel is a soft, elastic gel comprising proteins and optionally carbohydrates. In particular embodiments, the proteins used in the gel are fungal, plant or microbial proteins. In some embodiments, the proteins used in the fat replica might include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses, or genetically engineered microorganisms, or synthesized in vitro. The proteins may be artificially designed to emulate physical properties of animal fat. The proteins may be artificially designed to emulate physical properties of animal fat.

The fat droplets used in some embodiments of the present disclosure can be from a variety of sources. In some embodiments, the sources are non-animal sources. In particular embodiments, the sources are plant or fungal sources. Nondimiting examples of oils include com oil, olive oil, soy oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil, flax seed oil, algal oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, oils produced by bacteria, algae, archaea or fungi or genetically engineered bacteria, algae, archaea or fungi, triglycerides, monoglycerides, diglycerides, sphingosides, glycolipids, lecithin, lysolecithin, phophatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, conjugated oleic acid, or esters of: oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid, or glycerol esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid, or triglyceride derivatives of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid.

In some embodiments, fat droplets are derived from pulp or seed oil. In other embodiments, the source may be yeast or mold. For instance, in one embodiment the fat droplets comprise triglycerides derived from Mortierella isabellina.

In some embodiments plant or fungal oils are modified to resemble animal fats. The plant or fungal oils can be modified with flavoring or other agents to recapitulate the taste and smell of meat during and after cooking. Accordingly, some aspects of the disclosure involve methods for testing the qualitative similarity between the cooking properties of animal fat and the cooking properties of plant or fungal oils in the food product.

In some embodiments, the fat replica comprises a protein component comprising one or more isolated, purified proteins. The purified proteins contribute to the taste and texture of the meat replica. In some embodiments purified proteins can stabilize emulsified fats. In some embodiments the purified proteins can form gels upon denaturation or enzymatic crosslinking, which replicate the appearance and texture of animal fat. Examples and embodiments of the one or more isolated, purified proteins are described herein. In particular embodiments, the one or more isolated proteins comprise a protein isolated from fungi. Non-limiting examples of fungi are described herein, although variations with other fungi are possible. In some embodiments, the fungus is a filamentous fungus. In some embodiments the isolated purified proteins stabilize emulsions. In some embodiments the isolated purified proteins form gels upon crosslinking or enzymatic crosslinking. In some embodiments, the isolated, purified proteins comprise seed storage proteins. In some embodiments, the isolated, purified proteins comprise albumin. In some embodiments, the isolated, purified proteins comprise globulin. In a particular embodiment, the isolated, purified protein is a purified pea albumin protein. In another particular embodiment, the isolated, purified protein is a purified pea globulin protein. In another particular embodiment the isolate purified protein is a Moong bean 8S globulin. In another particular embodiment, the isolated, purified protein is an oleosin. In another particular embodiment, the isolated, purified protein is a caloleosin. In another particular embodiment, the isolated, purified protein is Rubisco. In some embodiments, the protein component comprises about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the fat replica by dry weight or total weight, or any subrange between 0.1% and 90%. In some embodiments, the protein component comprises about 0.1-5%, about 0.5-10%, about 1-20%, about 5-30%, about 10-50%, about 20-70%, or about 30-90% or more of the fat replica by dry weight or total weight. In some embodiments, the protein component comprises a solution containing one or more isolated, purified proteins.

In some embodiments, the fat replica comprises cross-linking enzymes that catalyze reactions leading to covalent crosslinks between proteins. Cross-linking enzymes can be used to create or stabilize the desired structure and texture of the adipose tissue replica, to mimic the desired texture of an equivalent desired animal fat. Non-limiting examples of cross-linking enzymes include, e.g., transglutaminase, lysyl oxidases, or other amine oxidases (e.g. Pichia pastoris lysyl oxidase). In some embodiments, the cross-linking enzymes are isolated and purified from a non-animal source, examples and embodiments of which are described herein. In some embodiments, the fat replica comprises at least 0.0001%, or at least 0.001%, or at least 0.01%, or at least 0.1%, or at least 1% (wt/vol) of a cross-linking enzyme. In particular embodiments, the cross-linking enzyme is transglutaminase.

In another aspect, the disclosure provides methods for making a fat replica. In some embodiments, the fat droplets are suspended in a gel. In some embodiments the present disclosure provides for methods for producing droplets of fat suspended in the gel. The fat can be isolated and homogenized. For example, an organic solvent mixture can be used to help mix a lipid. The solvent can then be removed. At this point the lipid can be frozen, lyophilized, or stored. So in some aspects the disclosure provides for a method for isolating and storing a lipid which has been selected to have characteristics similar to animal fat. The lipid fdm or cake can then be hydrated. The hydration can utilize agitation or temperature changes. The hydration can occur in a precursor solution to a gel. After hydration the lipid suspension can be sonicated or extruded to further alter the properties of the lipid in the solution.

In some embodiments, the fat replica is assembled to approximate the organization adipose tissue in meat. In some embodiments some or all of the components of the fat replica are suspended in a gel. In various embodiments the gel can be a proteinaceous gel, a hydrogel, an organogel, or a xerogel. In some embodiments, the gel can be thickened to a desired consistency using an agent based on polysaccharides or proteins. For example fecula, arrowroot, cornstarch, katakuri starch, potato starch, sago, tapioca, alginin, guar gum, locust bean gum, xanthan gum, collagen, egg whites, furcellaran, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, acadia gum, konjac, starch, pectin, amylopectin or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, of fungi can be used alone or in combination to thicken the gel, forming an architecture or structure for the food product.

In particular embodiments, the fat replica is an emulsion comprising a solution of one or more proteins and one or more fats suspended therein as droplets. In some embodiments, the emulsion is stabilized by one or more cross-linking enzymes into a gel. In some embodiments, the one or more proteins in solution are isolated, purified proteins. In some embodiments, the isolated, purified proteins comprise a purified pea albumin enriched fraction. In some embodiments, the isolated, purified proteins comprise a purified pea globulin enriched fraction. In some embodiments, the isolated, purified proteins comprise a purified Moong bean 8S globulin enriched fraction. In some embodiments, the isolated, purified proteins comprise a Rubisco enriched fraction. In some embodiments, the one or more fats are derived from plant- or fungus-based oils. In some embodiments, the one or more fats are derived from one or more of: com oil, olive oil, soy oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil, safflower oil, sunflower oil, flax seed oil, algal oil, palm oil, palm kernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, oils produced by bacteria, algae, archaea or fungi or genetically engineered bacteria, algae, archaea or fungi, triglycerides, monoglycerides, diglycerides, sphingosides, glycolipids, lecithin, lysolecithin, phophatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, conjugated oleic acid, or esters of: oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid, or glycerol esters of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid, or triglyceride derivatives of oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20: 1 eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugated linoleic acid, or conjugated oleic acid. In yet even more particular embodiments, the one or more fats is a rice bran oil. In another particular embodiment, the one or more fats is a canola oil. In some embodiments, the cross-linking enzyme is transglutaminase, lysyl oxidase, or other amine oxidase. In some embodiments, the cross-linking enzyme is transglutaminase. In particular embodiments, the fat replica is a high fat emulsion comprising a protein solution of purified pea albumin emulsified with 40-80% rice bran oil, stabilized with 0.5-5% (wt/vol) transglutaminase into a gel. In some embodiments, the fat replica is a high fat emulsion comprising a protein solution of partially-purified moong bean 8S globulin emulsified with 40-80%) rice bran oil, stabilized with 0.5-5% (wt/vol) transglutaminase into a gel. In some embodiments, the fat replica is a high fat emulsion comprising a protein solution of partially-purified moong bean 8S globulin emulsified with 40-80%) canola oil, stabilized with 0.5- 5% (wt/vol) transglutaminase into a gel. In some embodiments, the fat replica is a high fat emulsion comprising a protein solution of purified pea albumin emulsified with 40-80%> rice bran oil, stabilized with 0.0001-1%) (wt/vol) transglutaminase into a gel. In some embodiments, the fat replica is a high fat emulsion comprising a protein solution of partially-purified moong bean 8S globulin emulsified with 40-80%) rice bran oil, stabilized with 0.0001-1%) (wt/vol) transglutaminase into a gel. In some embodiments, the fat replica is a high fat emulsion comprising a protein solution of partially-purified moong bean 8S globulin emulsified with 40-80%) canola oil, stabilized with 0.0001-1%) (wt/vol) transglutaminase into a gel.

Connective Tissue Replica

Animal connective tissue provides key textural features that are an important component of the experience of eating meat. Accordingly, the present disclosure provides a composition derived from non-animal sources which recapitulates key features of animal connective tissue. In another aspect, the present disclosure provides a meat substitute product that comprises a composition derived from non-animal sources which recapitulates important textural and visual features of animal connective tissue. Such a composition is termed herein as “connective tissue replica”. In some embodiments, the connective tissue replica and/or meat substitute product comprising the connective tissue replica are partially derived from animal sources. Animal connective tissue can generally be divided into fascia-type and cartilage-type tissue. Fascia-type tissue is highly fibrous, resistant against extension (has high elastic modulus), and has a high protein content, a moderate water content (ca. 50%), and low-to-none fat and polysaccharide content. Accordingly, the present disclosure provides a connective tissue replica that recapitulates key features of fascia type tissue. In some embodiments, the connective tissue replica comprises about 50% protein by total weight, about 50% by liquid weight, and has a low fat and polysaccharide component.

The protein content of most fascia-type connective tissue is comprised mainly of collagen. Collagen is characterized by a high fraction of proline and alanine, and also is assembled into characteristic elongated fibrils or rod-like, flexible structures. Prolamins are one family of proteins found in non-animal sources, such as plant sources. Prolamins are highly abundant in plants and are similar in amino acid composition to collagen. Among proteins we tested for this purpose, prolamins were particularly favorable because of their low cost and their ability to readily form fibers or sheets when spun or extruded. Non-limiting examples of prolamin family proteins include, e.g., zein (found in corn), these include hordein from barley, gliadin from wheat, secalin, extensins from rye, kafirin from sorghum, avenin from oats. In fascia-type connective tissue, the prolamin family of proteins, individually or combinations thereof, demonstrates suitability for the protein component because they are highly abundant, similar in global amino acid composition to collagen (high fraction of proline and alanine), and amenable to processing into films and fibers. In addition to zein (found in com), these include hordein from barley, gliadin from wheat, secalin, extensins from rye, kafirin from sorghum, avenin from oats. Other proteins may be necessary to supplement prolamins in order to achieve targets specifications for physicochemical and nutritional properties. The list of potential candidates here is essentially open and may include Rubisco, any major seed storage proteins, proteins isolated from fungi, bacteria, archaea, viruses, or genetically engineered microorganisms, or synthesized in vitro. The proteins may be artificially designed to emulate physical properties of animal connective tissue, animal-derived or recombinant collagen, extensins (hydroxyproline-rich glycoproteins abundant in cell walls e.g. Arabidopsis thaliana, monomers of which are “collagen-like” rod-like flexible molecules). The proteins may be artificially designed to emulate physical properties of animal connective tissue.

Methods for forming fascia-type connective tissue will be as those practiced in the art with a bias towards methods producing fibrous or fibrous-like structures by biological, chemical, or physical means, individually or in combination, serially or in parallel, before final forming. These methods may include extrusion or spinning.

