WO2025072350A1

WO2025072350A1 - Fungal food products and methods of production

Info

Publication number: WO2025072350A1
Application number: PCT/US2024/048436
Authority: WO
Inventors: Debbie Yaver; Seth D'IMPERIO
Original assignee: Fynder Group Inc
Current assignee: Fynder Group Inc
Priority date: 2023-09-29
Filing date: 2024-09-25
Publication date: 2025-04-03
Anticipated expiration: 2026-03-29

Abstract

Disclosed herein are fungal food compositions derived from mycelial biomass produced from fungi of the genus Cunninghamella, Rhizopus, and/or Monascus, and methods of making the same. Methods of making suitable feedstocks for growing the mycelial biomass are also provided, as are methods of making fungal inocula from fungi of the genus Cunninghamella, Rhizopus, and/or Monascus.

Description

FUNGAL FOOD PRODUCTS AND METHODS OF PRODUCTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisional patent application 63/586,938, filed 29 September 2023, the entirety of which is hereby incorporated by reference in its entirety, for all purposes.

BACKGROUND

There are many challenges facing rural and low-income populations with respect to food and income security in areas around the globe. Lack of agricultural infrastructure and environmental conditions hinder the production of nutritious sources of food, and the effects of global warming make crop production even more difficult. This contributes to low access to nutritious food and also has economic impacts on farmers and others who would like to grow food. Additionally, in emergency situations, for example in the wake of a natural disaster, readily available sources of nutritious food can be difficult to come by. A lack of nutritious food can contribute to the mortality rate associated with emergency situations.

There is a need to increase income opportunities for small scale producers by leveraging their crop inputs in local protein production via decentralized fermentation, such as with a modular mycelial protein production technology that can be deployed quickly to produce fermented proteins using locally available crops and materials as feedstocks.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a block diagram depicting processes for producing biomass according to the present disclosure.

Figure 2 provides block diagrams depicting processes for preparing feedstocks from rice (FIG. 2A) and potato (FIG. 2B) according to the present disclosure.

Figure 3 shows a graph (FIG. 3A) and pictures (FIGs 3B and 3C) with characteristics of mycelial biomats grown at low pH.

Figure 4 shows the amino acid content of fungi grown on rice and potato according to embodiments of the disclosed methods.

Figure 5 shows biomats grown in fermentation equipment that have been treated with varying sanitation techniques.

Figure 6 shows biomats and food compositions containing fungus grown on rice feedstocks. FIGs 6A, 6B, and 6C are photographs of biomats grown on high viscosity rice porridge. FIG. 6D is a photograph of biomats grown on low viscosity rice medium. FIGs. 6E and 6F are photographs showing the food compositions containing fungus grown on high viscosity rice porridge. Figure 7 shows the biomass and total protein content of fungus grown on media composed largely of rice, potato, and a combination of rice and potato, via methods provided by the present disclosure.

Figure 8 depicts how variance in viscosity of a feedstock can change the overall yield of a resulting biomass, as described in Example 9.

Figure 9 depicts how variance in inoculum type (wet vs. dry) can change the overall yield of a resulting biomass, as described in Example 10.

Figure 10 depicts the particle size distribution of ground, de-hulled paddy rice.

Figure 11 provides graphs depicting the effect of urea and DAP addition on final protein content of fermented rice according to some embodiments provided by the present disclosure. Maxima are indicated by dashed lines.

DETAILED DESCRIPTION

The current disclosure provides fungal food compositions, and methods of making the same, that are fast to produce and require little infrastructure. The infrastructure can be established anywhere in the world with minimal effort and upkeep, and the disclosed methods are designed specifically to utilize local materials, such as grains or similar crops, as feedstocks suitable to support the growth of the disclosed fungi. Generally speaking, the present disclosure provides information that will empower people to utilize their own local crops and materials to quickly and efficiently produce highly nutritious food products, without the need to build and maintain large food production facilities. The disclosed methods and compositions are capable of producing clean and nutritious food products that are suitable for human consumption, either on their own or as a component or ingredient in other food products. By utilizing local crops and materials as feedstocks, the present disclosure also provides ways for people to reduce or eliminate food waste while maintaining a nutritious, balanced diet. The disclosed methods and equipment are also simple, but elegantly so in that they are capable of effective use by a single person, or by a group of people.

As used herein, the phrase "mycelial biomass" refers to a biomass of filamentous fungus in which fungal mycelium makes up about 30 wt% - about 100 wt% of the biomass. In embodiments, fungal mycelium makes up at least about 50 wt%, at least about 55 wt%, at least about 60 wt%, at least about 65 wt%, at least about 70 wt%, at least about 75 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt%, at least about 96 wt%, at least about 97 wt%, at least about 98 wt%, at least about 99 wt%, or substantially all of the biomass. The remainder (/.e., the non-mycelial portion) of the mycelial biomass may contain other fungal tissues (conidia, fruiting bodies, etc.). "Mycelial biomass" specifically includes biomass produced by any fermentation method and thus encompasses biomass produced by submerged fermentation methods and non-submerged fermentation methods. In some embodiments, a mycelial biomass is produced by any one or more submerged fermentation methods known in the art, such as, for example, those described in U.S. Patent 7,635,492 to Finnigan et al.. In other embodiments, a mycelial biomass is a "cohesive mycelial biomass" produced by a fermentation process other than a submerged fermentation process. A "cohesive mycelial biomass" has sufficient tensile strength and structural integrity to be picked up and moved by hand without disintegrating or tearing and is produced by any one or more fungal fermentation methods in which the filamentous fungus grows in such a way as to form a mass of interwoven mycelia, e.g., methods in which a fungal mycelium is grown in air or a controlled atmosphere out of a growth medium or feedstock (e.g., liquid surface fermentation, fermentation on the surface of a membrane or mesh scaffold, solid substrate fermentations in which mycelial biomass grows free of solid substrate, etc.). Examples of methods of producing a "cohesive mycelial biomass" are described in PCT Application Publications 2020/176758, 2019/099474, and 2018/014004. Generally speaking, mycelial biomasses recovered from submerged fermentation processes are paste-like substances with poor tensile strength and structural integrity and thus are not cohesive mycelial biomasses.

In that regard, in one aspect, the current disclosure provides methods that allow one or more individuals to culture filamentous fungus and produce food products with minimal required space, effort, time, and/or infrastructure. Feedstocks can be placed directly on the ground, in a housing or structure, or both. Feedstocks are contacted with filamentous fungal mycelium and cultured in a manner to allow the fungus to grow on, in, and/or around the feedstock producing a composition that comprises both feedstock and mycelial biomass. The disclosed fungal strains and species are capable of growing at high temperature and/or at low pH, which facilitates fungal growth in high temperature environments and also minimizes the risk of contamination. Methods for inoculating and culturing filamentous fungus on, in, and/or around solid and/or semi-solid feedstocks are disclosed herein. Feedstocks and methods for preparing feedstocks are also provided herein. In some embodiments, the feedstock is edible. In some embodiments, the filamentous fungus acts on a feedstock in order to improve its edibility (/.e., its fitness for consumption as food by humans or animals) and/or nutritional content. Food compositions and products produced from the disclosed fungi and feedstocks are also provided herein. In some embodiments, the food compositions and products contain mycelial biomass and edible feedstock, and in some embodiments the compositions and products do not include the feedstocks.

An advantage of the currently disclosed methods, and the resulting compositions, is that they require little to no infrastructure. No irrigation or sunlight is required. No special containers are required (though containers may be used if desired), and the fungus can be grown with feedstock placed directly on the ground or placed in a simple container, for example a tray, tub, or the like. A container, for example a tray, can be covered with a lid or utilized without one. A building is not required, but in embodiments the fungus can be grown in a building. The building can be a simple one with just walls and/or a roof, or more complex, e.g., containing fans, climate controls, and the like. What little equipment is needed is also simple to clean and maintain. This permits one or more individuals to produce fungal food products with improved nutritional value for animal consumption, human consumption, and/or for use in aquaculture without the cost and/or inconvenience of providing significant infrastructure. In some embodiments, the fungal food products also demonstrate improved bioavailability.

Useful Environments

The disclosed methods, and resulting compositions, are well suited to a variety of global environments. For example, the disclosed methods can be readily adapted for use in temperate forests, tropical rain forests, deserts, grasslands, the taiga, the tundra, the chaparral, the plains, and so on. As disclosed in detail herein, the methods, and the fungi utilized in them, are capable of use at high or low temperature, in high humidity environments, in arid environments, in areas with plentiful sunlight, and in areas without regular sunlight. Many low to middle income countries often lack the infrastructure required to grow and produce a variety of types of food. For example, many such countries may lack proper irrigation, farmland, farm equipment and machinery, distribution systems, and so on. The disclosed methods, and resulting compositions, overcome many of these deficiencies. The present disclosure provides methods that produce fungal food that can be grown, for example, without irrigation, outside of dedicated farmland, without the need for farm equipment or machinery, and that can be grown in the dark.

In some embodiments, the fungal mycelium grows into and/or around one or more edible feedstocks. This provides food that is ready to eat or requires little or no preparation prior to use itself as a food product or as a food composition component of a food product. In some embodiments, the fungal mycelium grows separately from, for example on top of, one or more edible feedstocks. The feedstock is then separated from the fungus after fermentation, to provide food that is ready to eat or requires little or no further preparation prior to use itself as a food product, or as a food composition, or as a component of a food product. In some embodiments, the fungal mycelium grows separately from one or more edible feedstocks and then the feedstock and fungus are combined after fermentation in order to provide food that is ready to eat or requires little or no further preparation prior to use itself as a food product, or as a food composition, or as a component of a food product. The disclosed methods and compositions are well suited for use in conditions and/or environments that are not tightly controlled, such as modern farms, and may be implemented and used in environments that are nomadic in nature, such as military or emergency healthcare installments.

Additionally, the disclosed methods and compositions are suitable for use in containerized systems. The container systems can be utilized outdoors or indoors, much like container gardening, in order to culture the disclosed fungi. In embodiments, containerized systems can have climate control features that assist with culturing filamentous fungus. Climate control can include regulation of temperature, air content (nitrogen, oxygen, carbon dioxide, and/or other components of air), air flow, humidity, and/or similar characteristics. For example, control of carbon dioxide levels (e.g., reduction or elimination) can help prevent fruiting body formation for fungi that fruit. One or more containerized systems can be used in a building, or a building itself can be a containerized system.

Methods

The current disclosure provides, inter alia, methods of making a mycelial biomass.

In one aspect, the methods include contacting an edible feedstock with an edible filamentous fungus and culturing the fungus and feedstock together (e.g., via fermentation), thereby producing the mycelial biomass. Fungi suitable for use in the disclosed methods meet one or more of the inclusion criteria disclosed herein, which include the capability of growth at high temperature, the capability of growth at low pH, and the inability to produce one or more mycotoxins at a detectable level (for example, via the use of a commercially available assay or test kit). In some embodiments, the fungus used meets all three inclusion criteria. In some embodiments, culturing includes growth of the biomass in such a way that it is separated from the feedstock as it grows and readily removed therefrom.

In other embodiments, culturing includes growth of the biomass such that it infiltrates the feedstock and produces an edible biomass-feedstock amalgamation that is suitable for production of a food composition. In some of these embodiments, the resulting amalgamation of biomass and feedstock is a cohesive mycelial biomass. In that regard, fungi that meet the inclusion criteria provided herein are capable of converting a non-cohesive system such as a fermentation system containing a feedstock that, in some embodiments, is liquid or viscous, into a cohesive system - a cohesive mycelial biomass.