Cartilage-type tissue can be macroscopically homogenous, resistant against compression, has higher water content (up to 80%), lower protein (collagen) content, and higher polysaccharide (proteoglycans) contents (ca. 10% each).

Compositionally, cartilage-type connective tissue can be very similar to fascia-type tissue with the relative ratios of each adjusted to more closely mimic ‘meat’ connective tissue.

Methods for forming cartilage-type connective tissue can be similar to those for fascia-type connective tissue, but with a bias towards methods producing isotropically homogenous structures.

The fat can be suspended in a gel. In some embodiments the present disclosure provides for methods for producing droplets of fat suspended in the proteinaceous gel. The fat can be isolated from plant or fungus tissues and emulsified. The emulsification can utilize high-speed blending, homogenization, agitation or temperature changes. The lipid suspension can be sonicated or extruded to further alter the properties of the lipid in the solution. At this point, in some embodiments other components of the food product are added to the solution followed by a gelling agent. In some embodiments crosslinking agents (e.g. transglutaminase or lysyl oxidase) are added to bind the components of the food product. In other embodiments the gelling agent is added and the lipid/gel suspension is later combined with additional components of the food product. In fascia-type connective tissue, the prolamin family of proteins, individually or combinations thereof, demonstrates suitability for the protein component because they are highly abundant, similar in global amino acid composition to collagen (high fraction of proline and alanine), and amenable to processing into films. In addition to zein (found in com), these include hordein from barley, gliadin from wheat, secalin, extensions from rye, kafirin from sorghum, avenin from oats. Other proteins may be necessary to supplement prolamins in order to achieve targets specifications for physicochemical and nutritional properties. The list of potential candidates here is essentially open and may include any major seed storage proteins, animal-derived or recombinant collagen, extensins (hydroxyproline-rich glycoproteins abundant in cell walls e.g. Arabidopsis thaliana, monomers of which are “collagen-like” rod-like flexible molecules).

In some embodiments some or all of the components of the food product are suspended in a gel. In various embodiments the gel can be a hydrogel, an organogel, or a xerogel. The gel can be made thick using an agent based on polysaccharides or proteins. For example fecula, arrowroot, cornstarch, katakuri starch, potato starch, sago, tapioca, alginin, guar gum, locust bean gum, xanthan gum, collagen, egg whites, furcellaran, gelatin, agar, carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, acadia gum, konjac, starch, pectin, amylopectin or proteins derived from legumes, grains, nuts, other seeds, leaves, algae, bacteria, of fungi can be used alone or in combination to thicken the gel, forming an architecture or structure for the food product. Enzymes that catalyze reactions leading to covalent crosslinks between proteins can also be used alone or in combination to form an architecture or structure for the food product. For example, transglutaminase, lysyl oxidases, or other amine oxidases (e.g. Pichia pastoris lysyl oxidase (PPLO)) can be used alone or in combination to form an architecture or structure for the food product. In some embodiments multiple gels with different components are combined to form the food product. For example, a gel containing a plant- or fungus-based protein can be associated with a gel containing a plant- or fungus-based fat. In some embodiments fibers or stings of proteins are oriented parallel to one another and then held in place by the application of a gel containing plant- or fungus-based fats.

The compositions of the disclosure can be puffed or expanded by heating, such as frying, baking, microwave heating, heating in a forced air system, heating in an air tunnel, and the like.

In some embodiments multiple gels with different components are combined to form the food product. For example, a gel containing a plant- or fungus-based protein can be associated with a gel containing a plant- or fungus-based fat. In some embodiments fibers or strings of proteins are oriented parallel to one another and then held in place by the application of a gel containing plant- or fungus-based fats.

In some embodiments the meat replica contains no animal products, less than 1% wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no soy protein isolate, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no soy protein concentrate, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no soy protein, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no tofu, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products, no tofu, and no wheat gluten. In some embodiments the meat replica contains no animal products, no soy protein, and no wheat gluten. In some embodiments the meat replica contains no methylcellulose, no carrageenan, no caramel color, no Konjac flour, no gum Arabic, and no acacia gum. In some embodiments the meat replica contains no animal products and less than 5% carbohydrates.

In some embodiments the meat replica contains no animal products, no soy protein, no wheat gluten, no methylcellulose, no carrageenan, no caramel color and no Konjac flour, no gum Arabic, and no acacia gum and less than 5% carbohydrates. In some embodiments the meat replica contains no animal products, and less than 1% cellulose. In some embodiments the meat replica contains no animal products, and less than 5% insoluble carbohydrates. In some embodiments the meat replica contains no animal products, no soy protein, and less than 1% cellulose. In some embodiments the meat replica contains no animal products, no soy protein, and less than 5% insoluble carbohydrates. In some embodiments the meat replica contains no animal products, no wheat gluten, and less than 1% cellulose. In some embodiments the meat replica contains no animal products, no wheat gluten, and less than 5% insoluble carbohydrates.

The percentage of different components may also be controlled. For example, non-animal- based substitutes for muscle, fat tissue, connective tissue, and blood components can be combined in different ratios and physical organizations to best approximate the look and feel of meat. The various can also components can be arranged to insure consistency between bites of the food product. The components can be arranged to insure that no waste is generated from the food product. For example, while a traditional cut of meat may have portions that are not typically eaten, a meat replicate can improve upon meat by not including these inedible portions. Such an improvement allows for all of the product made or shipped to be consumed, which cuts down on waste and shipping costs. Alternatively, a meat replica may include inedible portions to mimic the experience of meat consumption. Such portions can include bone, cartilage, connective tissue, or other materials commonly referred to as gristle, or materials included simulating these components. In some embodiments the food product may contain simulated inedible portions of meat products which are designed to serve secondary functions. For example, a simulated bone can be designed to disperse heat during cooking, making the cooking of the food product faster or more uniform than meat. In other embodiments a simulated bone may also serve to keep the food product at a constant temperature during shipping. In other embodiments, the simulated inedible portions may be biodegradable.

In some embodiments the meat substitute compositions contain no animal protein, comprising between 10-30% protein, between 5-80% water, between 5-70% fat, comprising one or more isolated purified proteins. In particular embodiments, the meat substitute compositions comprise transglutaminase. In some embodiments the food product contains components to replicate the components of meat. The main component of meat is typically skeletal muscle. Skeletal muscle typically consists of roughly 75 percent water, 19 percent protein, 2.5 percent intramuscular fat, 1.2 percent carbohydrates and 2.3 percent other soluble non-protein substances. These include organic acids, sulfur compounds, nitrogenous compounds, such as amino acids and nucleotides, and inorganic substances such as minerals.

In some instances, a meat replica is designed so that, when cooked, the percentages of components are similar to cooked meat. So, in some embodiments, the uncooked food product has different percentages of components than uncooked meat, but when cooked the food product is similar to cooked meat. For example, a meat replica may be made with a higher than typical water content for raw meat, but when cooked in a microwave the resulting product has percentages of components similar to meat cooked over a fire.

In some embodiments the food product is a meat replica with a lower that typical water content for meat. In some embodiments the disclosures provide for methods for hydrating a meat replica to cause the meat replica to have a content similar to meat. For example, a meat replica with a water content that would be low for meat, for example 1%, 10%, 20%, 30%, 40% or 50% water, is hydrated to roughly 75% water. Once hydrated, in some embodiments, the meat replica is then cooked for human consumption.

Production of Filamentous Fungi and Food Products Made Thereof

Edible filamentous fungi can be used as a nutrition source, such as for protein, either alone or incorporated into foodstuffs. The fruiting bodies of Basidiomycota and Ascomycota filamentous fungi are commonly used in foodstuffs, however, there are only a few products primarily comprising the vegetative mycelia of either the Basidiomycota or Ascomycota filamentous fungi. This is due, in part, to mycelia typically being either subterraneous or largely inseparable from the matter on which it grows.

Yet under particular conditions, filamentous fungi can be grown in a manner that the primary fungal biomass is filamentous fungal mycelial biomass. For example, filamentous fungi can form fungal biomats, that can consist essentially of mycelial biomass, via surface fermentation under anaerobic, microaerobic, or aerobic conditions or a combination thereof. Alternatively, fungal mycelial biomass can be produced by solid state fermentation, including in a form that is primarily, predominantly, or consists essentially of, mycelial biomass. Fungal mycelial biomass can also be produced by submerged fermentation. As such, filamentous fungal biomass can comprise the fungal species and/or strain and/or progeny thereof primarily in the form of mycelia, fragments of mycelia, hyphae, fragments of hyphae, and to a lesser extent contain conidia, microconidia, macroconidia, or any and all combinations thereof and in some cases can also contain pycnidia and chlamydospores.

Typically, the filamentous fungal mycelial biomasses are primarily comprised of mycelia; that is, a complex network of interwoven vegetative hyphae filaments. The average length of nonbroken filaments within the biomass is generally at least 0.1 mm, such as between 0.1 mm - 100 cm, or any range defined by any two whole numbers between 1 mm and 100 cm. In some embodiments, the average length can be at least 0.1 mm, 0.25 mm, 0.5 mm, , 1.0 mm, 1.4 mm 1.6 mm, 1.7 mm, 1.8 mm, 2 mm, 2.5 mm, 5 mm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 85 cm, or 100 cm, or any number in between.

Described herein are food materials comprising particles of edible filamentous fungi and particularly ones that are grown as a cohesive filamentous fungal mycelial biomass before being processed into particles.

The filamentous fungi suitable for use in the invention may be selected from the phyla or divisions basidiomycota or ascomycota. The phylum (or division) basidiomycota comprises, inter alia, the orders Agaricales, Russulales, Polyporales and Ustilaginales, and the phylum ascomycota comprises, inter alia, the orders Pezizales and Hypocreales. The particles of edible filamentous fungi of the present invention belong to an order selected from Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales and Hypocreales. In some embodiments, the filamentous fungi of the order Ustilaginales are selected from the family Ustilaginaceae. In some embodiments, the filamentous fungi of the order Russulales are selected from the family Hericiaceae. In some embodiments, the filamentous fungi of the order Polyporales are selected from the families Polyporaceae or Grifolaceae. In some embodiments, the filamentous fungi of the order Agaricales are selected from the families Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, or Omphalotaceae. In some embodiments, the filamentous fungi of the order Pezizales are selected from the families Tuberaceae or Morchellaceae. In some embodiments, the filamentous fungi of the order Hypocreales are selected from the families Nectriaceae or Cordycipitaceae. In some embodiments, the filamentous fungi are selected from the families Ophiocordycipitaceae (order Hypocreales) or Irpicaceae (order Polyprales).

Examples of the species of filamentous fungi include, without limitation, Ustilago esculenta, Hericululm erinaceus, Polyporous squamosus, Grifola fondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (elm oyster) Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoanmilata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (pearl), Pleurotus ostreatus var. columbinus (Blue oyster), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (cauliflower), Fusarium venenatum, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), Disciotis venosa, Cordyceps militans, Ganoderma lucidum (reishi), Flammulina velutipes, Lentinula edodes, Ophiocordyceps sinensis. Additional examples include, without limitation, Trametes versicolor, Ceriporia lacerate, Pholiota adiposa, Leucoagaricus holosericeus, Pleurotus djamor, Calvatia fragilis, and Handkea utriformis.

In some embodiments, the filamentous fungus is a Fusarium species. In some embodiments, the filamentous fungus is Fusarium strain flavolapis (ATCC Accession DepositNo. PTA- 10698 deposited with the American Type Culture Collection, 1081 University Boulevard, Manassas, Virginia, USA). Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698) was previously reported to be a Fusarium oxysporum strain. However, it has subsequently been identified as not being an oxysporum strain. In some embodiments, the filamentous fungus is the Fusarium strain Fusarium venenatum.

Fungal biomass in the food materials of the disclosure may be produced by a method to produce a cohesive mycelial biomass (e.g., by a surface fermentation process, a membrane fermentation process, or a solid-state fermentation process) or by a submerged fermentation process. The filamentous fungal biomass can consist essentially of fungal mycelium. For filamentous fungi that form fruiting bodies, the fungal biomass can be completely or substantially completely formed of fruiting bodies. Further, the filamentous fungal biomass can comprise conidia. In addition, the filamentous fungal biomass can comprise a mixture of mycelium, conidia, and fruiting body material in any proportions.

In embodiments, the fungal biomass in the food materials/products of the present disclosure may be a cohesive fungal biomass, and particularly may be a cohesive fungal biomass produced by a liquid surface fermentation process, a membrane fermentation process, and/or a solid-state fermentation process such that the cohesive fungal biomass comprises a substantial proportion (in embodiments, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt%, or at least about 95 wt% on a dry basis) of fungal mycelial matter that includes vegetative and/or aerial hyphae. As further described throughout this disclosure, one advantage of the use of a cohesive fungal mycelial biomass to produce food materials/products is that these biomasses have a surprising propensity and ability to take up aqueous solutions of one or more heme proteins, such that the heme proteins are well-absorbed by, well-adsorbed on a surface of, and/or effectively coated on a surface of the fungal biomass.

As described in detail herein, the filamentous fungi of the present invention have a surprisingly high protein content. It is noted that the filamentous fungi that grow naturally or in the wild or by prior art methods do not possess such high protein contents, whereas filamentous fungi grown or cultured as disclosed herein have a high protein content. For example, protein contents of filamentous fungi described herein refer to the protein contents of the filamentous fungi as grown as a cohesive fungal mycelial biomass according to the present disclosure. Consequently, food materials of the invention have high protein contents based on the filamentous fungi components of the materials without the need for and/or in the absence of protein content from a non-filamentous fungal source. Thus, in various embodiments, food materials of the invention do not contain or have an absence of protein content from a non-filamentous fungal source.

In some embodiments, the filamentous fungi comprise at least about 30 wt. % protein content. Unless specified otherwise herein, percentages of components, such as proteins, RNA or lipids, of fungal mycelial biomass, are given as a dry weight percent basis. For example, fungal mycelial biomass can be dried for 2 days at 99°C and further air dried for a few days, at the end of which the biomass is expected to contain about 5 wt. % or less moisture, such as less than 4 wt. %, less than 3 wt. %, less than 2 wt. %, less than 1 wt. %, less than 0.5 wt. %, less than 0.1 wt. % moisture. The total protein content in dried biomass samples can be measured using total nitrogen analysis method for estimating proteins.

In some embodiments, the filamentous fungi comprise at least about 30%, at least about 3 Iwt. %, at least about 32 wt. %, at least about 33 wt. %, at least about 34 wt. %, at least about 35 wt. %, at least about 36 wt. %, at least about 37 wt. %, at least about 38 wt. %, at least about 39 wt. %, at least about 40 wt. %, at least about 41 wt. %, at least about 42 wt. %, at least about 43 wt. %, at least about 44 wt. %, at least about 45 wt. %, at least about 46 wt. %, at least about 47 wt. %, at least about 48 wt. %, at least about 49 wt. %, at least about 50 wt. %, at least about 51 wt. %, at least about 52 wt. %, at least about 53 wt. %, at least about 54 wt. %, at least about 55 wt. %, at least about 56 wt. %, at least about 57 wt. %, at least about 58 wt. %, at least about 59 wt. %, at least about 60 wt. % protein content, at least about 61 wt. %, at least about 62 wt. %, at least about 63 wt. %, at least about 64 wt. %, at least about 65 wt. %, at least about 66 wt. %, at least about 67 wt. %, at least about 68 wt. %, at least about 69 wt. %, at least about 70 wt. % protein content, at least about 71 wt. %, at least about 72 wt. %, at least about 73 wt. %, at least about 74 wt. %, at least about 77 wt. %, at least about 76 wt. %, at least about 77 wt. %, at least about 78 wt. %, at least about 79 wt. %, or at least about 80 wt. % protein content. Alternatively, in embodiments of the invention, filamentous fungi can comprise protein in a range between 30 wt. % and 80 wt. % or in any whole number percentage range between 30 wt. % and 80 wt. %. In some particular embodiments, a protein content of the fungal mycelial biomass on a dry basis may be from about 35 wt% to about 60 wt%, or any value in any subrange having a lower bound of any whole number of percentage points from 35 wt% to 60 wt% and an upper bound of any other whole number of percentage points from 35 wt% to 60 wt%.

In some embodiments, a carbohydrate content of the fungal mycelial biomass on a dry basis may be from about 25 wt% to about 55 wt%, or any value in any subrange having a lower bound of any whole number of percentage points from 25 wt% to 55 wt% and an upper bound of any other whole number of percentage points from 25 wt% to 55 wt%. In some embodiments, a dietary fiber content of the fungal mycelial biomass on a dry basis may be from about 20 wt% to about 40 wt%, or any value in any subrange having a lower bound of any whole number of percentage points from 20 wt% to 40 wt% and an upper bound of any other whole number of percentage points from 20 wt% to 40 wt%. In some embodiments, a lipid content of the fungal mycelial biomass on a dry basis may be from about 2.3 wt% to about 7.0 wt%, or any value in any subrange having a lower bound of any whole number of tenths of a percentage point from 2.3 wt% to 7.0 wt% and an upper bound of any other whole number of tenths of a percentage point from 2.3 wt% to 7.0 wt%. In some embodiments, a c/.s.c/.s-poly unsaturated fatty acid content of the fungal mycelial biomass on a dry basis may be from about 1.2 wt% to about 2.6 wt%, or any value in any subrange having a lower bound of any whole number of tenths of a percentage point from 1.2 wt% to 2.6 wt% and an upper bound of any other whole number of percentage points from 1.2 wt% to 2.6 wt%. In some embodiments, a c/.s-monounsaturated fatty acid content of the fungal mycelial biomass on a dry basis may be from about 0.1 wt% to about 0.6 wt%, or any value in any subrange having a lower bound of any whole number of tenths of a percentage point from 0.1 wt% to 0.6 wt% and an upper bound of any other whole number of percentage points from 0.1 wt% to 0.6 wt%. In some embodiments, a saturated fatty acid content of the fungal mycelial biomass on a dry basis may be from about 0.6 wt% to about 1.0 wt%, or any value in any subrange having a lower bound of any whole number of tenths of a percentage point from 0.6 wt% to 1.0 wt% and an upper bound of any other whole number of percentage points from 0.6 wt% to 1.0 wt%. In some embodiments, the fungal mycelial biomass may be substantially free of trans fatty acids.

The filamentous fungi of the present invention also can have a surprisingly low RNA content. High amounts of RNA in food have been shown to have adverse health or physiological effects. For example, diets that are high in purines (present in RNA) are associated with incidence of gout. It is noted that the filamentous fungi grown or cultured as disclosed herein have intrinsically low RNA content and do not require additional or supplemental treatment to modify or lower the RNA content. Thus, in various embodiments, food materials of the invention do not contain components that have significant levels of RNA and/or have been treated for the purpose of modifying or lowering the RNA content of the components or food materials.

In some embodiments, the filamentous fungi comprise less than about 8 wt. % RNA content. The wt. % RNA content is given on a dry weight basis. For example, the total RNA content in dried biomass samples can be measured using the purine analysis method.

In some embodiments, the RNA content in the filamentous fungi is less than about 8.0 wt. % RNA content, less than about 7.9 wt. % RNA content, less than about 7.8 wt. % RNA content, less than about 7.7 wt. % RNA content, less than about 7.6 wt. % RNA content, less than about 7.5 wt. % RNA content, less than about 7.4 wt. % RNA content, less than about 7.3 wt. % RNA content, less than about 7.2 wt. % RNA content, less than about 7.1 wt. % RNA content, less than about 7 wt. % RNA content, less than about less than about 6.9 wt. % RNA content, less than about 6.8 wt. % RNA content, less than about 6.7 wt. % RNA content, less than about 6.6 wt. % RNA content, less than about 6.5 wt. % RNA content, less than about 6.4 wt. % RNA content, less than about 6.3 wt. % RNA content, less than about 6.2 wt. % RNA content, less than about 6.1 wt. % RNA content, less than about 6 wt. % RNA content, less than about 5.9 wt. % RNA content, less than about 5.8 wt. % RNA content, less than about 5.7 wt. % RNA content, less than about 5.6 wt. % RNA content, less than about 5.5 wt. % RNA content, less than about 5.4 wt. % RNA content, less than about 5.3 wt. % RNA content, less than about 5.2 wt. % RNA content, less than about 5.1 wt. % RNA content, less than about 5.0 wt. % RNA content, less than about 4.9 wt. % RNA content, less than about 4.8 wt. % RNA content, less than about 4.7 wt. % RNA content, less than about 4.6 wt. % RNA content, less than about 4.5 wt. % RNA content, less than about 4.4 wt. % RNA content, less than about 4.3 wt. % RNA content, less than about 4.2 wt. % RNA content, less than about 4.1 wt. % RNA content, less than about 4 wt. % RNA content, less than about 3.9 wt. % RNA content, less than about 3.8 wt. % RNA content, less than about 3.7 wt. % RNA content, less than about 3.6 wt. % RNA content, less than about 3.5 wt. % RNA content, less than about 3.4 wt. % RNA content, less than about 3.3 wt. % RNA content, less than about 3.2 wt. % RNA content, less than about 3.1 wt. % RNA content, less than about 3 wt. % RNA content, less than about 2.9 wt. % RNA content, less than about 2.8 wt. % RNA content, less than about 2.7 wt. % RNA content, less than about 2.6 wt. % RNA content, less than about 2.5 wt. % RNA content, less than about 2.4 wt. % RNA content, less than about 2.3 wt. % RNA content, less than about 2.2 wt. % RNA content, less than about 2.1 wt. % RNA content, less than about 2 wt. % RNA content, less than about 1.9 wt. % RNA content, less than about 1.8 wt. % RNA content, less than about 1.7 wt. % RNA content, less than about 1.6 wt. % RNA content, less than about 1.5 wt. % RNA content, less than about 1.4 wt. % RNA content, less than about 1.3 wt. % RNA content, less than about 1.2 wt. % RNA content, less than about 1.1 wt. % RNA content, less than about 1 wt. % RNA content, less than about 1 wt. % RNA content, less than about 0.9 wt. % RNA content, less than about 0.8 wt. % RNA content, less than about 0.7 wt. % RNA content, less than about 0.6 wt. % RNA content, or less than about 0.5 wt. % RNA content. Alternatively, in embodiments of the invention, filamentous fungi can comprise RNA in a range between 0.5 wt. % and 8 wt. % or any sub -range thereof.