As is evident from the above, the feedstock is preferably edible/suitable for consumption by both humans and animals/aquaculture. For example, the feedstock can be produced from grains, tubers, legumes, or similar crops, or combinations of any of the foregoing. In some embodiments, the feedstock can be produced from one or more secondary products, byproducts, and/or waste streams derived from primary food production processes. In some embodiments, the feedstock can be produced from overgrowth that is not sold or eaten, and/or would otherwise become food waste.

As per this aspect, the resulting biomass and/or biomass-feedstock amalgamation is edible. It can be eaten on its own, as a rich protein source with a favorable nutritional profile, or it can be used as a component in the production of other food products. The disclosed methods produce biomass quickly and efficiently.

In another aspect, the present disclosure provides methods of making food compositions from mycelial biomass, where the biomass is produced as described herein. After growth, the biomass can be used as a component in the generation of numerous food products (see below). The biomass can be used directly, or it can be subjected to one or more post-growth processing techniques that make it more suitable for a particular application. For example, the biomass may be inactivated, size reduced, separated from any remaining feedstock and/or growth media (if desired), dehydrated, made into a colloid, made into a liquid dispersion, dried, or any combination of the foregoing, all as described in detail herein. In some embodiments, the biomass is cohesive, such that it can be lifted with one hand and moved, without tearing. In other embodiments, the biomass is less structured. Either way, biomass produced by the present disclosure is suitable for use in the production of food products. Additionally, given that the biomass can be produced in suitable quantities for the production of food products in as little as 1-2 days, the disclosed methods provide means for aid workers to quickly and inexpensively produce nutritious food in an area in emergency situations, as well as means for individuals to produce nutritious food for their families and communities quickly and easily, and means for people to grow food in challenging environmental conditions, among other things.

As provided in greater detail herein, food products produced from the disclosed biomass can include a milk analog that can used in dairy applications, a mycelial flour, a tempeh analog, a meat analog, a tofu analog, and/or textured vegetable protein (e.g., soy) analog.

In a third aspect, the current disclosure provides methods of preparing a fungal inoculum suitable for use in the disclosed methods. Such methods can include, for example, preparing a feedstock by wetting and/or boiling it to ensure that it is both sterile and a suitable environment to initiate the growth of one or more fungi. Thereafter the feedstock is contacted with a fungal culture that meets the inclusion criteria disclosed herein, which is then fermented to produce a fungal biomass. Once a desired amount of fungal biomass is produced, it is dehydrated, dried, and/or frozen before it is size-reduced for storage. Dehydration and/or drying can occur, for example, via spray drying, baking, air drying or other suitable means as disclosed herein. Size-reducing can occur in a variety of ways, for example by grinding, chopping, tearing, or the like. In this method, the biomass is not inactivated, but is instead placed into a state of dormancy so that it will reactivate later, once inoculated into a feedstock for the production of biomass as described herein. Once the foregoing is completed, the inoculum is shelf stable and capable of long-term storage. To reactivate it, it simply needs to be brought into contact with a disclosed feedstock and cultured, as described herein.

Inclusion Criteria

Fungal species that are capable of satisfying the following inclusion criteria are suitable for use in the disclosed methods. That is, in various aspects, fungal strains having certain characteristics, or demonstrating certain properties, are suitable for use in the disclosed methods. In various embodiments, fungal strains suitable for use in the disclosed methods, and thus for the production of the disclosed compositions, may be capable of growing into a mycelial biomass at high temperature, at low pH, may not produce one or more mycotoxins, and/or may grow on a grain- or tuber-based feedstock (e.g., a rice- or potato-based feedstock). In some embodiments, the fungal strains may grow as a cohesive mycelial biomass.

Temperature

In some embodiments, fungal strains suitable for use in the disclosed methods are capable of growing at high temperature. This minimizes, if not eliminates, the need for external cooling sources during the fermentation process, reducing the amount of infrastructure required to practice the disclosed methods. In some embodiments, fungal growth occurs at a temperature of 30 - 75 °C, 30 - 70 °C, 30 - 60 °C, 30 - 50 °C, 30 - 45 °C, 30 - 40 °C, or any subrange within any of the foregoing.

In some embodiments, fungal strains suitable for use in the disclosed methods are capable of growing (via fermentation) at low pH. This minimizes risk of contamination by other microorganisms during fermentation and also reduces the materials and effort required for food production. Growth at low pH also minimizes the risk of contamination in the resulting food compositions and/or products. In some embodiments, fungal growth occurs at a pH of 1.0 - 7.0, 1.0 - 6.0, 1.0 - 5.0, 2.0 - 6.0, 2.0 - 5.0, 2.0 - 4.0, or any subrange within any of the foregoing.

Mycotoxins

In some embodiments, fungal strains suitable for use in the disclosed methods present minimal safety concerns, for example by producing little to no mycotoxins, and/or by not exhibiting any properties of pathogenic fungal organisms. In some embodiments, the fungal strains do not produce mycotoxins or produce one or more of them at levels low enough to be considered safe for consumption. In some embodiments, the mycotoxins are selected from an aflatoxin, a fumonisin, an ochratoxin, a deoxynivalenol, an acetyldeoxynivalenol, a fusarenon, a nivalenol, a T- 2 toxin, an HT-2 toxin, a neosolaniol, a diacetoxyscirpenol, a zearalenone. In some embodiments, the aflatoxin is aflatoxin Bl, aflatoxin B2, aflatoxin Gl, aflatoxin G2, and combinations thereof. In some embodiments, the fumonisin is selected from fumonisin Bl, fumonisin B2, fumonisin B3, and combinations thereof. In some embodiments, the ochratoxin is ochratoxin A. In some embodiments, the fusarenon is fusarenon X.

In embodiments, the fungal strains produce very low levels of one or more mycotoxins, each of which independently measuring at an amount of 0.1 ppb - 2.0 ppb, 0.1 ppb - 1.7 ppb, or 0.1 ppb - 1.5 ppb. In some embodiments, the amount of a mycotoxin produced by a mycelial biomass is undetectable. In some embodiments, the fungus does not produce any mycotoxins.

In some embodiments, the fungus does not cause disease at all and/or does not cause disease when subjected to the growth conditions disclosed herein. For example, the fungus does not grow and/or reproduce in and/or on an animal (e.g., a human), the fungus does not produce unsafe levels of a mycotoxin (e.g., when grown under conditions disclosed herein) or does not produce mycotoxins at all, and/or is not unsafe to individuals with reduced immune activity (e.g., individuals with one or more immune disorders and/or affected by immune-suppressing treatment).

Fungal strains

Strains that meet the criteria above are suitable for use in the disclosed methods and as part of the disclosed compositions.

Fungi of the genus Cunninghamella are frequently used to study drug metabolism but have not been described as a food composition or food product. The present disclosure changes that and affirmatively discloses the use of fungi of the genus Cunninghamella, in the disclosed methods, as compositions suitable for use as food on their own and/or in the production of food products. Three Cunninghamella species were analyzed in simple, easily performed, and non-time consuming assays of fungal growth at high temperature and low pH: Cunninghamella blakesleeana (strain NF4), Cunninghamella echinulate (strain NF30, NRRL No. 1387 provided by the USDA-ARS Culture Collection (NRRL)), and Cunninghamella elegans (strain NF31, NRRL No. 2310 provided by the USDA-ARS Culture Collection (NRRL)). All three strains produced biomass at high yield under low pH conditions, and one Cunninghamella echinulate (strain NF30) was even able to produce biomass at high yield at a pH of 3. All three species were able to be successfully used to produce food and/or food products. Cunninghamella species are thus excellent fungi for use the production of food and food products.

Fungi of the genera Rhizopus and Monascus are also suitable for such uses. The filamentous fungi can also be selected from the phyla or divisions zygomycota, glomermycota, chytridiomycota, basidiomycota or ascomycota. The phylum (or division) basidiomycota comprises, inter alia, the orders Agaricales, Russulales, Polyporales and Ustilaginales; the phylum ascomycota comprises, inter alia, the orders Pezizales and Hypocreales; and the phylum zygomycota comprises, inter alia, the order Mucorales. The edible filamentous fungi may belong to an order selected from Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, Hypocreales and Mucorales.

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 Mucorales are selected from the family Mucoraceae.

In some embodiments, the filamentous fungi may be selected from the genera Fusarium, Aspergillus, Trichoderma, Rhizopus, Ustilago, Hericululm, Polyporous, Grifola, Hypsizygus, Calocybe, Pholiota, Calvatia, Stropharia, Agaricus, Hypholoma, Pleurotus, Morchella, Sparassis, Disciotis, Cordyceps, Ganoderma, Flammulina, Lentinula, Ophiocordyceps, Trametes, Ceriporia, Leucoagaricus, Handkea, Monascus and Neurospora.

Examples of the species of filamentous fungi include, without limitation, Ustilago esculenta, Hericululm erinaceus, Polyporous sguamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmariuos (elm oyster) Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (pearl), Pleurotus ostreatus van col um bin us (Blue oyster), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (cauliflower), Fusarium venenatum, Disciotis venosa, Cordyceps militaris, Ganoderma lucidum (reishi), Flammulina velutipes, Lentinula edodes, Ophiocordyceps sinensis. Additional examples include, without limitation, Trametes versicolor, Ceriporia lacerate, Pholiota gigantea, Leucoagaricus holosericeus, Pleurotus djamor, Calvatia fragilis, Handkea utriformis, Rhizopus oligosporus, and Neurospora crassa.

Feedstocks

Source materials A variety of feedstocks produced from different source materials can be used in the currently disclosed methods and compositions. Generally speaking, suitable feedstocks are edible on their own and/or the growth of the fungus on the feedstock can improve their edibility and/or nutritional value. In one aspect, the fungus grows with and into the feedstock, incorporating the feedstock into a final composition. In another aspect, the feedstock is readily separable from the mycelial biomass and does not form a part of a final food composition.

Suitable feedstocks for use as described herein can be or be prepared from readily available substances, including grains and/or tubers. In some embodiments the feedstocks are prepared from grains, for example a cereal, a legume, or combinations thereof. Suitable cereals can include, for example, rice, wheat, rye, oats, barley, millet, maize (e.g., corn), sorghum, and the like. Broken rice or other byproducts of rice packaging and processing, unbroken rice, or a combination thereof can also be suitable cereals. Suitable legumes can include, for example, beans, soybeans, chickpeas, peanuts, lentils, lupins, grass peas, mesquite, carob, tamarind, alfalfa, clover, and the like. Suitable tubers can include, for example, potatoes, yams, sweet potatoes, cassava (including sweet cassava), dahlias, and the like. In some embodiments, the grain is rice and/or the tuber is potato. In various embodiments, the feedstock can be produced from a single source material, for example a single grain or a single tuber.

In other embodiments, suitable feedstocks can be prepared from a mixture of any of the foregoing source materials. In some embodiments, more than one source material is used to produce the feedstock. Using more than one source material to produce the feedstock can be quite beneficial in the disclosed methods. For example, using more than one source material for producing the feedstock can positively affect fungal growth (e.g., biomass production), and/or can help increase the protein content of the fungus in the resulting compositions or products. In some embodiments, a combination of one or more grains and one or more tubers produces surprising effects, e.g., by increasing fungal growth (e.g., biomass production) and/or protein content. In some embodiments, using a combination of rice and potato as a feedstock surprisingly increases fungal growth (e.g., biomass production), making the fungal strain a high yield strain, and increases the protein content of the fungal strain. In some embodiments, using a combination of rice and potato as a feedstock to grow fungi of the genus Cunninghamella surprisingly increases growth of the fungi of this genus (e.g., biomass production), making the Cunninghamella high yield, and increases the protein content of the Cunninghamella fungus.