In some embodiments, the filamentous fungus comprises a high protein content combined with a low RNA content as described above. For example, in some embodiments the filamentous fungus may comprise greater than 45 wt. % protein, greater than 50 wt. % protein, greater than 55 wt. % protein, or greater than 60 wt. % protein and less than about 8 wt. % RNA content, less than about 5 wt. % RNA content, less than about 4 wt. % RNA content, less than about 3 wt. % RNA content, or less than about 2 wt. % RNA content.

The filamentous fungi of the present invention and related food materials can also be characterized as having surprisingly low mycotoxin content. Known mycotoxins include Alfatoxin Bl, Alfatoxin B2, Alfatoxin Gl, Alfatoxin G2, Fumonisin Bl, Fumonisin B2, Fumonisin B3, Ochratoxin A, Nival enol, Deoxynivalenol, Acetyl deoxy nival enol, Fusarenon X, T-2 Toxin, HT-

2 Toxin, Neosolaniol, Diacetoxyscirpenol and zearalenone. In some embodiments, the total amount of mycotoxins and/or the total amount of any one of or subset of the above-listed mycotoxins in a filamentous fungi, biomass or food material of the invention is less than about 10 ppm. In other embodiments, the total amount of mycotoxins and/or the total amount of any one of or subset of the above-listed mycotoxins is less than about 9 ppm, less than about 8 ppm, less than about 7 ppm, less than about 6 ppm, less than about 5 ppm, less than about 4 ppm, less than about

3 ppm, less than about 2 ppm, or less than about 1 ppm.

The filamentous fungi of the present invention also have a surprisingly high branched amino acid content. Branched amino acids refer to leucine, isoleucine and valine. In some embodiments, the total amount of branched amino acids is greater than about 10 wt. %, greater than about 11 wt. %, greater than about 12 wt. %, greater than about 13 wt. %, greater than about 14 wt. %, greater than about 15 wt. %, greater than about 16 wt. %, greater than about 17 wt. %, greater than about 18 wt. %, greater than about 19 wt. %, greater than about 20 wt. %, greater than about 21 wt. %, greater than about 22 wt. %, greater than about 23 wt. %, greater than about 24 wt. %, greater than about 25 wt. %, greater than about 26 wt. %, greater than about 27 wt. %, greater than about 28 wt. %, greater than about 29 wt. %, greater than about 30 wt. %. Additionally or alternatively, the filamentous fungi may be a “complete” protein source, i.e., may include all of the essential amino acids. Growing and harvesting filamentous fungal biomats

A filamentous fungal cohesive mycelial biomass produced by surface fermentation is referred to herein as a “biomat.” This involves inoculating liquid media containing a carbon source and a nitrogen source with filamentous fungal cells. Suitable carbon sources are sugars (e.g. sucrose, maltose, glucose, fructose, Japan rare sugars, etc.), sugar alcohols (e.g. glycerol, polyol, etc.), starch (e.g. corn starch, etc.), starch derivative (e.g. maltodextrin, cyclodextrin, glucose syrup, hydrolysates and modified starch), starch hydrolysates, hydrogenated starch hydrolysates (HSH; e.g. hydrogenated glucose syrups, maltitol syrups, sorbitol syrups, etc.), lignocellulosic pulp or feedstock (e.g. sugar beet pulp, agricultural pulp, lumber pulp, distiller dry grains, brewery waste, etc.), com steep liquors, acid whey, sweet whey, milk serum, wheat steep liquors, carbohydrates, food waste, olive oil processing waste, hydrolysate from lignocellulosic materials, and/or combinations thereof. The filamentous fungi generate biomats which are located on the surface of the growth media; that is, they essentially float atop the growth media.

Inoculation may be done with an inoculum comprising filamentous fungal cells, conidia, microconidia or macroconidia or spores, or fruiting bodies. In many cases, especially for Ascomycota fungi, growth media may be inoculated with an inoculum comprising filamentous fungal cells, conidia, microconidia or macroconidia. Ideally, the cells of the inoculum float on the surface of the growth media, such as those cells having a high lipid content, and result in increased growth rate. Cells or clumps of cells that are submersed within the growth media negatively affect the cells floating on the surface and the biomats they form.

In some embodiments, the inoculum may comprise spores. For example, in one embodiment, approximately 2 cc of sterile Basidiomycota spores suspended in deionized water from a spore syringe (e.g. MycoDirect, Huntley, IL) were used to inoculate approximately 75 mL of growth media in small Pyrex trays. Alternatively, 1 cc of spores suspended in deionized water from a spore syringe was plated on a container having malt extract agar media + CF (30 g dry malt extract, 20 g agar, 1000 mL water + 0.01% chloramphenicol) using standard sterile conditions. Containers were sealed with parafilm and incubated at room temperature until mycelium completely covered the surface of the agar. A segment of mycelium from the agar preparation approximately 2 cm in width cut into a wedge was then diced into the smallest size possible before transferring to a tube with growth media. Liquid culture tubes were sealed, incubated at room temperature, and shaken by hand or shaken by mechanical means (i.e. continuous shaking or a continuous stirred tank reactor) for about 1 minute at least five (5) times per day to break up mycelium as much as possible. Liquid cultures were incubated until visually turbid, typically three or more days. The liquid cultures were then used to inoculate growth medium in trays at a 10% or 15% of total growth medium volume.

In some embodiments, the inoculum may comprise fruiting bodies. For example, in some embodiments, Basidiomycota fruiting bodies were used to generate inoculum for initiating filamentous biomats. In some instances, inoculum was prepared by (a) surface sterilizing fruiting bodies, for example in a 5% bleach solution, (b) rinsing with sterile media, (c) grinding under sterile conditions to either less than 5 mm long aggregates or greater than 5 mm aggregates, depending on the final use, (d) surface sterilizing the ground mushroom biomass for example in a 5% bleach solution, and again rinsing with sterile media. 5 grams of the ground surface-sterilized fruiting body biomass was used directly as inoculum. In other instances, a pure culture derived from a fruiting body was used. Here, ~ 3 mm³ portions of fruiting body was placed on agar media containing 0.01% chloramphenicol and incubated at room temperature. After 2-5 days of growth, hyphae were transferred onto fresh agar + chloramphenicol media and grown for another 3-7 days. Culture purity was confirmed by extracting and purifying DNA (FastDNA Spin Kit, MP Biomedicals), sequencing the 18S rRNA sequence and/or ITS region, and performing phylogenetic classification of the sequences using Blast (NCBI database). Upon confirmation, hyphae were used to inoculate 50 mL of sterile liquid media and agitated/rotated at 185 rpm for approximately 5 days before using as inoculum at a ratio of about 7.5% inoculum to 92.5% liquid media.

While a number of different media can be used, some media may not be well adapted for growth of filamentous fungal biomats, such as Hansen’s media (per liter = 1.0 g peptone, 0.3 g KH2PO4 • 7H2O, 2.0 g MgSCL • 7H2O 5.0 g glucose with a C:N ratio of 26.9) upon which full, cohesive biomats were not produced. Those media which work exceptionally well include MK7A, MK7-1, MK7-3 (all described in WO 2017/151684), as well as the media presented below.

Osmotic concentrations can be determined by measurement of media with an Osmometer (e.g., Model 3250 SN: 17060594) capable of measuring up to 5000 mOsm. Three readings were taken for several media and provided the following results: Hansen’s = 39, 39, 38; Malt 001 = 169, 168, 169; MK-7 SF = 1389, 1386, 1387; Malt 001 + NH₄NO₃ = 288, 287, 286. As noted before, methods of the invention can result in increasing the protein content of the filamentous fungus. Without being bound by theory, this result is believed to be due in part to the media used to grow the fungus.

For example, while the natural protein content of the fruiting body of Blue Oyster mushrooms (Pleurotus ostreatus var. Columbinus) is reported to be about 16.32% (Ulziijargal and Mau (2011) Int J Medicinal Mushrooms, 13(4):343-49) or 24.65% (Stamets (2005) Int J Medicinal Mushrooms 7:103-110) Blue Oyster biomats grown according to the present invention on Malt 001 media have a higher moisture corrected protein content of 29.82%, an increase in protein content of 13.6% or 5.71%.

The protein content of the fruiting body of Pearl Oyster mushrooms (Pleurotus ostreatus) is reported to be about 23.85% (Ulziijargal and Mau (2011) Int J Medicinal Mushrooms, 13(4):343-49) or 27.25% (Stamets (2005) Int J Medicinal Mushrooms 7: 103-110); Pearl Oyster biomats grown according to the present invention have a higher moisture corrected protein content of 39.77% , an increase in protein content of at least 46% to a maximum of 67%.

The protein content of the fruiting body of Cauliflower mushrooms (Sparassis crispa) is reported to be about 13.4% (Kimura (2013) BioMed Research International); Cauliflower biomats grown according to the present invention have a higher moisture corrected protein content of 32.21% - 46.24%, an increase in protein content of least 140% to a maximum of 245%.

Other characteristics of the media that are believed to be important for the formation of biomats on the surface of a fermentation media are the osmotic pressure and the ionic strength of the media. In some embodiments, the osmotic pressure of the media for growth of biomats can be greater than about 3 atm, greater than about 10 atm, greater than about 20 atm, greater than about 30 atm, greater than about 40 atm, greater than about 50 atm, greater than about 60 atm, greater than about 70 atm, greater than about 80 atm, greater than about 90 atm, greater than about 100 atm, greater than about 110 atm, greater than about 120 atm, or greater than about 125 atm. In alternative embodiments, the osmotic pressure may range between about 3 atm to about 125 atm, between about 20 atm and about 100 atm or between any two whole number atm values between 3 and 125.

In some embodiments, the ionic strength of the media that can be used to grow biomats can be greater than about 0.02 M, greater than about 0.05 M, greater than about 0.10 M, greater than about 0.20 M, greater than about 0.30 M, greater than about 0.40 M, greater than about 0.50 M, greater than about 0.60 M, greater than about 0.70 M, greater than about 0.80 M, greater than about 0.90 M, or greater than about 1.0 M. In alternative embodiments, the ionic strength may range between about 0.02 M to about 1 .0 M, between about 0. 10 M and about 0.50 M or between any two number molar concentration values between 0.01 and 1.0.

Harvesting of biomats can occur at any time a sufficiently thick biomat has formed. Harvesting typically occurs after 2-3 days of growth, although in some instances longer growth periods are desirable, such as when thicker or denser biomats are desired/required. For example, harvesting can occur after growth of between 2 days and 60 days or any range of days or partial days (e.g., hours) between 2 days and 60 days. For example, such growth periods can be 3.5 - 4 days, 3-5 days, 4-6 days, 5-7 days, 6-9 days, 7-10 days, or 19-21 days. As used herein, the term “harvesting,” refers to any process or step that stops growth of a biomat (e.g., separation from a nutrient source or change in temperature conditions) and/or that modifies a physical characteristic of a biomat (e.g., converting a biomat into particles or strips).

Due to the cohesive structure of the filamentous biomats grown under surface fermentation conditions described in PCI7US2017/020050 and herein, the filamentous biomats have enough tensile strength to be lifted essentially intact from the surface of the media at the end of the growth period. For example, the filamentous biomats have enough tensile strength to be lifted with a single hand and remain intact when the biomat is at least about 500 cm², at least about 600 cm², at least about 700 cm², at least about 800 cm², at least about 900 cm², or at least about 1000 cm². In various embodiments, biomats of the invention can have a tensile strength of at least about 30 g/cm², at least about 40 g/cm², at least about 50 g/cm², at least about 60 g/cm², at least about 70 g/cm², at least about 80 g/cm², at least about 90 g/cm², at least about 100 g/cm², at least about 150 g/cm², at least about 200 g/cm², at least about 250 g/cm², at least about 300 g/cm², at least about 350 g/cm², at least about 400 g/cm², at least about 450 g/cm², at least about 500 g/cm², at least about 550 g/cm², or at least about 600 g/cm², or at least about 650 g/cm², or at least about 700 g/cm², or at least about 750 g/cm², or at least about 800 g/cm², or at least about 850 g/cm², or at least about 900 g/cm², or at least about 950 g/cm², or at least about 1000 g/cm², or at least about 1500 g/cm², or at least about 2000 g/cm², or at least about 2500 g/cm², or at least about 3000 g/cm², or at least about 3500 g/cm², or at least about 4000 g/cm², In other embodiments, biomats of the invention can have a tensile strength of greater than any whole number greater than 30 g/cm². Alternatively, the tensile strength of biomats of the invention can be in a range of between about 30 g/cm² and about 4000 g/cm² or any whole number range between about 30 g/cm² and about 4000 g/cm².

In various embodiments, biomats of the invention can have a thickness ranging from about 0.05 cm to about 30 cm, or any subrange thereof.

Surface fermentation can be carried out under various conditions, including static media conditions (as described in PCT Publication WO 2017/151684, which is incorporated herein by reference in its entirety), semi-static media conditions, and continuous media flow conditions.

Growth under semi-static media conditions means that at least a portion of the medium is replaced before the filamentous fungal biomat is harvested. These conditions allow linear dry biomass production over an extended period of time demonstrating the suitability of this system to operate as a continuous production system. For example, in one experiment, linear dry biomass production was achieved from day 4 through day 18 (r² = 0.995), after which biomass weight stabilized at about 2.5 Kg dry/m².

Biomats can also be produced under continuous media flow conditions where biomat growth is confined to the surface of the growth media where the medium underneath the mat is continuously refreshed or semi -continuously refreshed.

In some cases, UVB light (290-320 nm) can trigger pigment production by filamentous fungi, such as for Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), producing a pigmented biomat. In addition to a color change, which can be useful for creating various food effects, treatment with UVB converts ergosterol present in the fungal cell membranes into vitamin D2 and increases production of carotenoids, such as beta carotene and astaxanthin. Consequently, irradiating filamentous fungi, such as Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), with UVB can be used to increase vitamin D2 and carotenoids in the resulting biomats.

In some cases, the filamentous fungal biomats formed are composed of layers which are uniform in appearance, one surface of the filamentous biomat in contact with the air and one surface in contact with the synthetic media. In other cases, at least two distinct layers are present: an aerial hyphae layer at the top surface and a dense multicellular bottom layer in contact with the synthetic media. Oftentimes three distinct layers are present: (a) an aerial hyphae layer at the top surface, (b) a dense bottom layer and (c) a transitional layer between the top and bottom layers. The transitional layer may be only loosely attached to the dense bottom layer, in those cases enabling easy separation of the bottom layer from the rest of the biomat. Filament densities of the transitional layer range from slightly less dense than the bottom layer in the zone where the two layers meet, to a density that is comparable to the aerial hyphae near the top of the biomat.

Inactivation of filamentous fungal biomass

Filamentous fungal biomass of the invention is inactivated after it is produced. This step is conducted to eliminate cell viability and further growth and/or to inactivate enzymes produced by the filamentous fungus. This process will depend on the manner in which the filamentous fungus is produced, such as by liquid surface fermentation, membrane fermentation, solid state fermentation, submerged fermentation or other processes.

The inactivation process begins with biomass harvested after cultivation. While biomass can be rinsed to remove excess growth media, biomass rinsing is not required, although in some cases the removal of growth media or excess growth media is preferable. Similarly, biomass can be squeezed to remove excess growth media, again not required, but which may be preferable for some applications.

Elimination of cell viability and the potential of further biomass growth is desired particularly for use of the biomass as a stand-alone protein source or a protein ingredient in foodstuffs. This can be accomplished by heating, irradiation, and/or steaming.

For the heating process, filamentous fungal biomass can be treated according to WO 95/23843 or British Patent No 1,440,642, for example, or incubated at temperatures that destroy the vast majority of the organism’s RNA without adversely affecting the organism’s protein composition.

In irradiation, filamentous fungal biomass is exposed to ionizing energy, such as that produced by ⁶⁰Co (or infrequently by ¹³⁷Cs) radioisotopes, X-rays generated by machines operated below a nominal energy of 5 MeV, and accelerated electrons generated by machines operated below a nominal energy of 10 MeV.

Steaming can also be used for inactivating some filamentous fungal biomass as steaming can also remove some specific metabolites from the biomass construct if those metabolites are produced. Here, biomass is placed such that biomass excreted liquids and condensed steam can easily drip away from the biomass. Suitable biomass holding systems include porous plastic mesh and porous trays. Other biomass holding systems include, but are not limited to, systems that secure the biomass in a vertical position, such as systems with a clamping mechanism that clamps at least one end of a cohesive biomass while the remaining end(s) of the biomass hang from said clamp and mesh systems which clamp at least two sides of the biomass, to name but a few.

The biomass can be positioned within a steamer such that heated steam, such as steam of a temperature greater than 85 °C or 95 °C, comes into contact with the biomass. In those cases where multiple trays or containers are placed in a single steamer, for example one above the other, it is preferred to protect a lower positioned biomass from the drippings of a higher positioned biomass. Protection should be of a form which allows steam to contact biomass, thereby de-activating biomass viability, and to also deflect biomass excreted liquids and condensed steam produced at a higher level in the steamer from contacting biomass positioned at a lower level in the steamer. In one embodiment, a cone is positioned between an upper tray and a lower tray to accomplish this result. In other embodiments, separation between upper and lower trays also include at least one other geometric shape such as a cylinder, a cube and/or cuboid, a pyramid, a sphere, a torus, and/or other platonic solids. In yet another embodiment, trays are separated using at least one cylinder, cube and/or cuboid, pyramid, sphere, tori, other platonic solid, or combinations thereof.

Biomass is steamed at least to the point where biomass viability is reduced such that further biomass growth and/or cellular reproduction within a biomass is negligible. Biomass viability is a function of the original substrate, biomass development, steam/heat transfer characteristics, biomass position in a steamer and biomass orientation relative to evolved steam. As an example, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698) biomass grown on a glycerol or acid whey substrate are non-viable after 5 minutes, and in some cases less than 5 minutes, of steaming. Steamed cohesive biomasses can be rinsed and/or squeezed to remove excretions and condensed steam.

Particles of cohesive fungal mycelial biomass

The inactivated edible cohesive fungal mycelial biomass can also be size reduced for use as a protein source in foodstuffs. The size reduction can occur by mechanical means such as cutting, chopping, dicing, mincing, grinding, blending, etc. or via sonication and is conducted prior to mixing with other ingredients or liquids. Size reduced particles can be uniform in size or variable.

Typically, the length of the sized reduced particles is between 0.05-500 mm, the width is between 0.03 -7 mm, and height is between 0.03- 1.0 mm. For example, particles may range between 0.03 mm and 0.4 mm, or between 100 mm and 500, etc. Larger size particles can be produced. For example, biomats have been grown in inflatable pools (66” in diameter) producing a single biomat 66” in diameter and completely round. Larger vessels can be used to grow even larger mats.

The number of size reduced particles produced from a cohesive fungal biomass is dependent on the initial biomass size and the purpose for which the biomass size reduced particles will be used.

Large particles

In some embodiments, the inactivated edible filamentous fungal cohesive biomass is reduced to particles, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles have a particle length of about 0.05 mm to about 500 mm, a particle width of about 0.03 mm to about 7 mm, and a particle height of about 0.03 mm to about 1.0 mm, or alternatively in any subranges within these ranges. For example, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

In some embodiments, the inactivated edible filamentous fungal cohesive biomass is reduced to particles, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the mass of the particles have a particle length of about 0.05 mm to about 500 mm, a particle width of about 0.03 mm to about 7 mm, and a particle height of about 0.03 mm to about 1.0 mm, or alternatively in any subranges within these ranges. For example, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the mass of the particles may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

For example, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

Such particles mimic the texture and chewiness of meat products such as chicken nuggets or hamburgers, and are useful in the preparation of such products, such as a filler or extender of meat products, or their vegetarian versions. In the case of use of particles of the invention as a filler or extender of meat product, the ratio of filamentous fungal particles to meat can range from 10:90 to 90: 10 or any ratio in between.

For example, in some embodiments, the filamentous fungal particles comprise particles having at least 90% of the particles with lengths less than about 1.5 mm and the majority of lengths being 1 mm or less, widths of less than about 1 mm, and heights of less than about 0.75 mm. Food materials comprising such particles is characterized as having a higher perceived density in the mouth, is easier to chew, offers a creamy mouth feel and a more refined food experience, and such particles may be used to prepare a food material that resembles a hamburger found in fine dining establishments.

In some embodiments, the filamentous fungal particles comprise particles having at least about 90% of the particles with lengths between about 4 mm and about 10 mm, widths of about 1.0 mm to about 3 mm, and heights of less than 0.75 mm. Food materials comprising such particles is found to lead a more heartier food experience similar to the type of burger prepared commonly found in burger restaurants or BBQ’s.

Fine particles

In some embodiments, the inactivated edible filamentous fungal cohesive biomass is reduced to fine particles. In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles fall within the range of 0.03 mm to about 0.4 mm, or alternatively in any subrange within this range, such as about 0.03 mm to 0.07 mm, about 0.07 mm to about 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about 2.0, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm or 0.2 mm to about 0.4 mm. In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles fall within the range of 0.075 mm to about 0.12 mm.

In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the mass of the particles fall within the range of 0.03 mm to about 0.4 mm, or alternatively in any subrange within this range, such as about 0.03 mm to 0.07 mm, about 0.07 mm to about 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about 2.0, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm or 0.2 mm to about 0.4 mm. In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the mass of the particles fall within the range of 0.075 mm to about 0.12 mm.

The size reduction may be done using a mill, grinder or other conventional equipment for size reduction.

In some embodiments, the moisture content of fine particulate material of the invention is less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1%. The low moisture levels aid to prevent clumping of the particles. In some embodiments, moisture may be removed from particulate fungal material by spray-drying, and/or by any other suitable method for removing moisture from edible materials as may be known in the art. As disclosed herein, spray-dried fungal mycelial biomass, and/or other forms of biomass with low moisture content, may in some embodiments exhibit hydrophobic properties that would cause the biomass to repel solutions of heme protein in water or other aqueous ingredients. Accordingly, in some embodiments, food products according to the present disclosure may include one or more food-grade surfactants (e.g., alkyl glycosides, carrageenans, cholesterols, lanolins, lecithins, monoglycerides, phytosterols, proteins, tea saponin extracts, sorbitan derivatives, organic sodium sulfonates and sulfosuccinates, etc.) in any suitable amount to aid the dispersibility of aqueous ingredients into the fungal mycelial biomass.