Pretreatment

Source materials can be pretreated prior to use as a feedstock. Such pretreatment can improve edibility, ease of digestion, nutritional content, biomass production, and/or other characteristics. For example, in some embodiments source materials can be wetted. In some embodiments, rice and/or paddy rice is wetted prior to its use as a feedstock. In some embodiments, source materials can be processed to produce a porridge, e.g., a rice porridge with water and/or one or more other useful liquids. In some embodiments, rice is processed to produce a porridge. In some embodiments, paddy rice is the source material, which can be wetted and/or processed to produce a porridge. In some embodiments, source materials can be processed to remove a husk, a seed coat, and/or a seedling. In some embodiments, source materials can be mashed, chopped, and/or shredded. For example, a tuber can be mashed, e.g., a mashed potato. In some embodiments, dry grain source materials can be pretreated by grinding, sieving to remove fines, boiling to reduce contaminants, and the like.

Use

When in use as a means of producing mycelial biomass, feedstocks can be dry, maintained in saturated aqueous media, supplemented with a nitrogen source, for example urea, potassium nitrate, yeast extract, or one or more other nitrogen sources, or combinations of the foregoing.

Additionally, the pH of the feedstock can be optionally reduced during use in order to reduce the possibility of contamination with one or more microorganisms.

In some embodiments, one or more grains are ground, sieved to remove fines, boiled, and maintained in saturated aqueous media supplemented with a nitrogen source when used as a feedstock to produce a mycelial biomass. In such embodiments, the pH may also be optionally reduced, for example to a pH of between about 3 and about 6. In some embodiments, rice is ground, sieved to remove fines, boiled, and maintained in saturated aqueous media supplemented with a nitrogen source, and the pH is adjusted, for example to a pH of between about 3 and about 6. Such a feedstock is then used to produce a mycelial biomass. In some embodiments, rice is ground such that the majority of the rice has a particle size of at least 250 pm, 500 pm, 600 pm, 700 pm, or 800 pm, up to 850 pm, 900 pm, 1 mm, 1.25 mm, 1.5 mm, or 2 mm. In some embodiments, rice is ground such that the majority of the rice has a particle size of 800 pm - 1 mm. In some embodiments, rice is ground such that the size of at least about 70% of the total particles is about 200 pm - about 1100 pm. In some embodiments, rice is ground such that the size of at least about 80% of the total particles is about 200 pm - about 1100 pm. In some embodiments, rice is ground such that the size of at least about 90% of the total particles is about 200 pm - about 1100 pm. In some embodiments, rice is ground such that the size of at least about 95% of the total particles is about 200 pm - about 1100 pm. In some embodiments, rice is ground such that the size of about 90% to about 95% of the particles is about 200 pm - about 700 pm, and the size of about 5% to about 10% of the particles is about 1100 pm (see, e.g., Figure 10). In various embodiments, one or more nitrogen-containing salts can be added to a feedstock before, during, or after the growth of mycelial biomass. Nitrogen-containing salts can be included to help improve the total protein content of the final biomass. For example, in embodiments a mycelium species disclosed herein can be grown on a feedstock that contains one or more nitrogencontaining salts in order to increase the total protein content of the feedstock (e.g., rice) during fermentation. In some embodiments, the nitrogen-containing salt is selected from urea, diammonium phosphate (DAP), or both urea and DAP. In such embodiments, the nitrogencontaining salt(s) is/are added to a feedstock that has been suitably processed, as provided herein, and is ready for fermentation. The amount of urea added to the feedstock can vary. In some embodiments, the amount of urea added is about 9 g/kg - about 22 g/kg, about 9 g/kg - about 11.5 g/kg, in some embodiments about 10 g/kg - about 11 g/kg, in some embodiments is about 20.52 g/kg, and in some embodiments is about 10.26 g/kg. The amount of DAP added to the feedstock can vary. In some embodiments, the amount of DAP added is about 4 g/kg - about 14 g/kg, in some embodiments about 6 g/kg - about 13 g/kg, in some embodiments is about 6.14 g/kg, in some embodiments is about 9.4 g/kg, and in some embodiments is about 12.28 g/kg. In some embodiments, the amount of urea is about 19 - about 21 g/kg and the amount of DAP is about 8 - about 10 g/kg, in some embodiments the amount of urea is about 20 - about 21 g/kg and the amount of DAP is about 9 - about 10 g/kg, and in some embodiments the amount of urea is about 20.52 g/kg and the amount of DAP is about 9.4 g/kg.

In such embodiments, the total protein content of the mycelial biomass is increased with the addition of the nitrogen-containing salt(s). The protein content can increase by as much as up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, or up to about 5% total protein content. In such embodiments, "up to" include some measurable increase, with the lower limit not being zero.

Feedstocks for mycelial biomass production

Feedstocks can be evaluated for their ability to support the production of mycelial biomass by contacting a desired feedstock with a desired fungus. The feedstock can be contacted with conidia (e.g., spores), a dried mycelial grain or wedge, or a liquid inoculant of the fungus. Feedstock, inoculum, and optionally a nitrogen source such as, for example, urea, may be added together and mixed (e.g., by hand or with an electric mixer) for up to about 20 seconds, up to about 30 seconds, up to about 1 minute, or longer before being distributed to a fermentation vessel such as a tray. Mixing may allow for homogenous distribution of nitrogen and inoculum throughout the feedstock. The feedstock and fungus are then incubated at an appropriate temperature for the fungal species, so that growth of mycelial biomass can be evaluated. Alternatively or in addition, a fermentation vessel (such as a tray) containing feedstock and inoculum may be mixed part way through the fermentation process. In various embodiments, feedstocks capable of supporting rapid production of mycelial biomass, at high yield and high protein content, are preferred.

Feedstocks and harvesting

"Harvesting," refers to any process or step that stops the growth of a mycelial biomass, such as separation from a nutrient source or change in temperature conditions, and/or that modifies a physical characteristic of a mycelial biomass in order to make it suitable for consumption or suitable for use in the production of a food product (e.g., converting a biomat into particles or strips). Harvesting of the mycelial biomass can occur at any time sufficient mycelial growth has occurred, either on its own or with and into the feedstock. One of the benefits of the disclosed methods is that harvesting of either the resulting mycelial biomass or the biomass/feedstock composition can occur after 1 - 3 days of growth, although in some instances longer growth periods are desirable, such as when thicker or denser mycelial biomasses are desired/required. In various embodiments, harvesting can occur after 2 - 60 days of growth, or any subrange therein. In some embodiments, harvesting also includes inactivation of the mycelial biomass as described herein.

Feedstocks capable of supporting the production of mycelial biomass that are also easy to harvest are advantageous. In some embodiments, feedstocks capable of supporting biomass production are also easily separated from the biomass are utilized in the disclosed methods. In other embodiments, the biomass grows with and into the feedstock, producing an edible composition that is an amalgamation of biomass and feedstock. In such embodiments, the amalgamation of biomass and feedstock can contain about 5 wt% - about 90 wt% (on a dry weight basis) of mycelial biomass, or any subrange thereof. Also in those embodiments where such an amalgamation is produced, the mycelial biomass improves the nutritional quality of the feedstock. For example, as compared to a feedstock made from a grain, a tuber, or combination thereof, the amalgamated biomass and feedstock demonstrates an increase in total protein percentage and improved amino acid composition. In one embodiment, an amalgamation of biomass and rice feedstock has a total protein percentage selected from about 15%-20% and about 16% as compared to the rice feedstock alone, which has a total protein percentage selected from about 6%-10% and about 7.5%. In one embodiment, an amalgamation of biomass and potato feedstock has a total protein percentage selected from about 15%-25% and about 19.8% as compared to the potato feedstock alone, which has a total protein percentage selected from about l%-5% and about 2.2%. Additionally, in some embodiments the amalgamated biomass and feedstock has increased Lysine, Threonine, Leucine, and/or Isoleucine content as compared to a feedstock made from plain paddy rice, potato, or both. All four of these amino acids cannot be synthesized in humans or other animals, are largely deficient in rice and potato-based diets, and are required for several basic metabolic functions including uptake and utilization of other amino acids. In some embodiments, an amalgamation of biomass and rice feedstock has a total protein percentage that is about 7% - about 28% higher than that of rice feedstock alone. In some embodiments, an amalgamation of biomass and sweet cassava feedstock has a total protein percentage that is about 3-fold - about 5- fold higher than that of sweet cassava feedstock alone. In some embodiments, the amalgamation of biomass and sweet cassava feedstock has undetectable levels of cyanide. In some embodiments, an amalgamation of biomass and millet feedstock has a protein percentage that is greater than about 2-fold higher than that of millet alone. In embodiments, an amalgamation of biomass and feedstock has increased fiber, vitamin B6, folate and/or other nutrient(s) as compared to the feedstock alone.

While biomass can be rinsed to remove excess growth media, rinsing is not required, although in some cases the removal of liquid (e.g., water) or excess liquid is desirable. In some embodiments, biomass can be either squeezed, heated, spun, or some combination of any of the foregoing, in order to remove excess liquid, again not required, but which may be desirable for some applications.

Mycelial biomass

Characteristics of biomass

Mycelial biomass produced by growth of a fungus in contact with a feedstock as disclosed herein has a high protein content. This is in contrast to filamentous fungi that grow naturally or by prior art methods. In some embodiments, mycelial biomass produced by the disclosed methods comprise at least about 30 wt% protein. Unless specified otherwise herein, percentages of components, such as proteins, RNA or lipids, of biomats or filamentous fungi particles, are given as a dry weight percent basis. In the context of protein content, for example, biomass can be dried for 2 days at 99°C and then air dried for at least 2 days; at the end of this time, the biomass is expected to contain about 5 wt% moisture, or less. The total protein content in dried biomass samples can then be measured using a total nitrogen analysis method for estimating protein content. In some embodiments, mycelial biomass produced by the disclosed methods comprises 30 wt% - 80 wt% protein, or any subrange within such range.

The disclosed biomass also has 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. Mycelial biomass produced according to the disclosed methods 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 compositions produced from the disclosed biomass have low levels of RNA as compared to food compositions that do not contain such biomass and/or that have not been treated for the purpose of modifying or lowering the RNA content. In some embodiments, mycelial biomass produced by the disclosed methods comprises less than about 8 wt% RNA on a dry weight basis. In some embodiments, mycelial biomass produced by the disclosed methods comprises 0.5 wt% - 8 wt% RNA, or any sub-range thereof.

In some embodiments, mycelial biomass produced by the disclosed methods comprises both a high protein content and a low RNA content, as described above.

Mycelial biomass produced according to the present disclosure also have a 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 30 wt%, or any subrange in between.

Mycelial biomass produced according to the present disclosure may also represent a "complete" protein source by providing all nine essential amino acids and/or all 20 proteinogenic amino acids. Nine amino acids— histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine— are not synthesized by mammals and are therefore considered to be dietarily essential or indispensable nutrients. The disclosed biomass is an excellent source of these amino acids.

Inoculation

In various aspects, fungal growth occurring according to the present disclosure proceeds when a feedstock is inoculated with a fungus. Inoculation occurs when a feedstock is brought into contact with a fungal inoculum. A fungal inoculum can be conidia (e.g., spores), a dried mycelial grain or wedge, or a liquid inoculant. In some embodiments, a feedstock is contacted with dry spawn of a fungus. The dry spawn can include conidia, mycelium, and/or other parts of the fungus. Dry spawn can be produced by fermentation using a variety of feedstocks, such as those disclosed herein.

Generally speaking a feedstock is prepared for inoculation by pretreating it as described above and contacting it with a fungal inoculum. By way of example, a grain and/or a tuber can be wetted with water and boiled, excess water is removed, and the boiled feedstock is contacted with a fungal inoculum. The feedstock and fungus are then fermented. Following fermentation, the resulting biomass, which may or may not include the feedstock incorporated into the mycelial biomass, is ready for use as either a food product in and of itself, or as a component of a food product. If production of a fungal inoculum from the biomass is desired, all or a portion of the biomass is then dehydrated and/or lyophilized. The dehydrated biomass is then ground to produce a dry spawn.