Liquid Dispersion

One aspect of introducing protein into a foodstuff is to use a liquid dispersion made from the filamentous fungal cohesive biomass. The liquid dispersion comprises particles of filamentous fungal cohesive biomass dispersed in an aqueous medium.

The size of the filamentous fungal cohesive biomass particles suitable for use in liquid dispersions is typically smaller than about 10 microns. In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles in a liquid dispersion fall within the range of about 1 microns to about 10 microns, or alternatively in any subrange within this range. In some embodiments, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles are less than 10 microns, less than 9 microns, less than 8 microns, less than 7 microns, less than 6 microns, less than 5 microns, less than 4 microns, less than 3 microns, less than 2 microns, or less than 1 micron. The liquid dispersion can be prepared by combining and blending a filamentous fungal cohesive biomass with an aqueous phase, such as water. The blended mixture can be heated gradually, such as to a boiling temperature. The heated mixture is then allowed to cool. In some embodiments, a liquid dispersion can be produced under nitrogen. This process results in a creamier consistency of liquid dispersion with less fungal scent. Production under nitrogen can be accomplished by bubbling with nitrogen in a closed vessel such that nitrogen replaces most all of the available oxygen, either during blending, such as with a Vitamix, or in the heat cycle.

The filamentous fungal cohesive biomass to water ratio can be adjusted to produce a liquid dispersion of the appropriate consistency and density. The ratio of the cohesive biomass to water can range from about 1 :5 to about 10: 1 or any range of ratios in between. In some embodiments, the ratio of the biomass to water can be about 1:5, about 1 :4, about 1:3, about 1 :2, about 1 : 1, about 2:1, about 3:1, about 4: 1, about 5:1, about 6:1, about 7: 1, about 8:1, about 9: 1 about 10:1.

In various embodiments, a liquid dispersion of the invention is stable such that the particulates of filamentous fungus do not readily separate from the liquid medium in which they are dispersed. For example, upon forming the dispersion, the formed liquid appears to be homogeneous in appearance and does not visibly separate into distinct phases. For example, no visibly discernable or significant sediment forms on the bottom of the container holding the dispersion. In some embodiments, the liquid dispersion remains stable for at least about 1, 2, 3, 4, 5, 6, 9, 12, 15,18, 21, or 24 hours or alternatively, it can remain stable for at least about 1, 2, 3, 4, 5, 6, or 7 days, or 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, or 6 months. In these embodiments, the dispersion can either be at room temperature or at refrigerated temperatures, such as at about 35°F (1.6°C).

In some embodiments, a liquid dispersion of cohesive fungal mycelial biomass can remain undisturbed in a refrigerator for at least about 5 days, 10 days, 15 days, 20 days, 25 days, or 30 days, with no visible separation was observed and/or with no degradation of flavor, smell, and/or color.

In some embodiments, the dispersion comprises at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% solids. In other embodiments, a liquid dispersion of the invention will have a solids content of between about 4% and about 30% or any sub-range between 4% and 30%.

In some embodiments, the liquid dispersion can be used to form a stable foam, in that it forms a foam that does not collapse spontaneously immediately upon cessation of the foaming process. The foaming process can include whipping with a whipping appliance, incorporation of compressed gases or other conventional foaming processes. The foam is smooth and creamy in appearance and shows the presence of bubbles in a distribution of sizes. The larger bubbles tend to pop after sitting or being poured, but the smaller bubbles stay in suspension for a long time to form a stable foam product. A foam product of the invention has the compositional characteristics of a liquid dispersion and additionally has air or other gas incorporated into the foam in a stable manner. For example, a foamed material of the invention can have an increased volume (i.e., overrun) by incorporation of air of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, as compared to the starting volume of the liquid dispersion prior to foaming. In various embodiments, a foamed material is stable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, or at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, or at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days. In some embodiments, the liquid dispersion remains stable for at least about one month, at least about two months, or at least about three months. As used in reference to a foam, stability refers to retaining at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% of its initial foamed volume.

In some embodiments, the food material comprises a spreadable or gelatinous food product having a savory or meaty flavor profile, c. ., a pate analog food product, a gefilte fish analog food product, or an aspic analog food product (hereinafter referred to as a “soft meat analog food product”), comprising the particles of the filamentous fungal biomass of the present invention dispersed in an aqueous medium. In some embodiments of the soft meat analog, the ratio of filamentous fungal particles to water may range from about 1 :3 to about 2: 1. A higher ratio of filamentous fungal particles to water is expected to increase the texture and reduce runniness of the soft meat analog food product. In some embodiments, the ratio of filamentous fungal particles to water may be about 1 :3, 1:2, 1:1 or 2: 1.

In some embodiments, the soft meat analog food product comprises a thickening or gelling agent. Such agents are known in the art and include but are not limited to: agar, gelatin, starches (i.e. arrowroot, tapioca, com, potato), higher fat liquids (coconut milk), fat (i.e. coconut flakes, deodorized or otherwise), chickpea water, flax seeds, xanthan gum, guar gum, psyllium husk, ground chia seed, nut / seed butters, pumpkin puree, cooked mashed yams/ sweet potato, applesauce, mashed overripe bananas or plantains, pureed dates or prunes, soaked and simmered figs, shredded fruit/vegetables, shredded coconut, gluten free flours (e g. teff flour, buckwheat flour, amaranth flour, chickpea flour, sorghum flour, almond flour), cooked pureed beans, cocoa Powder, vegetable gums, polysaccharides, vegetable mucilage, seaweed derivatives, pectin, gluten, soy and egg analogs. A thickening agent may be a fat, which may be a liquid such as coconut milk, or a solid such as deodorized coconut flakes.

In some embodiments, the cells of the filamentous fungi are lysed, which releases more protein and leads to increased thickening and potentially greater bioavailability of the nutrients. The lysis may be effected by any methods known in the art such as sonication.

Particles of the filamentous fungal cohesive biomass can be added as a protein or other nutritional source to augment the nutritional content of a foodstuff or can be, for example, the sole protein component. For foods composed entirely of filamentous fungal cohesive biomass, the size reduced particles can be optimized for particular textures, mouth feel, and chewiness. The ability to alter texture, mouth feel, and chewiness allow customization to accommodate individuals having particular dietary needs, such as those that have trouble chewing, or who require/desire softer foods while still providing the same nutritional and taste experience or those who desired food with more texture, more mouthfeel and more mastication. Because of the ability to easily control the particle size, foods augmented with filamentous fungal cohesive biomass or made solely from filamentous fungal cohesive biomass have textures very similar to the standard protein foods that they emulate.

Particles of the filamentous fungal cohesive biomass can be used as sole protein component in a food material or can be used to augment protein content of other food materials. Examples of foods that can be produced using only the reduced particle size of the filamentous fungal cohesive biomass, with or without added flavorings, include without limitation meat-like vegetarian or vegan products (e.g., ground beef, ground chicken, ground turkey, chicken nuggets, fish sticks or patties, jerky).

Foods augmented with the reduced particle size of the filamentous fungal cohesive biomass can significantly increase the protein content, which is particularly important for individuals following a vegan diet. For example, the protein contents of soups, drinks or smoothies may be augmented by the addition of a liquid dispersion of particles of fungal mycelial biomass, such as, by way of non-limiting example, particles of Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698).

Whether cohesive biomass particles of reduced size are used to augment the protein content of food or is used as the sole protein component, in some instances binders are helpful in achieving the desired texture. Approved foodstuff binders are envisaged, such as egg albumen, gluten, chickpea flour, vegetarian binders, arrowroot, gelatin, pectin, guar gum, carrageenan, xanthan gum, whey, chick pea water, ground flax seeds, egg replacer, flour, agar-agar, Chia seeds, psyllium, etc. which can be used singularly or in combination. In addition to foodstuff binders, the reduced particle size of the fdamentous fungal biomass can also be mixed with approved flavors, spices, flavor enhancers, fats, fat replacers, preservatives, sweeteners, color additives, nutrients, emulsifiers, stabilizers, thickeners, pH control agents, acidulants, leavening agents, anti-caking agents, humectants, yeast nutrients, dough strengtheners, dough conditioners, firming agents, enzyme preparations, gasses, and combinations thereof. Typically, binders, flavors, spices, etc. are selected to meet the demands of a particular population. For example, milk and/or milk solids are not used to accommodate individuals with dairy allergies/sensitivities, wheat flour may not be used to accommodate those with gluten allergies/sensitivities, etc.

In some applications, a single type of reduced particle size filamentous fungal cohesive biomass can be used or a variety of reduced particle sizes. Similarly, the reduced particle sizes can be from a single source of filamentous fungal cohesive biomass or from a combination of different sources; e.g. Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698) alone or Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698) and Fusarium venenatum, or Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698) and Fusarium venenatum and Giant Puffball biomats, etc. In some cases, the filamentous fungal cohesive biomass can be used as a source of oil, for example, truffle oil produced from surface fermentation edible fungal biomats of Tuber species.

Aspects of the present disclosure are further described by way of the following nonlimiting, illustrative experimental Examples. Example 1:

Colorimetry of Fungal Burger Analog Materials

Fourteen burger analog fungal food products were made by combining various non-fungal ingredients with (1) diced 1 mm pieces of surface fermentation-derived cohesive mycelial biomass of Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), (2) a “dough” of the same strain produced by submerged fermentation, and/or (3) particles of the same strain obtained via submerged fermentation and then spray-dried to produce a “flour,” in the amounts shown in Tables 1A and IB below. All values given are weight percentages of the overall burger composition before cooking.

Table 1A

Table IB

The procedure for making each of the burger analog materials was identical. For each burger material, all “dry” non-fungal ingredients, except for sodium chloride and Arcon U118 soy protein, were mixed together, and all “wet” non-fungal ingredients were separately mixed together. The salt was sprinkled over the fungal biomass and mixed into the biomass with a spatula. The Arcon soy protein was then mixed into to the fungal biomass/salt mixture, and this mixture was allowed to hydrate for 30 minutes. After hydration, the dry ingredients were mixed into the fungal/salt/ Arcon mixture in a stand mixer until the combined mixture texturally resembled ground beef crumbles. To these crumbles, the wet ingredients were added and mixed well; the combined mixture was then allowed to hydrate for a further 30 minutes and finally formed into “slider patties” (50 g) and/or “burger patties” (100 g).

Samples of each burger material were analyzed in a colorimeter and Hunter Lab color values for each material in its “raw” form were obtained. Each material was then cooked to an internal temperature of 160 °F on a 300 °F pan and then allowed to rest for 1 hour, whereupon the colorimetry analysis was repeated for the “cooked” material. The raw and cooked color values of each burger material are given in Tables 2 and 3, respectively; the values given in Tables 2 and 3 are averages of analysis of three samples of each material.

Figure 1 is a photograph of the raw (topmost and bottommost rows) and cooked (central rows) burger materials. (In Figure 1, burger materials 1-7 are in that order, left to right, in the top two rows; burger materials 8-10 and 12 are in that order, left to right, as the four leftmost columns in the bottom two rows; burger materials 13 and 14 are in that order, left to right, as the two rightmost columns in the bottom two rows; and burger material 11 is not pictured. The burger material in the third column from the right in the bottom two rows of Figure 1 is not described in this Example.)

As Figure 1 illustrates, usage of 15 wt% heme protein solution (0.45 wt% total heme protein content) in the burger materials showed the darkest pink/brown color and maintained the dark color at all three levels (30 wt%, 45 wt% and 70 wt%) of fungal material usage. As would be expected, burger materials including 5 wt% heme protein solution (0.15 wt% total heme protein) were characterized by lighter pink colors when raw and lighter brown colors when cooked, and materials that included no heme were generally tan or beige when raw and yellow to light brown when cooked, regardless of the protein sources used.

Three competitive burger products — an 80% lean/20% fat beef hamburger patty, a Beyond™ plant-based burger analog patty, and an Impossible™ plant-based burger analog patty — were also obtained and cooked by the same procedures. Pre- and post-cooking Hunter Lab colorimetry data for these patties were also obtained and are presented in Tables 2 and 3, respectively. Table 2

Table 3

Visual observation of the burger materials before, during, and after cooking also revealed various patterns. In the burger materials that utilized heme and diced cohesive biomass pieces as the fungal mycelial biomass (IDs #4-11), the heme solution appeared to permeate substantially throughout the biomass, rather than merely coating the biomass on its surface; this conclusion is based on the fact that none of the patties made from these materials exuded a red liquid (the heme solution) during cooking. Although it is possible the heme solution was being absorbed by some of the “dry” ingredients rather than the fungal biomass, even the burger material that utilized 70 wt% cohesive biomass pieces and 15% heme solution (ID #10), and therefore had minimal other ingredients available to absorb the heme solution, yielded no red exudate, strongly suggesting that it is the fungal biomass itself that absorbed the heme solution. The burger material that utilized the submerged fermentation-derived “dough” (ID #12) exhibited similar heme solution absorption characteristics.

By contrast, the burger material in which the fungal mycelial biomass was a “flour” (i.e., fine spray-dried particles of submerged fermentation-derived biomass, ID #13) exhibited different absorption behavior. The biomass itself was observed to be extremely dry prior to mixing with other ingredients, and once mixed with other wet ingredients, it exhibited hydrophobic tendencies and strongly repelled the heme solution, with droplets of the heme solution forming on the surface of the biomass. As a result, the burger material of ID #13 was “spotted” and did not have a uniform color. In the material that utilized a mix of cohesive biomass pieces and flour (ID #14), it was possible to incorporate the heme solution more uniformly throughout the material, but the flour still demonstrated some degree of hydrophobicity that made absorption more challenging. These phenomena were investigated in more detail in Example 3 below.

As shown in these results, the fungal-based burgers compared favorably to the comparative examples in terms of color analysis, particularly when analyzed in a cooked state.

Example 2:

Sensory Evaluation of Fungal Burger Analog Materials

A panel of taste testers tasted each of the fourteen burger materials prepared in Example 1 after the patties were cooked. The qualitative sensory perceptions of the taste testers are recorded in Table 4 below. Table 4

As Table 4 illustrates, the negative control material (ID #2) had very minimal flavor besides plant protein off-notes. Burger materials that utilized 30 wt% diced cohesive biomass pieces as the protein source and included 5 wt% or 15 wt% heme protein solution (0.15 wt% or 0.45 wt% heme protein) (ID #5 and #8) produced off-flavors, such as a “marine” or seaweed taste, a iron-like tang, and burnt mushrooms; however, an overall umami flavor was still present. At a cohesive biomass piece usage level of 45 wt% and 5 or 15 wt% heme protein solution (ID #6, #9, and #11), the overall flavor profile achieved good balance and the panelists agreed that these materials were most similar to a meat/beef product. Usage of 70 wt% fungal cohesive biomass pieces (ID #7 and #10) resulted in increased off-flavors, such as marine-seaweed and earthy /dirty flavors, due to higher moisture levels within the product and, possibly, off-flavors within the cohesive biomass itself. The use of submerged fermentation-derived “dough” or “flour” with 5 wt% heme protein solution (ID #12, #13, and #14) imparted metallic and marine off-flavors, and, although the distribution of color in the materials that used a fungal “dough” was improved (see the results for ID #12 in Example 1) relative to the use of “flour,” the flavor of the heme protein was not apparent in this burger material. The materials that used a fungal “flour,” meanwhile (ID #13 and #14) exhibited a raw flour flavor and minimal or no savory or umami flavor; a slight grill note was detected in the 20 wt% flour burger (ID #13), but this was quickly masked by an bitter aftertaste. Overall, the panelists agreed that burger materials utilizing cohesive fungal biomass between the 30 wt% and 70 wt% at the 45 wt% level as the fungal mycelial material and 5 or 15 wt% of the heme protein solution (ID #6, #9, and #11) provided a desirable balance of umami and savory flavors and had largely acceptable characteristics for a burger analog food product.

Example 3 :

Water Holding Capacity of Fungal Mycelial Biomasses

Two of the fungal mycelial biomasses described in the preceding two Examples — diced pieces of cohesive filamentous fungal mycelial biomass obtained by a surface fermentation process, and a submerged fermentation-derived fungal “dough,” both of Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698) — were evaluated for their water holding capacity. Each biomass was soaked in five times its own mass of either water or a 3% solution of beef myoglobin in water, to determine whether the presence of the heme protein affected the ability of the fungal mycelial biomass to take up the solution. The biomass was then removed from the fluid and an 18 mm circular sample was cut from the biomass using the mouth of a 50 m centrifuge tube. The mass of the freshly cut mat was recorded. The biomass sample was then placed in the centrifuge tube on top of a filter paper and centrifuged at 4,000 rpm for 20 minutes, whereupon the mass of the biomass was recorded again. The water holding capacity was defined as the mass of the sample after centrifugation divided by the mass of the mat before centrifugation. The results are given in Table 5 below.

Table 5

The results above indicate that, although heme does not have a significant impact on the water holding capacity of a cohesive fungal mycelial biomass compared to pure water, it has a significant negative effect on the water holding capacity of a submerged fermentation-derived “dough,” as compared to pure water. This result suggests that cohesive fungal mycelial biomasses, e.g., surface fermentation-derived biomats, are better suited for use in certain food products according to the present disclosure than non-cohesive fungal mycelial biomasses. The present disclosure, in various aspects, embodiments, and configurations, includes components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various aspects, embodiments, configurations, sub-combinations, and subsets thereof. Those of skill in the art will understand how to make and use the various aspects, aspects, embodiments, and configurations, after understanding the present disclosure. The present disclosure, in various aspects, embodiments, and configurations, includes providing devices and processes in the absence of items not depicted and/or described herein or in various aspects, embodiments, and configurations hereof, including in the absence of such items as may have been used in previous devices or processes, e.g., for improving performance, achieving ease and\or reducing cost of implementation.

The foregoing discussion of the disclosure has been presented for purposes of illustration and description. The foregoing is not intended to limit the disclosure to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the disclosure are grouped together in one or more, aspects, embodiments, and configurations for the purpose of streamlining the disclosure. The features of the aspects, embodiments, and configurations of the disclosure may be combined in alternate aspects, embodiments, and configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the claimed disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed aspects, embodiments, and configurations. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate preferred embodiment of the disclosure.

Moreover, though the description of the disclosure has included description of one or more aspects, embodiments, or configurations and certain variations and modifications, other variations, combinations, and modifications are within the scope of the disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative aspects, embodiments, and configurations to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

1. A food product, comprising: a) edible fungal mycelial matter comprising one or more fungal proteins; and b) at least one heme protein that is exogenous to the edible fungal mycelial matter.

2. The food product of claim 1, wherein the food product comprises no meat or other animal-derived products.

3. The food product of claim 1 or claim 2, wherein, before, during, and/or after a cooking process, a sensory perception of the food product is substantially similar to a sensory perception of a raw, cooking, or cooked meat product.

4. The food product of claim 3, wherein the sensory perceptions are selected from the group consisting of visual perceptions, auditory perceptions, olfactory perceptions, tactile perceptions, and gustatory perceptions.

5. The food product of claim 4, wherein the sensory perceptions are visual perceptions, wherein the visual perception of the food product before the cooking process is substantially similar to a visual perception of a raw meat product.

6. The food product of claim 4 or claim 5, wherein the sensory perceptions are visual perceptions, wherein the visual perception of the food product during and/or after the cooking process is substantially similar to a visual perception of a cooking or cooked meat product.

7. The food product of any one of claims 3-6, wherein the meat product is a ground beef product.

8. The food product of any one of claims 3-7, wherein the cooking process comprises exposing the food product to a temperature of at least about 150 °C for about 3 to about 5 minutes.

9. The food product of any one of claims 1-8, wherein the at least one heme protein is a globin, a cytochrome, or a methemalbumin.

10. The food product of any one of claims 1-9, wherein at least one fungal protein is a textured fungal protein.

11. A food material, comprising a heme polypeptide and a fungal mycelial biomass of a filamentous fungus belonging to an order selected from the group consisting of Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the fungal mycelial biomass comprises greater than about 40 wt. % protein content and less than about 8 wt. % RNA content.

12. The food material of claim 11, wherein the heme polypeptide is produced by a yeast belonging to the order Saccharomycetales .

13. The food material of claim 11 or claim 12, wherein the filamentous fungus belongs to a family selected from the group consisting of Ustilaginaceae , Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae , Strophariaceae , Lycoperdaceae , Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae,Morchellaceae, Sparassidaceae, Nectriaceae and Cordycipitaceae .

14. The food material of any one of claims 11-13, wherein the filamentous fungus belongs to a species selected from the group consisting of Ustilago esculenta Hericululm erinaceus Polyporous squamosus Grifola fondosa Hypsizygus marmoreus Hypsizygus ulmarius Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus ostreatus var. columbinus Tuber borchii. Morchella esculenta, Morchella conica. Morchella importuna, Sparassis crispa, Fusarium venenatum, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698), Disciotis venosa, and Cordyceps militaris.

15. The food material of any one of claims 11-14, wherein the filamentous fungus is a Fusarium species.

16. The food material of any one of claims 11-15, wherein the filamentous fungus is Fusarium venenatum.

17. The food material of any one of claims 11-16, wherein the filamentous fungus is Fusarium strain flavolapis (ATCC Accession Deposit No. PTA-10698).

18. The food material of any one of claims 11-17, wherein at least one of the following is true:

(a) the filamentous fungus comprises greater than about 45 wt. % protein content; and

(b) the filamentous fungus comprises less than about 5 wt. % RNA content.

19. The food material of any one of claims 11-18, wherein the filamentous fungus comprises less than about 10 ppm of a mycotoxin selected from the group consisting of Alfatoxin Bl, Alfatoxin B2, Alfatoxin Gl, Alfatoxin G2, Fumonison Bl, Fumonison B2, Fumonison B3, Ochratoxin A, Nival enol, Deoxynivalenol, Acetyl deoxy nival enol, Fusarenon X, T-2 Toxin, HT- 2 Toxin, Neosolaniol, Diacetoxyscirpenol zearalenone, and any combinations thereof.