In another example, a feedstock can be prepared from, e.g., potato and/or rice by wetting the potato and/or rice, boiling it, and removing excess water. In some embodiments, the pH during boiling can be lowered to about 3 to about 5, to help reduce the risk of contamination. The boiled potato and/or rice is then cooled and contacted with a fungal stock and fermented, allowing the fungus to grow. Thereafter, the resulting biomass, which may or may not incorporate the feedstock, is harvested and dehydrated, for example by spray drying or lyophilization. The dehydrated biomass is then ground to produce a dry spawn.

A dry spawn is highly versatile in terms of the methods in which it can be used to inoculate a feedstock. Methods of using the dry spawn as inoculum can be extremely simple, such as sprinkling pieces of dry spawn onto a feedstock, where one or more pieces of dry spawn can be used to inoculate a feedstock and generate a biomass. Dry spawn can be easily spread on or into a feedstock because the lack of water reduces or prevents clumping of the spawn. Dry inoculum can constitute at least about 0.05%, about 0.1%, about 0.15%, or about 0.2% of the total weight of the feedstock/inoculum combination, or from about 0.05% - about 0.5%, about 0.1% - about 0.5%, about 0.15% - about 0.5%, about 0.2% - about 0.5%, about 0.3% - about 0.5%, or about 0.4% - about 0.5% of the total weight of the feedstock/inoculum mixture, or any subrange within any of the foregoing.

Dry spawn has several additional advantages. It can be stored for long periods of time, on the order of weeks (e.g., about 1 - 8 weeks), months (e.g., about 2 - 11 months), or about 1 year or more. Dry spawn does not require freezing or refrigeration and it is resistant to contamination because it is not wet. Dry spawn are also easy to handle because the lack of water prevents spilling and/or leakage from a container of liquid. Dry spawn are also advantageous because the amount of inoculum can be increased, e.g., to achieve higher yields and/or faster growth, without significantly changing the volume and/or moisture content of the feedstock.

Inoculation can also be accomplished via an inoculum comprising planktonic filamentous fungal cells, conidia, microconidia or macroconidia or spores, or fruiting bodies and may be either a dry or a wet inoculum. In many cases, growth media may be inoculated with an inoculum comprising planktonic filamentous fungal cells, conidia, microconidia or macroconidia. In some embodiments, the cells of the inoculum float on the surface of the growth media, which can result in increased growth rate.

Growth of biomass Mycelial biomass can be grown by several fermentation processes.

In some embodiments, mycelial biomass can be grown by liquid surface fermentation. Liquid surface fermentation refers to those fermentations in which the microorganisms employed grow on the surface of the fermentation media without any further support. The media is typically a free-flowing aqueous media. Without being bound by theory, it is thought that this method can be used to preferentially grow a cohesive mycelial biomass as a result of some combination of aerobic, microaerobic and/or anaerobic metabolism. For example, an exposed surface of the biomass is thought to rely on aerobic respiration while the opposite side, which is in contact with the growth media, may be microaerobic to highly anaerobic.

Filamentous fungi can form a cohesive mycelial biomass via surface fermentation under anaerobic, microaerobic, or aerobic conditions or a combination thereof. Here, the cohesive mycelial biomass comprises 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, chlamydospores, and portions of extracellular matrix.

In some embodiments, mycelial biomass can be grown by solid substrate surface fermentation. Solid substrate surface fermentation refers to surface fermentations in which the fungi employed grow on the surface of the fermentation media using carbon and nutrients supplied by solids that are submerged in fermentation media. In some embodiments, some portion of the resulting biomass may be partially submerged.

In some embodiments, mycelial biomass can be grown by submerged fermentation. As its name implies, submerged fermentation refers to fermentations wherein the fungi employed grow in a submerged state within fermentation media.

In some embodiments, mycelial biomass can be grown by solid surface or solid-state fermentation. Solid surface or solid-state fermentation refers to the culture of fungi grown on a solid support selected for the purpose. For example, in some embodiments a solid culture substrate, such as rice or wheat bran, is contacted with a fungus and fermented. Solid-state fermentation typically uses culture substrates with low water levels (reduced water activity). In this regard, the medium (e.g., rice or wheat bran) is saturated with water, but little of it is free flowing. The solid medium comprises both the substrate and the solid support on which the fermentation takes place.

In some embodiments, mycelial biomass can be grown by "supported fermentation," or fermentation that occurs on a solid support material such as a membrane, mesh, cloth or other solid support. For example, fungi can be inoculated directly onto a solid support and still be placed into contact with growth media. In this regard, the media supports the growth of the mycelial biomass on the surface of the solid support. Advantageously, growth on a solid support allows for fermentation waste products to be easily moved away from the growing biomass. Solid supports can also facilitate post-growth processing, for example inactivation by steaming can be made easier by simply moving the solid support, with the biomass on it, to a steaming location.

The above fermentation techniques can be accomplished using media of any viscosity that is suitable for the method employed. Low viscosity media are free-flowing and can be produced by size reducing the feedstock, for example by grinding. High viscosity media have larger granules and can be of a similar viscosity as porridge. For example, whole grains, such as rice grains and/or dehusked rice grains, can be included in high viscosity media. Mashed potatoes can also be used in either low or high viscosity media by controlling the extent of mashing to produce more or less free flowing media. In various embodiments, mycelial biomass produced according to the present disclosure is grown on high viscosity media. In some embodiments, the high viscosity media is a porridge. In some embodiments, the porridge is produced from a grain or a tuber, in some embodiments from both a grain and a tuber.

In some embodiments, high yields of biomass production can be achieved by use of growth media having an appropriate viscosity. More particularly, in some embodiments, the viscosity of the growth media is between about 1500 cP and about 6500 cP, between about 1600 cP and about 6000 cP, between about 1600 cP and about 4500 cP, or within any subrange between 1500 cP and about 6500 cP.

Mycelial growth via any fermentation technique typically proceeds in the presence of a growth medium. Growth media may be liquid or solid and characterized by a desired or preselected mass ratio of carbon to nitrogen ("C:N ratio"). Modification of the C:N ratio during mycelial growth allows one to drive growth toward the production of protein or the production of fats/fatty acids by the mycelium. Generally speaking, increasing the C:N ratio can lead to greater fat production, whereas decreasing the C:N ratio can lead to greater protein production. In some embodiments, in order to decrease the C:N ratio, non-protein nitrogen can be added, for example urea and/or ammonium sulfate, which are not proteins but can be converted into proteins by mycelia. Typically, the C:N ratio of liquid growth media may have a C:N ratio of between about 1:1 and about 50:1, or alternatively a ratio of the form X:2 where X is an integer between about 2 and about 100.

In some embodiments, during growth of a mycelial biomass UVB light (290-320 nm) can be used to trigger pigment production by filamentous fungi, producing a pigmented biomass. 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 growing biomass with UVB can be used to increase vitamin D2 and carotenoids in the resulting biomass. In some embodiments, exposure to UVB occurs for a period of time from 2 seconds - 20 minutes, or any subrange in between.

In some embodiments, a fermentation vessel, such as a tray, is covered with a lid during the fermentation process. The lid may be breathable (/.e., it may have holes or other passages to permit air flow) or it may be solid. In some embodiments, the lid may be a mesh. The lid may be made of any composition, for example cloth, plastic, wood, ceramic, aluminum, and/or stainless steel. The lid may be made of a material that can withstand being autoclaved. Use of a lid to cover the fermentation vessel during fermentation can help to maintain a desired humidity level in the vessel and/or help to reduce contamination. In some embodiments, solid-state fermentation occurs using a rice feedstock in a tray maintained at from about 60%, about 70%, or about 75%, about 80%, about 90% or about 95% humidity, or from about 60% - about 80%, from about 60% - about 90%, from about 60% - about 95%, from about 70% - about 80%, from about 70% - about 90%, from about 70% - about 95%, from about 75% - about 80%, from about 75% - about 90%, or from about 75% - about 95% humidity. Desired humidity can be achieved by any of several means including, for example, using a breathable lid on the fermentation vessel, and/or optimizing initial moisture content of the feedstock. In some embodiments, ambient humidity may be used to help maintain a desirable level of humidity in the fermentation vessel.

Inactivation of biomass

Once growth of the biomass is complete, elimination of cell viability, and thus elimination of the potential of further biomass growth can occur. Inactivation is desired in some embodiments, for example for use of the biomass as a stand-alone food and/or protein source or an ingredient in the production of other foodstuffs. Inactivation can be accomplished by heating, irradiation, contact with ethanol and/or steaming. As used herein, unless otherwise specified, the term "inactivated" refers to: a biomass in which the fungal cells have been rendered nonviable (/.e., dead); a state where or enzymes capable of degrading or causing biochemical transformations within the biomass have been deactivated; or both. By extension, the term "inactivation" refers to any method or process by which a filamentous fungal biomass may be inactivated, such as, by way of non-limiting example, pressure treatment, rinsing, size reduction, steaming, and temperature cycling. As contrasted with dormancy, which can be accomplished by dehydrating or freezing fungi, an inactivated fungus is no longer capable of metabolizing, growing, or reproducing and is not capable of being reactivated to metabolize, grow, or reproduce. In some embodiments, inactivation occurs via heating, wherein filamentous fungal biomass is 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 RNA without adversely affecting the organism's protein composition.

In some embodiments, inactivation occurs via irradiation, whereby 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/or accelerated electrons generated by machines operated below a nominal energy of 10 MeV.

In some embodiments, inactivation occurs via steaming. Steaming has an advantage in that it can remove certain metabolites from the biomass if they are produced. In some embodiments, biomass is positioned within a steamer and contacted with hot water vapor (e.g., heated to a temperature greater than 85 °C - 95 °C). In some embodiments, it is preferred to protect the biomass from wastewater coming off of it during steaming. Biomats are steamed at least to the point where biomat viability is reduced such that further biomat growth and/or cellular reproduction within a biomat is negligible, for example for a period of time from about 2 - about 25 minutes.

Inactivated biomass can be used directly as a protein source, for example by consuming the biomass itself, which may or may not incorporate the feedstock on which it was grown, or by using the biomass as a component in the preparation of other foodstuffs, for example food products comparable to tempeh, tofu, bacon, paneer cheese, and jerky, to name but a few.

Processing of biomass

Particles made from mycelial biomass

After growth, mycelial biomass, which in some embodiments is amalgamated with a feedstock and/or may be inactivated, can be size reduced into a plurality of particles for use as a protein source on its own or in foodstuffs. The size reduction can occur by mechanical means such as cutting, chopping, dicing, mincing, grinding, blending, tearing, etc., or via sonication, and in some embodiments is conducted prior to mixing with other ingredients or liquids. Size reduced particles can be uniform in size or can vary in size to create texture.

In various embodiments, 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 small enough to mimic flour (e.g., for baking applications) typically range between 0.03 mm and 0.4 mm, particle of suitable size to generate meat analogs such as jerky range between 100 mm and 500, etc. Larger size particles can also be produced, such as pieces of mycelial biomass that approximate the size and shape of an animal-based product of which the mycelial biomass product is an analog of, e.g., a bacon slice or a chicken tender.

The number of size reduced particles produced per unit biomass is dependent on the initial volume of biomass and the purpose for which the size-reduced particles will be used.

Such particles can be eaten on their own and/or are useful in the preparation of food compositions.

Food Products

Described herein are food products comprising and/or derived from a mycelial biomass produced according to the present disclosure. In various embodiments, a food product may be prepared from such a mycelial biomass.