20. The food material of any one of claims 11-19, wherein the filamentous fungus comprises less than about 10 ppm total mycotoxin content.

21. The food material of claim 20, wherein the filamentous fungus comprises less than about 5 ppm total mycotoxin content.

22. The food material of any one of claims 11-21, wherein the filamentous fungus comprises greater than about 15 wt. % of branched chain amino acids.

23. The food material of any one of claims 11-22, wherein the food material is a meat analog food product.

24. The food material of any one of claims 11-23, wherein the food material is vegetarian.

25. The food material of claim 24, wherein the food material is vegan.

26. The food material of any one of claims 11-23, comprising meat, wherein the meat comprises a heme protein.

27. The food material of claim 26, wherein the heme protein that is exogenous to the edible fungal mycelial matter consists of, or consists essentially of, the heme protein of the meat.

28. The food material of claim 26, wherein the heme protein that is exogenous to the edible fungal mycelial matter comprises a heme protein exogenous to the meat.

29. The food material of any one of claims 11-28, wherein the heme polypeptide and one or more fungal proteins present in the filamentous fungus collectively comprise all essential amino acids.

30. The food material of claim 29, wherein the one or more fungal proteins present in the filamentous fungus comprise all essential amino acids.

31. The food material of any one of claims 11-30, wherein the filamentous fungus is nonviable.

32. A food composition, comprising: fungal mycelial biomass, in an amount from about 30 wt% to about 70 wt%; and a heme protein that is exogenous to the fungal mycelial biomass, in an amount from about 0.1 wt% to about 5 wt%.

33. The food composition of claim 32, wherein the fungal mycelial biomass is a cohesive fungal mycelial biomass.

34. The food composition of claim 33, wherein the fungal mycelial biomass is produced by a liquid surface fermentation process, a solid-state fermentation process, or a membrane fermentation process.

35. The food composition of claim 32, wherein the fungal mycelial biomass is produced by a submerged fermentation process.

36. The food composition of any one of claims 32-35, wherein the heme protein is dispersed in the fungal mycelial biomass.

37. The food composition of claim 36, wherein the food composition is produced by contacting a liquid dispersion of the heme protein in water with the fungal mycelial biomass, wherein the heme protein is dissolved, colloidally dispersed, or suspended in the water.

38. The food composition of claim 37, wherein a solubility of the heme protein in water is at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 50 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, or at least about 250 g/L.

39. The food composition of claim 37 or claim 38, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the heme protein is dissolved in the water of the liquid dispersion.

40. The food composition of any one of claims 36-39, wherein a mass of the liquid dispersion adsorbed by, adsorbed on a surface of, or coating the fungal mycelial biomass is from about 23 wt% to about 51 wt% of a mass of the fungal mycelial biomass in the absence of the liquid dispersion.

41. The food composition of any one of claims 32-40, wherein the fungal mycelial biomass is not spray-dried.

42. The food composition of any one of claims 32-40, wherein the fungal mycelial biomass is spray-dried.

43. The food composition of any one of claims 32-42, wherein the fungal mycelial biomass comprises at least one filamentous fungus belonging to an order selected from the group consisting of Ustilaginales Russulales Polyporales Agaricales , Pezizales and Hypocreales.

44. The food composition of claim 43, wherein the filamentous fungus belongs to a family selected from the group consisting of Ustilaginaceae Hericiaceae. Polyporaceae Grifolaceae Lyophyllaceae Strophariaceae , Lycoperdaceae , Agaricaceae Pleurotaceae Physalacriaceae, Omphalotaceae, Tuberaceae,Morchellaceae, Sparassidaceae, Nectriaceae, and Cordycipitacea .

45. The food composition of claim 44, wherein the filamentous fungus belongs to the genus Fusarium.

46. The food composition of claim 44, wherein the filamentous fungus belongs to a species selected from the group consisting of Ustilago esculenta Hericululm erinaceus Polyporous squamosus, Grifola fondosa, Hypsizygus marmoreus, Hypsizygus ulmarius, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii Pleurotus oslrealus. Pleurotus ostreatus var. columbinus, Tuber borchii. Morchella esculenta Morchella conica, Morchella importuna Sparassis crispa, Fusarium venenatum, Fusarium strain flavolapis (ATCC Accession Deposit No. PTA- 10698), Disciotis venosa. and Cordyceps militaris.

47. The food composition of any one of claims 32-46, wherein the fungal mycelial biomass comprises from about 35 wt% to about 60 wt% protein on a dry basis.

48. The food composition of any one of claims 32-47, wherein the fungal mycelial biomass comprises from about 25 wt% to about 55 wt% carbohydrates on a dry basis.

49. The food composition of any one of claims 32-48, wherein the fungal mycelial biomass comprises from about 20 wt% to about 40 wt% dietary fiber on a dry basis.

50. The food composition of any one of claims 32-49, wherein the fungal mycelial biomass comprises from about 2.3 wt% to about 7.0 wt% lipids on a dry basis.

51. The food composition of any one of claims 32-50, wherein at least one of the following is true:

(i) the fungal mycelial biomass comprises from about 1.2 wt% to about 2.6 wt% cis,cis- polyunsaturated fatty acids on a dry basis;

(ii) the fungal mycelial biomass comprises from about 0.1 wt% to about 0.6 wt% cis- monounsaturated fatty acids on a dry basis;

(iii) the fungal mycelial biomass comprises from about 0.6 wt% to about 1.0 wt% saturated fatty acids on a dry basis; and

(iv) the fungal mycelial biomass is substantially free of trans fatty acids.

52. The food composition of any one of claims 32-51, wherein the food composition is a meat analog.

53. The food composition of any one of claims 32-52, wherein the food composition is selected from the group consisting of an animal feed, a pet food, and an aquaculture feed.

54. The food composition of claim 52, wherein the food composition is selected from the group consisting of a hot dog analog, a burger analog, a ground meat analog, a sausage analog, a steak analog, a filet analog, a roast analog, a meatball analog, a meatloaf analog, and a bacon analog.

55. The food composition of any one of claims 32-54, wherein the heme protein is selected from the group consisting of androglobin, cytoglobin, globin E, globin X, globin Y, hemoglobin, myoglobin, leghemoglobin, erythrocruorin, beta hemoglobin, alpha hemoglobin, non-symbiotic hemoglobin, flavohemoglobin, protoglobin, cyanoglobin, Hell’s gate globin I, bacterial hemoglobin, ciliate myoglobin, histoglobin, neuroglobins, truncated 2/2 globin, HbN, HbO, Glb3, a heme peroxidase, a heme ligninase, a heme cytochrome, a heme oxidoreductase or catalase, and combinations thereof.

56. The food composition of any one of claims 32-55, further comprising one or more non-heme proteins exogenous to the fungal mycelial biomass.

57. The food composition of claim 56, wherein the one or more non-heme proteins are derived from a vegetarian source.

58. The food composition of claim 57, wherein the vegetarian source is a vegan source.

59. The food composition of claim 58, wherein the one or more non-heme proteins are selected from the group consisting of seed proteins, legume proteins, tuber proteins, and combinations thereof.

60. The food composition of claim 59, wherein the one or more non-heme proteins are selected from the group consisting of pea proteins, potato proteins, soy proteins, and combinations thereof.

61. The food composition of any one of claims 56-60, wherein the one or more non- heme proteins are present in an amount from about 6.5 wt% to about 33.5 wt% of the food composition.

62. The food composition of any one of claims 32-61 , further comprising carbohydrates exogenous to the fungal mycelial biomass and selected from the group consisting of starch, dietary fiber, and combinations thereof.

63. The food composition of claim 62, wherein the carbohydrates are present in an amount from about 0.1 wt% to about 10 wt% of the food composition.

64. The food composition of any one of claims 32-63, further comprising at least one binder or gelling agent.

65. The food composition of claim 64, wherein the at least one binder or gelling agent is selected from the group consisting of methyl cellulose, hydrocolloids, carrageenans, calcium chloride, and combinations thereof.

66. The food composition of claim 64 or claim 65, wherein the at least one binder or gelling agent is present in an amount from about 0.1 wt% to about 10 wt% of the food composition.

67. The food composition of any one of claims 32-66, further comprising at least one flavor, spice, or seasoning.

68. The food composition of claim 67, wherein the at least one flavor, spice, or seasoning is selected from the group consisting of sodium chloride, a natural meat flavor additive, an artificial meat flavor additive, and combinations thereof.

69. The food composition of claim 67 or claim 68, wherein the at least one flavor, spice, or seasoning is present in an amount from about 0.1 wt% to about 5 wt% of the food composition.

70. The food composition of any one of claims 32-69, further comprising at least one food coloring.

71. The food composition of claim 70, wherein the at least one food coloring is present in an amount from about 0.01 wt% to about 1 wt% of the food composition.

72. The food composition of any one of claims 32-71, further comprising at least one cooking fat or oil.

73. The food composition of claim 72, wherein the at least one cooking fat or oil is selected from the group consisting of sunflower oil, coconut oil, and combinations thereof.

74. The food composition of claim 72 or claim 73, wherein the at least one cooking fat or oil is present in an amount from about 1 wt% to about 15 wt% of the food composition.

75. The food composition of any one of claims 32-74, wherein at least one of the following is true when the food composition is raw or uncooked: (i) a Hunter L color value of the food composition is from about 44 to about 66;

(ii) a Hunter a color value of the food composition is from about 5 to about 19; and

(iii) a Hunter b color value of the food composition is from about 15 to about 24.

76. The food composition of claim 75, wherein at least two of (i), (ii), and (iii) are true.

77. The food composition of claim 76, wherein all three of (i), (ii), and (iii) are true.

78. The food composition of any one of claims 32-77, wherein at least one of the following is true one hour after the food composition is cooked to an internal temperature of 160 °F:

(i) a Hunter L color value of the food composition is from about 34 to about 53;

(ii) a Hunter a value of the food composition is from about 7 to about 13; and

(iii) a Hunter b value of the food composition is from about 14 to about 26.

79. The food composition of claim 78, wherein at least two of (i), (ii), and (iii) are true.

80. The food composition of claim 79, wherein all three of (i), (ii), and (iii) are true.

81. A method for making the food composition of any one of claims 32-80, comprising: contacting a cohesive fungal mycelial biomass with a proteinaceous composition comprising at least one heme protein, whereby at least a portion of the proteinaceous composition is absorbed by, adsorbs onto a surface of, or coats the cohesive fungal mycelial biomass.

82. The method of claim 81, wherein the proteinaceous composition is a liquid dispersion of the heme protein.

83. The method of claim 82, wherein the liquid dispersion comprises the heme protein and water, wherein the heme protein is dissolved, colloidally dispersed, or suspended in the water.

84. The method of claim 83, wherein a solubility of the heme protein in water is at least about 5 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 50 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, or at least about 250 g/L.

85. The method of claim 83 or claim 84, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the heme protein is dissolved in the water of the liquid dispersion.

86. The method of any one of claims 83-85, wherein a mass of the portion of the liquid dispersion adsorbed by, adsorbed on a surface of, or coating the fungal mycelial biomass is from about 23 wt% to about 51 wt% of a mass of the fungal mycelial biomass in the absence of the liquid dispersion.

87. The method of claim 81, wherein the proteinaceous composition is a powder or an aerosol.