In various aspects, the present disclosure provides a mycelial biomass prepared from a filamentous fungal strain that satisfies the above inclusion criteria and is grown in contact with a feedstock as described herein. In such aspects, the biomass is edible and suitable for consumption on its own and/or as a part of a food product. Such a biomass can be processed into various formats including, without limitation, (1) protein chunks (soy chunk or paneer replacements); (2) protein rich flakes (staple breakfast replacement); (3) protein candy/confectionary bar (chikki replacement); or (4) protein rich savory snacks (extruded and popped snack replacement). Additional formats include a liquid dispersion to form a milk analog that can used in dairy applications, a fine particulate mycelial flour that can be used in baked goods in the same manner as traditional flours, particles of larger size that can be used as analogs of tempeh, meat, tofu, textured vegetable protein, and similar products.

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., ice cream, mayonnaise, 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."

Embodiments include compositions comprising mycelial biomass produced as disclosed herein, which in some embodiments is amalgamated with a feedstock, typically compositions comprising edible filamentous fungal biomass, and most typically filamentous fungal food compositions, i.e., edible filamentous fungal compositions that are adapted for consumption by humans or domesticated, farmed (e.g., agriculture or aquaculture), or livestock animals, that include mycelial biomass. In some embodiments, the food composition may be a food product that is analogous to a conventional or known food product comprising meat or another animal-derived ingredient, wherein the mycelial biomass is provided in addition to or in lieu of the animal-derived ingredient.

Water content

Embodiments include food products prepared from a food composition comprising mycelial biomass and a solution, commonly but not always an aqueous solution, comprising one or more food additives, such as, by way of non-limiting examples, salt, flavorings, vitamins, and nutritional components (e.g. carbohydrates, fats, proteins, etc.), in which the solution comprises about 5 wt% - about 50 wt%, or any range between 5 wt% and 50 wt%, of the total food composition.

One significant advantage and benefit is that the mycelial biomass present in the food compositions may have significantly greater ability to absorb or otherwise take up a solution, e.g., a "marinade" or similar solution applied to the biomass to achieve a seasoning, flavoring, and/or nutritional objective and/or to define a textural property (e.g., chewiness, elasticity, mechanical behavior characteristics such as tendency to rip or tear, etc.), than conventional ingredients; without wishing to be bound by any theory, the present inventors hypothesize that this effect is due to differences between the mycelial structure of a filamentous fungal biomat and the myofibrillar structure of meat, in that the mycelial structure is more amenable to changes in the ratio of bound water to unbound water (and thus to reabsorption of the marinade) than meat. Because the biomass itself may have a moisture content of as much as 80 wt%, higher than most whole cuts of meat, even before a solution is applied thereto, the water content of food compositions, and particularly meat analog food products, may be advantageously high.

Embodiments further include methods of forming a food composition comprising mycelial biomass and a solution as described above (e.g., by contacting biomass with the solution and allowing the biomass to absorb or otherwise take up the solution), and food compositions made by such methods. "Food compositions," as that term is used herein unless otherwise specified, may be "final food compositions" (i.e., food products that are compositions intended to be consumed by a human or a domesticated, farmed, or livestock animal without further processing required) or "intermediate" food compositions (i.e., compositions that are intended to be further processed and consumed by a human or a domesticated, farmed, or livestock animal only after further processing). Processing steps by which "intermediate" food compositions may become "final" food compositions include, but are not limited to, size reduction and/or dehydration.

In some embodiments, the mycelial biomass may have a moisture content of at least about 30 wt% - at least about 80 wt%. In some embodiments, very little or none of the moisture present in the filamentous fungal biomass may be free water, or, in other words, the filamentous fungal biomass may have a total content of physically bound water and tightly bound water of at least about 30 wt% - at least about 80 wt%. Without wishing to be bound by any particular theory, it is believed that the ability of the filamentous fungal food compositions to absorb or otherwise take up a solution is related primarily to the content of physically bound and tightly bound water and not necessarily to the total moisture content; particularly, free water may be more easily removed during dehydration and replaced by a marinade or other liquid solution than bound water. Thus, even high-moisture biomass may suitably be used in filamentous fungal food compositions and may absorb or take up a solution in significant quantities, so long as the proportion of moisture that is present as free water is relatively low. In some embodiments, therefore, methods of the present disclosure may include a step of treating a biomass to remove at least a portion of the free water therefrom to increase the biomass's capacity for taking up a liquid solution in a subsequent treatment step; such a step may result in a longer shelf life for the resulting food product, as shelf life correlates with the proportion of moisture initially present as bound water due to stabilization within the biomass.

One particular advantage and benefit of a food product produced by mycelial biomass produced according to the present disclosure is that the product can have advantageously high shelf stability, or, in other words, an advantageously long shelf life, especially as compared to conventional food products to which the filamentous fungal food compositions may be analogous (e.g., meat products), while reducing, or in some embodiments even eliminating, the use of preservatives, stabilizers, and/or mold inhibitors (e.g., potassium sorbate) that may adversely affect the aesthetic or nutritional properties of the food product. In some embodiments, the food product may be substantially free of preservatives, stabilizers, and/or mold inhibitors (i.e., a component added to a food product that, in the quantity added, can materially affect the shelf life of the product), and/or methods for making the food product may not include any step of adding a preservative, stabilizer, and/or mold inhibitor. Without wishing to be bound by any particular theory, these advantages and benefits may be, in at least some embodiments, a result of either or both of (1) control over the moisture content of the biomass enabled by the disclosed methods, particularly, the ability to remove as much or as little of the free water within the biomass (and optionally to replace the removed free water with other chemistries) as desired, and (2) inactivation of the biomass as described herein.

By way of non-limiting example, conventional meat jerky products are extremely shelfstable and may, under ambient conditions, have a shelf life of many months, but achieve this stability only by including significant quantities of salt and/or sugar, which act as plasticizers and/or stabilizers for the jerky product. Additions of salt and sugar, however, have well-known nutritional drawbacks. The food compositions provided herein can overcome this drawback in any one of several ways, for example by substituting salt for sugar as a stabilizer (which may in some embodiments result in a more acceptable nutritional profile), by using stabilizers in lesser amounts than analogous conventional food products, or by virtue of having a lower fat content (or other nutritional advantage) relative to true meat. In some embodiments, food compositions comprise mycelial biomass and one or more food additives (e.g. salt, flavorings, vitamins, added carbohydrates, added fats, added proteins, etc.) and have a shelf life on the order of weeks, months or a year or more. In addition or alternatively, such embodiments can include food compositions including no more than 15 grams of sugar per 28 grams of food composition, and/or a sugar content of no more than about 10 wt%.

Particularly, food compositions may have an "elastic" or "chewy" consistency and texture, and/or a "juiciness" or perceived amount or extent of moisture, analogous to conventional meat products, while still possessing both an advantageously long shelf life and an advantageously low sugar content.

As set forth herein, in some embodiments the mycelial biomass is grown with and into the feedstock, forming an amalgamation comprising the biomass and feedstock. These embodiments provide significant advantages in food production, as the amalgamation provides advantageous physical parameters to food products. For example, as described herein, a feedstock can be made from a grain, or a tuber, or a combination of both. By themselves, grains (for example) provide limited options for food preparation (e.g., porridge, breads, cereal, pasta, etc.). However, combining a grain- or tuber-based feedstock with a mycelial biomass in an amalgamated fermentation product increases the cohesiveness of the starting material (feedstock), thereby increasing the total number of food products that may be made from the feedstock material. The amalgamated product will have a very different mouthfeel, and therefore palatability, and also enables frying, grilling, adding to soups, and the like without the amalgamated product falling apart. This makes the amalgamated food product easier to work with in a food preparation context, and it reduces food waste (consider rice on its own versus a rice:biomass amalgamation: it is difficult to put rice on a grill and cook it, or to put it in a sandwich on its own; a rice:biomass amalgamation changes that and makes both options a possibility). In addition, amalgamations comprising the biomass and feedstock can demonstrate increased water holding capacity, which not only improves shelf life in the short term, but can also improve the taste and texture of a food product made from the amalgamated product. Amalgamations can also demonstrate improved fat holding capacity, which would allow a food product made from such amalgamation to be more caloric and nutritious per unit volume than the feedstock material on its own.

Liquid dispersion - production

In some embodiments, the biomass is formulated into a liquid dispersion, where particles of the biomass are dispersed in an aqueous medium, thereby forming a milk analog product. Suitable particle sizes are typically less than about 10 microns. The liquid dispersion is formed by blending the particles with water, optionally while heating, whereby the milk analog is formed upon cooling. The ratio of biomass to water can range from about 1:10 to about 10:1, or any range of ratios in between.

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 a high- energy size reduction or milling process, or in the heat cycle.

In various embodiments, a liquid dispersion is stable such that the particulates of mycelial biomass 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 24 hours or alternatively for at least about 7 days, up to 4 weeks, or up to 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).

Uses of liquid dispersions

The liquid dispersion can be used as a drink or beverage, including as a substitute for any milk product such as dairy milk, almond milk, rice milk, soy milk, etc. It can be used in a number of recipes including soups, ice cream, yogurt, smoothies, fudge, and candies. In some cases, biomass produced from different fungal strains, different feedstocks, different carbon sources, and the like, can result in liquid dispersions having different flavors. For example, when the feedstock/carbon source is glycerol, the resulting liquid dispersion produced from the biomass is sweet, while a liquid dispersion resulting from biomass grown on an acid whey feedstock/carbon source will be more sour.

In some embodiments, the liquid dispersion can be used to form a stable foam that does not collapse spontaneously upon cessation of the foaming process. In such embodiments, the stable foam can be generated by the incorporation of one or more gases in order to form bubbles, for example nitrogen, carbon dioxide, air, or any other suitable, food grade gas. 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 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 can have an increased volume (/.e., overrun) by incorporation of air of at least about 10% - 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 up to about 30 days. In some embodiments, the liquid dispersion remains stable for at least up to 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.

Cultured food products

In some embodiments, the food composition is a cultured food product. As used herein, unless otherwise specified, the term "cultured food product" refers to a food product in which a microbial food culture, i.e. live bacteria, yeasts, or molds, is introduced to a mycelial biomass. By way of non-limiting example, fungal food compositions may be cultured with Lactobacillus spp. or other lactic acid bacteria (to make, e.g., a yogurt analog food product or other dairy analog food product), Saccharomyces cerevisiae or other yeasts used in brewing or baking (to make, e.g., a baked good analog food product or an alcoholic beverage analog food product), molds traditionally used to make sausages (e.g., Penicillium chyrsogenum or Penicillium nalgiovense, to make a sausage analog food product) or soy sauces (such as Aspergillus oryzae or Aspergillus sojae, to make a soy sauce analog food product), and so on. In some embodiments, cultured food products may be cultured with two or more microbial food cultures, either simultaneously or sequentially, to produce an analog of a food product that is made by fermentation of two or more microbial cultures; by way of non-limiting example, cultured food products may include semi-soft ripened cheese analog food products (made by subjecting a fungal material to a first culture by Lactobacillus spp. or other lactic acid bacteria and a second culture by a cheese ripening yeast), blue cheese analog food products (made by subjecting a fungal material to a first culture by Lactobacillus spp. or other lactic acid bacteria and a second culture by a mold such as Penicillium roqueforti), soft ripened cheese (e.g. Brie or Camembert) analog food products (made by subjecting a fungal material to a first culture by Lactobacillus spp. or other lactic acid bacteria and a second culture by Penicillium camembert!), etc.

In some embodiments, the food composition comprises a yogurt analog food product comprising particles of a mycelial biomass dispersed in an aqueous medium. In some embodiments of the yogurt analog, the ratio of fungal particles to water may range from about 1:10 to about 10:1. A higher ratio of ratio of fungal particles to water is expected to increase the texture and reduce runniness of the yogurt 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 yogurt analog food product comprises an invert sugar or inverted sugar. Invert sugar is resistant to crystallization and promotes retention of moisture, and is used commercially in various foods such as baked goods, confections, or fruit preserves and beverages to enhance flavor and texture and prolong shelf life. Examples include honey or a mixture of glucose and fructose that is obtained by hydrolysis of sucrose and is sweeter than sucrose.

In some embodiments, the yogurt 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 (/.e., arrowroot, tapioca, corn, 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 mycelial biomass 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.

In some embodiments, the yogurt analog food product comprises lactic acid bacteria (LAB). These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation. Examples of LAB include the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus. In some embodiments, it comprises the bacteria Lactobacillus bulgaricus and/or Streptococcus thermophilus.

In some embodiments, the yogurt analog food product further comprises a rennet. The rennet may be derived from an animal source, a vegetarian source or a microbial source. In vegetarian or vegan food products, the rennet is derived from a vegetarian source and/or a microbial source.

In some embodiments, the yogurt analog food product further comprises an enzymatic water. For example, an enzymatic water can be produced as follows. 100 gm of whole rye or durum wheat seeds (or other suitable whole cereal seeds) may be combined with 1 liter of water, and germinated for 2-4 hours. When seeds start to sprout and the first roots appear, the seeds may be placed into a clean jar with 1 liter of water. The jar may be covered with a permeable cloth (linen or cotton), and incubated at room temp for 24 hours, at the end of which the water in the jar changes color and odor. This water is referred to as enzymatic water and can be used in the production of yogurt and cheese.

In some embodiments, the yogurt analog food product further comprises a probiotic. Probiotics are mixtures of live micro-organisms such as bacteria and yeast that provide health benefits including improved digestion.

In some embodiments, the yogurt analog food product comprises milk solids derived from animal milk. In some embodiments, the yogurt analog food product is free of milk solids derived from animal milk, i.e., it does not contain any milk solids.

In addition to yogurt analog food products, food compositions may comprise any one or more other dairy analog food products. For example, food compositions may comprise a cheese analog food product, such as a hard cheese (e.g., Parmesan) analog food product, a semi-hard cheese (e.g., Gouda) analog food product, a semi-soft cheese (e.g., Havarti) analog food product, a soft or soft ripened cheese (e.g., Brie) analog food product, a cream cheese analog food product, a sour milk cheese analog food product, a blue cheese analog food product, a mascarpone cheese analog food product, a pasta fi lata (e.g., Mozzarella) cheese analog food product, a brined cheese (e.g., Feta) analog food product, a whey cheese (e.g., Ricotta or Brunost) analog food product, or a fresh cheese (e.g., cottage cheese) analog food product. Food compositions may alternatively comprise a butter analog food product, such as a raw cream butter analog food product, a butterfat analog food product, a clarified butter analog food product, a whey butter analog food product, a cultured butter analog food product, a mild cultured butter analog food product, a sweet cream butter analog food product, or a traditional buttermilk analog food product. By way of third nonlimiting example, food compositions may comprise a whey analog food product, such as a sour whey analog food product or a sweet whey analog food product. Food compositions may alternatively comprise a cream analog food product, such as a creme frafche analog food product, a smetana analog food product, a sour cream analog food product, a half-and-half analog food product, a table cream analog food product, a whipping cream analog food product, a double cream analog food product, a clotted cream analog food product, a soured cream analog food product, a pasteurized cream analog food product, or a condensed cream analog food product. Food compositions may alternatively comprise a sour milk analog food product, such as a quark analog food product, a cheese curd analog food product, a soured milk analog food product, a kefir analog food product, an organic yogurt or mild yogurt analog food product, a yogurt analog food product, a cream yogurt analog food product, or a cultured buttermilk analog food product. Food compositions may alternatively comprise a milk analog food product, such as a raw milk analog food product, a lowfat or skimmed raw milk analog food product, a pasteurized milk analog food product, a fresh whole milk analog food product, a lowfat milk analog food product, a skimmed milk analog food product, an extended shelf-life (ESL) milk analog food product, an ultra-high temperature processed (UHT) milk analog food product, a sterilized milk analog food product, a condensed or evaporated milk analog food product, a part-skim condensed milk analog food product, or a condensed skimmed milk analog food product. Food compositions may alternatively comprise a powdered dairy analog food product, such as a powdered whey analog food product, a powdered milk analog food product, or a powdered skimmed milk analog food product. Dairy analog food products may, in some embodiments, be vegan food products, i.e., food products that contain no animal products, and thus allow observers of vegan diets to incorporate such dairy analogs into their diet.

Other food products

In addition to the aforementioned drink products and cultured food products, the liquid dispersion may be used to prepare any of the following: egg analogs (e.g., scrambled egg analogs), hard cheese analogs (e.g., Parmesan), semi-hard cheese analogs (e.g., Gouda), semi-soft cheese analogs (e.g., Havarti), soft or soft ripened cheese analogs (e.g., Brie), cream cheese analogs, sour milk cheese analogs, blue cheese analogs, mascarpone cheese analogs, pasta fi lata analogs (e.g., Mozzarella), brined cheese analogs (e.g., Feta), whey cheese analogs (e.g., Ricotta or Brunost), fresh cheese analogs (e.g., cottage cheese), butter analogs (e.g., raw cream butter analogs, butterfat analogs, clarified butter analogs, whey butter analogs, cultured butter analogs, mild cultured butter analogs, sweet cream butter analogs, buttermilk analogs, etc.), whey analogs (e.g., sour whey analogs, sweet whey analogs, etc.), cream analogs (e.g., cuisine cream analogs, creme fraiche analogs, smetana analogs, sour cream analogs, half-and-half analogs, table cream analogs, whipping cream analogs, double cream analogs, clotted cream analogs, soured cream analogs, pasteurized cream analogs, condensed cream analogs), sour milk analogs (e.g., quark analogs, cheese curd analogs, soured milk analogs, kefir analogs, organic yogurt or mild yogurt analogs, yogurt analogs, cream yogurt analogs, cultured buttermilk analogs), milk analogs (e.g., raw milk analogs, low fat or skimmed raw milk analogs, pasteurized milk analogs, fresh whole milk analogs, low fat milk analogs, skimmed milk analogs, extended shelf-life (ESL) milk analogs, ultra-high temperature processed (UHT) milk analogs, sterilized milk analogs, condensed or evaporated milk analogs, part-skim condensed milk analogs, condensed skimmed milk analogs), powdered dairy analogs (e.g., powdered whey analogs, powdered milk analogs, powdered skimmed milk analogs), ice cream analogs, and the like.

In addition to the aforementioned drink products and cultured food products, the liquid dispersion may be used to prepare colloidal food products, where particles of the liquid dispersion are dispersed throughout a volume of a different substance. Embodiments provided by the present disclosure include colloidal suspensions of liquid dispersions of mycelial biomass, typically colloidal food compositions, i.e., edible colloidal compositions that are adapted for consumption by humans or domesticated, farmed (e.g., agriculture or aquaculture), or livestock animals, that include particles of mycelial biomass. In some embodiments, the colloidal food composition may be a food product that is analogous to a conventional or known food product comprising a dairy or otherwise animal-derived ingredient (milk, egg, etc.), wherein the particles of mycelial biomass in a liquid dispersion are provided in addition to, or in lieu of, one or more animal-derived ingredients. In some embodiments, the colloidal food composition may be a non-dairy composition or food product and may be a vegan (i.e., no animal-derived components) composition or food product. Embodiments of colloidal food compositions include, without limitation, blancmange, bread, butter, cake, creamers (e.g., for coffee and tea), custard, egg white foam, ice cream, jam, jelly, margarine, mayonnaise, meringue, milk, whipped cream, and analogs of any of the foregoing.

Protein and nutrition source

Particles of the mycelial 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 a mycelial biomass, or the size-reduced particles of such biomass, the particles can be optimized for particular textures, mouthfeel, 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 mycelial biomass or made solely from mycelial biomass have textures very similar to the standard protein foods that they emulate.

Particles of the mycelial biomass can also be used to augment protein content of other food compositions. Examples of foods that can be produced using only the reduced particle size of the mycelial 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), snacks (e.g. chips), soups, smoothies, beverages, milk analogs, breads, pastas, noodles, dumplings, pastries (e.g. Pate a Choux), cookies, cakes, pies, desserts, frozen desserts, ice cream analogues, yogurt, confections, and candy.

Foods augmented with the reduced particle size of the mycelial biomass can significantly increase the protein content, which is particularly important for infirm individuals and/or those following a vegan diet. For example, soups, drinks or smoothies can be augmented with mycelial biomass liquid dispersion.

Whether mycelial 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 suitable, 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 mycelial 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 substantially unimodal particle size distribution, i.e., in which all particles are approximately the same size, may be used, while in other applications a broad or multimodal distribution or combination of distributions of particle size may be used. Similarly, size- reduced particles can be derived from a single source of mycelial biomass or from a combination of different sources of mycelial biomass.

Flour use In some embodiments, the biomass is formulated into a flour, where the biomass is dehydrated and reduced in size to small, fine particles. Suitable particle sizes can vary depending on the desired application. In some embodiments, the biomass is size reduced so that 80-100% of the particles are distributed within the size ranges of 10-41 pm and/or 41-300 pm, and 2-11% of the particles are less than 10 pm. In some embodiments, the biomass is size reduced so that 80- 100% of the particles are 52-108 pm in size. In some embodiments, the biomass is size reduced so that at least 98% of the particles are less than 212-pm in size. In some embodiments, the biomass is size reduced such that the particles are less than about 1 micron in size. The flour is formed by dehydrating the biomass (e.g., via spray-drying) and size-reducing the biomass. In some embodiments, dry biomass is ground or otherwise size-reduced into relatively fine particles that resemble traditional flour, before being incorporated into a food product.

In some embodiments, the flour is used in the place of some or all of traditional flour in a baked food product (/.e., bread, etc.). Biomass flour is also suitable for use as an addition/supplement to other standard flours (e.g., all-purpose flour, self-rising flour, cake flour, bread flour, pastry flour, high-protein flour, etc.). Biomass flour can be used as a substitute for about 5% - about 30% traditional flour with no deleterious effects on taste, rising, texture, appearance, or smell. Biomass flour can be used to make, for example, bread of any kind (e.g., 7 grain, white, wheat, etc.), rolls, muffins, cakes, pastries (e.g., Pate a Choux), cookies, pies, pasta, dumplings and the like.

In some embodiments, the biomass is formulated into small particles, where the biomass is size-reduced in size to small particles and added to a meat product, as a suitable meat extender. In some embodiments, the biomass is formulated into small particles, where the biomass is size- reduced in size to small particles and added to a plant-based product (e.g., soy) or a vegetarian or vegan analog of a meat product, as a suitable vegetarian or vegan extender. Suitable particle sizes are typically less than about 10 mm. The ratio of biomass particles to meat can range from about 10:90 - about 90:10, or any ratio in between.

In such embodiments, the biomass is used to increase the amount of total food product by the addition of biomass particles to other meat products (e.g., beef, pork, poultry, fish, etc.). For example, a meat product can be extended by the addition of about 10% - about 50% of biomass particles. Smaller particle sizes produce denser, creamier textures, whereas larger particles produce more texture and mouthfeel, requiring more chewing.

In some embodiments, the biomass is formulated into particles that can be used as analogs of meat, tofu, textured vegetable protein, and similar products. In such embodiments, the biomass is processed to formulate an analog of a meat product, such as whole cuts of meat, bacon, ground meat (and derivative products, e.g., meatballs, meatloaves, burger patties, etc.), shredded meat, jerky, and the like. Here, biomass is size-reduced (e.g., by grinding, pulverizing, etc.) into coarse particles in which at least about 90% of the particles have a length of about 4 mm - about 10 mm, a width of about 1 mm - about 3 mm, and a height of up to about 0.75 mm, resembling ground or processed meat. From there, the biomass can be formulated into any number of meat analog food products.

Additionally, analogs of a wide variety of conventional meat products (e.g., smoked meat products, meat jerky, etc.) that are meat-free, lactose-free, egg-free, soy-free, dairy-free, and/or vegan, may be produced from the disclosed biomass. In some embodiments, certain ingredients that may pose health risks to certain individuals can be replaced by the disclosed biomass. For example, red meat or other tissues that may have adverse effects on the cardiovascular health of certain individuals can be replaced by the disclosed biomass.

Fungal forms for use in food products

Fungal food compositions may comprise any one or more forms or types of mycelial biomass. By way of non-limiting example, forms or types of mycelial biomass suitable for use in the food compositions include, but are not limited to, processed biomass, fungal paste formed from biomass, unprocessed or "raw" biomass, and combinations and mixtures of these. By way of further non-limiting example, it is possible to combine forms or types of mycelial biomass produced by two or more different processes, e.g. any two or more of a solid substrate surface fermentation process, a steady-state fermentation process, a solid surface fermentation process, an air-medium colloid fermentation process, a submerged fermentation process, etc.

Fungal food compositions may, in some embodiments, comprise a highly dense fungal biomass or portion thereof. Depending on the fungus and growth conditions, the biomass may also exhibit a fibrous texture, which is an important consideration when producing food compositions that require texture to simulate meat (i.e. a meat analog food product); in some embodiments, the fibrous structure of the biomass may be engineered or oriented to provide a material (and thus, in some cases, a food composition or product) that is relatively difficult or relatively easy to tear, or has selected portions that are relatively difficult or relatively easy to tear. The dense nature of the biomass can also enable easy harvesting, without the need for a concentration step (e.g., centrifugation, filtration) between harvesting of the biomass and processing into a food composition. The density of the biomass can range from about 0.01 g dry weight/cm³ to about 1 g/cm³, and any subrange within this range. In some embodiments, the density can be greater than about 0.01, greater than about 0.02, greater than about 0.03, greater than about 0.04, greater than about 0.05, greater than about 0.06, greater than about 0.07, greater than about 0.08, greater than about 0.09, greater than about 0.1, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, or greater than about 1 g/cm³.

In some embodiments, methods for preparing a mycelial biomass food composition comprise dehydrating a filamentous fungal biomass at relatively low temperature, e.g., no more than about 185 °F and most typically no more than about 165 °F, over a period of, e.g., between about 20 minutes and about 12 hours. Typically, a low-temperature drying oven or dehydrator may be employed to carry out dehydration of the mycelial biomass, as may a smoker, a vacuum dehydration system, tumble-drying (at either ambient pressure or under vacuum), or any other suitable apparatus or method for lowering the moisture content and water activity of the mycelial biomass while otherwise retaining the mycelial material's basic structure.

EXAMPLES

Example 1. Biomass production process

This non-limiting example of a biomass production process includes feedstock production, inoculum production, fermentation, post-processing, and waste aspects, as shown in Figure 1.

Feedstock Production

This Example uses paddy rice and/or potatoes as a feedstock. The paddy rice is ground and sieved, yielding a paddy rice stream and a stream of waste grain material. Water, nitrogencontaining salts (e.g., urea and/or ammonium phosphate), and HCI are added to the paddy rice stream. The paddy rice stream is then boiled to produce a grain feedstock.

Potatoes can also be used as a feedstock. Potatoes are sliced. Water, nitrogen-containing salts (e.g., urea and/or ammonium phosphate), and HCI are added to the potatoes. The potatoes are then boiled to produce a feedstock.

A paddy rice stream can also be combined with sliced potatoes. Water, nitrogen-containing salts (e.g., urea and/or ammonium phosphate), and HCI are added to the paddy rice stream and sliced potatoes. The rice stream and potatoes are then boiled to produce a feedstock.

Inoculum production

This Example uses millet as a component in the production of the dry spawn inoculum. Water is added to the millet, which is then boiled to produce millet and waste water streams. Fungal stock, such as Cunninghamella stock, is added to the millet stream and then fermented. The fermented material is then dehydrated and ground to produce a waste spawn material stream and the dry spawn.

Fermentation A tray, which can be vented, is sanitized with perchloric acid and/or ethanol to produce a vessel suitable for fermentation. Feedstock and Cunninghamella dry spawn are then added to the fermentation tray and fermented to produce a mycelial biomass.

Post-processing

The mycelial biomass can then be optionally inactivated, dried, and size-reduced to produce a food composition.

Example 2. Paddy Rice Porridge Feedstock Preparation

Paddy rice grain is stored at low humidity, for example about 8% to about 10% relative humidity. The rice is dehulled using mesh with pore size of about 595 micron (e.g., size 30 mesh) to produce a rice grain and bran stream and a hull waste stream. A typical yield is 74% uniform grains containing inorganic Nj in the rice grain and bran stream and 26% hulls in the hull waste stream. The rice grain and bran stream is then combined with water, urea, diammonium phosphate (DAP), and HCI and allowed to soak for about 18 hours at 30 degrees Celsius and pH of 3. The soaked rice grain and bran stream is then drained of excess liquid, removing about 80% of the water. The drained rice grain and bran stream, containing about 20% water, is then loaded into trays and steam sterilized at a pressure of about 103 kPA and a temperature of about 121 degrees Celsius. Sterilization proceeds for about one hour. After sterilization, the rice grain and bran stream is allowed to cool to about 50 degrees Celsius to produce a feedstock.

When the rice grain and bran stream has cooled to about 50 degrees Celsius, inoculum, such as dry spawn inoculum, can be added to the feedstock. The feedstock is contacted with dry spawn and allowed to ferment for about 72 hours at a temperature of about 30 degrees Celsius and a relative humidity of about 90%, to produce a mycelial biomass. Following fermentation, the mycelial biomass can be subjected to post-treatment.

Example 3. Mashed Potato Feedstock Preparation

Potatoes are chopped into 2mm cubes, loaded into trays and steam sterilized at a pressure of about 103 kPA and a temperature of about 121 degrees Celsius for about one hour. After sterilization, the potatoes are allowed to cool to about 55 degrees Celsius. Urea and DAP are then added to the potatoes to produce the feedstock.

The feedstock can then be contacted with inoculum, such as dry spawn inoculum. The inoculum and feedstock are fermented for about 72 hours at about 30 degrees Celsius and 90% relative humidity. Following fermentation, the resulting mycelial biomass can be subjected to posttreatment.

Example 4. Testing of Strain Growth at Low pH Three strains of the Cunninghamella genus were tested for growth at low pH: Cunninghamella blakesleeana, Cunninghamella echinulata, and Cunninghamella elegans. Strain NF30 of Cunninghamella echinulata produced mycelial biomass when grown at pH of 5, 4, and even 3. Strain NF31 of Cunninghamella elegans produced mycelial biomass when grown at pH of 5 or 4. Strain NF4 of Cunninghamella blakesleeana produced mycelial biomass when grown at pH 5. Cunninghamella echinulata was also able to produce mycelial biomass when grown at 40 degrees Celsius.

Mycotoxin Screening

Strain NF30 was grown on rice porridge supplemented with DAP and urea. The resulting mycelial biomass was tested for the production of many mycotoxins. The results of this testing are shown in Table 1, showing that all mycotoxins tested for were below the level of detection (LOD) of this sensitive assay. These data indicate that these Cunninghamella strains either do not produce any of the mycotoxins tested for when grown according to the present disclosure, or produce them in amounts that are so low as to not be detectable. Table 1. Mycotoxin levels in strain NF30 of Cunninghamella echinulata

Example 5. Amino Acid Content

The amino acid content of strains NF4 and NF30 (see Example 4) mycelial biomass was analyzed. Mycelial biomass produced from the two strains was grown on both paddy rice porridge or mashed potato feedstock. Both strains produced complete proteins with all nine essential amino acids that are enriched in valine, aspartic acid, and glutamic acid and also substantial amounts of lysine, threonine, leucine, and isoleucine. The two strains produced similar amino acid profiles and produced more protein when grown on potatoes.

Example 6. Methods of Open Air Inoculation

Growth of strain NF30 (see Example 4) was tested at high temperature and low pH, with varying sanitation steps performed, to test for production of mycelial biomass and inhibition of contamination. Three sanitation methods were tested: aseptic (sanitized tray and grown in aseptic environment), open air inoculation (no sanitation of tray and inoculation and growth without aseptic environment), and open air inoculation but with sanitized trays (top, middle, and bottom rows of Figure 5, respectively).

Feedstock was prepared at pH 4.0 and then boiled for 30 minutes with constant stirring. Nitrogen salts were added in the final 5 minutes of boiling. The feedstock was then allowed to cool to about 50 degrees Celsius with a lid. The trays for the aseptic and open air with sanitized trays groups were sanitized with 70% ethanol and allowed to air dry. Feedstock was poured into trays at about 50 degrees Celsius, and the feedstock was contacted with dry spawn inoculum once the feedstock cooled to about 40 degrees Celsius. The inoculated trays were then covered with an ethanol sanitized cloth and incubated for 3 days at about 30 degrees Celsius and 90% relative humidity.

As shown in Figure 5, all conditions enabled production of mycelial biomass.

Example 7. Biomass production

Paddy rice was dehusked, wetted to 40% moisture, and boiled. The resulting mixture was supplemented with DAP and urea and then dried to produce a feedstock. The feedstock was then contacted with dry spawn inoculum of strain NF30 (see Example 4) and allowed to grow for 72 hours.

The use of high viscosity rice porridge feedstock produced surprising and unexpected characteristics of the mycelial biomass. As shown in Figure 6, the fungus grew into and around the paddy rice porridge media. Mycelia infiltrated the media and produced a solid and strong but flexible mycelial biomass that included the feedstock amalgamated with mycelial biomass. The mycelial biomass contained substantially no residual liquid feedstock, which minimizes postprocessing required to produce food. The mycelial biomass had neutral taste and soft texture, demonstrating its suitability for use in a wide variety of foods with minimal required processing, if any. The mycelial biomass produced was a cohesive mycelial biomass having sufficiently high enough tensile strength and flexural strength to be lifted with one hand but was brittle enough that a piece can be broken off (see missing piece at top right of FIG. 6B, in which a corner piece was broken off the rest of the mycelial biomass). This eases handling of the mycelial biomass and eases production of food products that are or contain chunks of the advantageously nutritious of mycelial biomass, for example as protein chunks (Figures 6E and 6F).

Example 8. Synergistic Combination of Rice and Potato Media

This Example illustrates the synergistic combination of rice and potato mixtures, which unexpectedly increase biomass yield. A feedstock was prepared as provided in Examples 1, 2, and 3, using a combination of rice and potato, which does not increase total carbon load of the feedstock. As shown by the data in FIG. 7, biomass density and total protein content are vastly improved when the media contains both rice and protein. Use of potato alone as a feedstock led to biomass with almost 15 g/m² total protein and about 32 g/m² biomass. Use of rice alone as a feedstock led to biomass with about 15 g/m² total protein and about 45 g/m² biomass. Use of a combination of potato and rice resulted in growth of a biomass with almost 33 g/m² total protein and about 80 g/m² biomass.

Example 9. High viscosity feedstock increases biomass production

Individual samples of biomass were grown as provided in Example 1, but the viscosity of the feedstock was varied as indicated in FIG. 8. Biomass dry yield increased with increasing viscosity of feedstock, except at the highest viscosity tested (6650 cP). Peak dry yield was observed with feedstocks having viscosities of 1622, 2414, and 4421 cP. Example 10. Dry inoculum increases biomass production

Liquid and dry spawn inocula were prepared as described herein, and used as inoculum to grow biomass as provided in Example 1. Use of dry spawn inoculum unexpectedly led to increased wet biomass production, as compared to wet inoculum as demonstrated in FIG. 9.

Example 11. Effect of different nitrogen salts on protein content of rice

Cunninghamella echinulata is capable of fermenting cooked rice and increasing the total protein content of the rice. A matrix study was conducted to assess the effects of two nitrogen salts, urea and Diammonium Phosphate (DAP), on the final protein content of the rice mixture.

Paddy rice was mechanically de-hulled and ground to a coarse mixture with the particle size distribution shown in Error! Reference source not found.0. Tap water was added to the ground rice and the mixture was adjusted to pH 3.0 using concentrated hydrochloric acid. The mixture was allowed to soak at this pH for 18 hours at room temperature. The rice was removed from the soaking water and allowed to drain. Rice was distributed into mixing bowls and the soaking water was added back to the rice at 0.3% w/w. Mixing bowls with rice and soaking water were autoclaved for 30 minutes at 121 °C and allowed to cool for 60 minutes. Urea and Diammonium Phosphate (DAP) solutions were added to the cooled rice in each mixing bowl as shown in Table 2.

Rice and nitrogen salts were mixed for 30 seconds using an electric stand mixer. Dry Cunninghamella echinulata inoculum was added to each mixing bowl at a rate of 0.2% w/w and the mixture was again mixed for 30 seconds using an electric stand mixer. The inoculated rice mixture was distributed into 4 sterile glass dishes at a depth of 2.5 cm for each treatment. Glass trays were covered with sterile autoclave cloth and incubated for 72 hours at 30 °C, 80% relative humidity. Glass trays with fermented rice were autoclaved for 30 minutes at 121°C to inactivate the fungi. Fermented rice was dried at 65 °C for 24 hours and samples were analyzed for total protein on a Leco nitrogen analyzer (LECO Corporation, St. Joseph, Ml, USA).

The addition of urea or DAP to the rice mixture resulted in increased final protein concentrations in a quadratic fashion as compared to rice without nitrogen amendment. The impact of urea amendment on the final protein concentrations was more substantial than the addition of DAP alone. Maximum protein in the final material was achieved when a combination of urea and DAP was added to the rice mixture (Figure 11). Protein concentration was modeled to reach a maximum (38%) when urea and DAP were added at 20.52 g/kg and 9.4 g/kg, respectively.

Claims

1. A method of making a mycelial biomass, comprising: contacting an edible feedstock with an edible filamentous fungus; and culturing the fungus to produce the mycelial biomass; wherein the filamentous fungus is capable of growth at high temperature and/or low pH, and with no detectable mycotoxin production.

2. The method of claim 1, wherein the fungus grows separately from the feedstock and the mycelial biomass is capable of being separated from the feedstock.

3. The method of claim 1, wherein the fungus infiltrates the feedstock, producing an amalgamation of mycelial biomass and feedstock.

4. The method of any one of claims 1-3, wherein the feedstock comprises a grain, a tuber, or combinations thereof.

5. The method of claim 4, wherein the grain is rice or paddy rice.

6. The method of claim 4 or claim 5, wherein the tuber is potato.

7. The method of any one of claims 1-6, wherein the feedstock comprises rice and potato.

8. The method of any one of claims 1-7, wherein the fungus is of the genus Cunninghamella.

9. The method of any one of claims 1-8, wherein the fungus is selected from Cunninghamella blakesleeana, Cunninghamella echinulata, Cunninghamella elegans, and combinations thereof.

10. The method of any one of claims 1-9, wherein the temperature is about 30 - about 75 °C.

11. The method of any one of claims 1-10, wherein the pH is about 1.0 - about 6.0.

12. The method of any one of claims 1-11, wherein the mycelial biomass is a cohesive mycelial biomass.

13. The method of any one of claims 1-12, wherein the edible filamentous fungus is dehydrated prior to the contacting.

14. The method of any one of claims 1-13, wherein the edible filamentous fungus is conidia, mycelia, or combination thereof.

15. The method of any one of claims 1-14, wherein the mycotoxin is selected from the group consisting of an aflatoxin, a fumonisin, an ochratoxin, a deoxynivalenol, an acetyldeoxynivalenol, a fusarenon, a nivalenol, a T-2 toxin, an HT-2 toxin, a neosolaniol, a diacetoxyscirpenol, a zearalenone, and combinations thereof.

16. The method of claim 15, wherein the aflatoxin is selected from the group consisting of aflatoxin Bl, aflatoxin B2, aflatoxin Gl, aflatoxin G2, and combinations thereof.

17. The method of claim 15 or claim 16, wherein the fumonisin is selected from the group consisting of fumonisin Bl, fumonisin B2, fumonisin B3, and combinations thereof.

18. The method of any one of claims 15-17, wherein the ochratoxin is ochratoxin A.

19. The method of any one of claims 15-18, wherein the fusarenon is fusarenon X.

20. The method of claim 12, wherein the cohesive mycelial biomass can be lifted with one hand without tearing.

21. The method of any one of claims 1-20, wherein the fungus improves edibility and/or a nutritional value of the feedstock.

22. The method of any one of claims 1-21, wherein the feedstock is pretreated prior to contact with the fungus.

23. The method of claim 22, wherein the pretreatment is selected from the group consisting of wetting, boiling, mashing, grinding, dehusking, sieving, chopping, shredding, producing a porridge, and combinations thereof.

24. The method of claim 22 or claim 23, wherein the pretreatment improves the nutritional content and/or one or more physical characteristics of the feedstock.

25. The method of any one of claims 1-24, wherein the feedstock is supplemented with a nitrogen source and/or a nitrogen salt.

26. The method of any one of claims 1-25, wherein the mycelial biomass is produced in about 2 days.

27. The method of any one of claims 1-7 or 10-26, wherein the fungus is of a genus selected from the group consisting of Rhizopus, Monascus, and combinations thereof.

28. The method of any one of claims 1-27, further comprising size reducing the mycelial biomass.

29. The method of any one of claims 3-28, wherein the amalgamation of biomass and feedstock is a food composition.

30. The method of any one of claims 3-28, wherein the amalgamation of biomass and feedstock comprises about 5 wt% - about 90 wt% on a dry weight basis of mycelial biomass.

31. A method of making a food composition, comprising: contacting an edible feedstock with an edible filamentous fungus of the genus Cunninghamella that is capable of growth at high temperature and/or low pH, and with no detectable mycotoxin production; and culturing the fungus until it infiltrates the feedstock and produces the food composition.

32. The method of claim 31, wherein the Cunninghamella fungus is selected from Cunninghamella blakesleeana, Cunninghamella echinulata, Cunninghamella elegans, and combinations thereof.

33. The method of claim 31 or claim 32, wherein the feedstock comprises a grain, a tuber, or combination thereof.

34. The method of any one of claims 31-33, wherein the feedstock comprises rice and potato.

35. The method of any one of claims 31-34, wherein the temperature is about 30 - about 75 °C.

36. The method of any one of claims 31-35, wherein the pH is about 1.0 - about 6.0.

37. The method of any one of claims 31-36, wherein the food composition comprises a mycelial biomass.

38. The method of any one of claims 31-37, wherein the Cunninghamella fungus is dehydrated prior to the contacting.

39. The method of any one of claims 31-38, wherein the edible filamentous fungus is conidia, mycelia, or combination thereof.

40. The method of any one of claims 31-39, wherein the mycotoxin is selected from the group consisting of an aflatoxin, a fumonisin, an ochratoxin, a deoxynivalenol, an acetyldeoxynivalenol, a fusarenon, a nivalenol, a T-2 toxin, an HT-2 toxin, a neosolaniol, a diacetoxyscirpenol, a zearalenone, and combinations thereof.

41. The method of claim 40, wherein the aflatoxin is selected from the group consisting of aflatoxin Bl, aflatoxin B2, aflatoxin Gl, aflatoxin G2, and combinations thereof.

42. The method of claim 40 or claim 41, wherein the fumonisin is selected from the group consisting of fumonisin Bl, fumonisin B2, fumonisin B3, and combinations thereof.

43. The method of any one of claims 40-42, wherein the ochratoxin is ochratoxin A.

44. The method of any one of claims 40-43, wherein the fusarenon is fusarenon X.

45. The method of any one of claims 31-44, wherein the food composition comprises a cohesive mycelial biomass that can be lifted with one hand without tearing.

46. The method of any one of claims 31-45, wherein the Cunninghamella fungus improves edibility and/or a nutritional value of the feedstock.

47. The method of any one of claims 31-46, wherein the feedstock is pretreated prior to contact with the fungus.

48. The method of claim 47, wherein pretreatment is selected from the group consisting of wetting, boiling, mashing, grinding, dehusking, sieving, chopping, shredding, producing a porridge, and combinations thereof.

49. The method of claim 47 or claim 48, wherein the pretreatment improves the nutritional content and/or one or more physical characteristics of the feedstock.

50. The method of any one of claims 31-49, wherein the feedstock is supplemented with a nitrogen source and/or a nitrogen salt.

51. The method of any one of claims 31-50, wherein the food composition is produced in about 2 days.

52. The method of any one of claims 31-51, further comprising size reducing the food composition.

53. A method of making a food composition, comprising: contacting a feedstock with a fungus of the genus Cunninghamella; culturing the fungus to form a mycelial biomass; harvesting a mycelial biomass; and processing the mycelial biomass to produce the food composition.

54. The method of claim 53, wherein the Cunninghamella fungus is capable of growth at high temperature and/or low pH, and with no detectable mycotoxin production.

55. The method of claim 53 or claim 54, further comprising, prior to the processing, inactivating the mycelial biomass.

56. The method of any one of claims 53-55, wherein the fungus grows separately from the feedstock and the mycelial biomass is capable of being separated from the feedstock.

57. The method of any one of claims 53-55, wherein the fungus infiltrates the feedstock, producing an amalgamation of mycelial biomass and feedstock.

58. The method of any one of claims 53-57, wherein the culturing is selected from submerged fermentation, liquid surface fermentation, solid surface fermentation, and supported fermentation.

59. The method of any one of claims 53-58, wherein the food composition is essentially free of residual feedstock.

60. The method of any one of claims 53-59, wherein the food product is selected from a milk analog, a mycelial flour, a tempeh analog, a meat analog, a tofu analog, and textured vegetable protein analog.

61. The method of any one of claims 53-60, wherein the feedstock is supplemented with an organic nitrogen source and the food composition has high protein content.

62. A method of preparing a fungal inoculum, comprising: wetting a feedstock; boiling the feedstock; contacting the feedstock with a fungal culture; fermenting the fungal culture to produce a fungal biomass; dehydrating the fungal biomass; and size-reducing the fungal biomass.

63. The method of claim 62, further comprising removing excess liquid from the boiled feedstock.

64. The method of claim 62 or claim 63, wherein the dehydration occurs by spray drying.

65. The method of any one of claims 62-64, wherein the size-reducing comprises grinding the fungal biomass.

66. The method of any one of claims 62-65, wherein the fungal culture comprises an edible filamentous fungus of the genus Cunninghamella that is capable of growth at high temperature and/or low pH, and with no detectable mycotoxin production.

67. A food composition, comprising a fungus of the genus Cunninghamella.

68. The food composition of claim 67, further comprising a feedstock.

69. The food composition of claim 67 or claim 68, wherein the fungus has been size reduced.

70. The food composition of claim 69, wherein size is reduced by cutting, chopping, dicing, mincing, grinding, blending, sonicating, or combinations thereof.

71. The food composition of any one of claims 67-70, wherein the fungus comprises all essential amino acids.

72. The food composition of any one of claims 67-71, wherein the fungus displays no detectable mycotoxin production and the mycotoxin is selected from the group consisting of an aflatoxin, a fumonisin, an ochratoxin, a deoxynivalenol, an acetyldeoxynivalenol, a fusarenon, a nivalenol, a T- 2 toxin, an HT-2 toxin, a neosolaniol, a diacetoxyscirpenol, a zearalenone, and combinations thereof.