Food ingredients containing filamentous fungal particles and membrane bioreactor design

Abstract

We provide food ingredients that include edible filamentous fungi. [Solution] A food ingredient is provided that contains particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than approximately 40 wt.% and an RNA content of less than approximately 8 wt.%. Also provided is a method for producing an edible filamentous fungal biomat preparation and a disposable or reusable self-sufficient biomat reactor comprising at least one compartment, a container in which the compartment contains a supply material, fungal inoculant material, a gas permeable membrane, and optionally a liquid nutrient medium.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the benefit of priority based on U.S. Provisional Patent Application 62 / 811,421, filed Feb. 27, 2019, which is incorporated herein by reference in its entirety.

[0002] Technical field This application relates to edible filamentous fungi, and provides a method for producing edible fungi for use in food, liquid and solid products of edible fungi and associated uses and methods, foodstuffs containing edible filamentous fungi and methods and uses thereof.

Background Art

[0003] background The United Nations announced in August 2017 that the world's population reached 7.5 billion, and predicted that it will increase to 8 billion by 2023 and 10 billion by 2056. In a related report, the Food and Agriculture Organization of the United Nations (FAO) estimates that if the world's population increases to 9.1 billion by 2050, world food production needs to increase by 70%, and double in developing countries. This increase in food production needs to be achieved regardless of increased energy costs, reduced groundwater resources, loss of agricultural land due to unregulated urban expansion, and severe climate changes due to climate change (such as rising temperatures, increasing droughts, increasing floods, etc.). This is a particular challenge for countries such as those in Africa where protein intake was already insufficient according to 2009 figures, as well as countries such as China, India, Pakistan and Indonesia where there is a risk of insufficient protein intake. Furthermore, global demand is expected to increase by 60% for meat and 50% for dairy products by 2040.

[0004] However, not all protein sources are equally suitable for building complete proteins. Animal-based foods (meat, eggs, dairy products) provide "complete" proteins because they contain all the essential amino acids: methionine, leucine, isoleucine, phenylalanine, valine, threonine, histidine, tryptophan, and lysine. Plant-based foods, while containing some essential amino acids, generally do not provide a complete set. For example, the proteins found in starchy root vegetables lack the essential amino acid lysine and must therefore be obtained from other foods in the diet. Legumes and Fabaceae contain high levels of lysine but lack the essential amino acid methionine. While it is possible to construct complete proteins by combining plant foods, it is far easier to ensure a nutritionally balanced diet with complete proteins.

[0005] One non-animal source of complete protein is obtained from edible filamentous fungi such as Fusarium venenatum (formerly classified as Fusarium graminearum). However, to date, protein production from these resources requires enormous investments in energy resources and manufacturing equipment, such as capital-intensive bioreactors and centrifuges. The need for inexpensive cultivation, harvesting, and food production methods that require less energy and consume fewer natural resources remains unresolved. This invention solves these problems.

[0006] Furthermore, one area related to the reduction of logistical supplies associated with responding to natural disasters, logistically isolated environments, or military and / or space / extraterrestrial missions is the containment of life support loops, particularly waste spills, while providing essential products for operations such as nutritious and appetite-stimulating food, fuel, metabolite expression platforms, building materials, and / or microbial factories. These types of environments often lack or have limited sterile facilities available and / or require sealed sterile systems to adequately contain waste spills and / or food, fuel, and resulting materials. For example, the European Space Agency's study (Expeditions 25-28, Growth and Survival of Colored Fungi in Space (CFS-A)) showed that fungi can grow inside a space station and decompose food and other organic materials under humid conditions; here, containment of the fungal system is paramount to prevent accidental contamination of other supplies and surfaces. In addition to the need for food and waste decomposition in the developing field of space travel, these needs also exist when dealing with natural disasters, in theater military operations, wilderness activities, situations in the Third World where public health and refrigeration are unreliable, in enclosed spaces, in areas of logistical difficulty, and in certain agricultural / industrial activities. Self-sufficient sterile systems that operate efficiently with minimal space, energy, and maintenance are required.

[0007] A robust and efficient portable self-sufficient biomat reactor system capable of converting a wide range of waste spills into many valuable products addresses these problems. This invention describes a simple sterile bioreactor platform that produces a dense, easily harvestable textured biomat, requiring no agitation, active aeration, or external energy source (other than temperature control) during fermentation, producing little to no waste residue, and requiring minimal water. Furthermore, the self-sufficient biomat reactor system may be portable and / or scalable for larger, more intensive operations and / or groups. [Overview of the project]

[0008] overview One aspect of the present invention is to provide a food ingredient containing particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than approximately 40 wt.% and an RNA content of less than approximately 8 wt.%.

[0009] In one embodiment, filamentous fungi belong to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, and Pleurotaceae. It may belong to a family selected from the following: rotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

[0010] In one embodiment, the filamentous fungi include Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.It may belong to a group of species selected from the genus Leucoagaricus, which includes PTA-10698, Disciotis venosa, Clonostachys rosea, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and Leucoagaricus spp.

[0011] In one embodiment, the filamentous fungus may be a Fusarium species.

[0012] In one embodiment, the filamentous fungus may be Fusarium venenatum.

[0013] In one embodiment, the filamentous fungus may be strain MK7 (ATCC Accession Deposit No. PTA-10698).

[0014] In one embodiment, the filamentous fungus may have characteristics selected from the group consisting of (a) having a protein content of more than approximately 45 wt.%; (b) having a protein content of more than approximately 50 wt.%; (c) having a protein content of more than approximately 55 wt.%; (d) having a protein content of more than approximately 60 wt.%; (e) having an RNA content of less than approximately 5 wt.%; (f) having an RNA content of less than approximately 4 wt.%; (g) having an RNA content of less than approximately 3 wt.%; (h) having an RNA content of less than approximately 2 wt.%; and a combination of one of a-d and one of e-h.

[0015] In one embodiment, the filamentous fungus may contain less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zearalenone, beuvericin, fusarin C, fusaric acid, and any combination thereof.

[0016] The filamentous fungi may contain a mycotoxin content of less than approximately 10 ppm, less than approximately 9 ppm, less than approximately 8 ppm, less than approximately 7 ppm, less than approximately 6 ppm, less than approximately 5 ppm, less than approximately 4 ppm, less than approximately 3 ppm, less than approximately 2 ppm, less than approximately 1 ppm, less than approximately 0.9 ppm, less than approximately 0.8 ppm, less than approximately 0.7 ppm, or less than approximately 0.6 ppm, or equal to 1 / 10 ppm of any of these, or a total mycotoxin content of less than 10 ppm. In specific embodiments, the selected mycotoxin may be fumonisin or a combination of fumonisins, beuvericin, fusarin C, fusaric acid, or a combination thereof.

[0017] In one embodiment, the filamentous fungus may contain a total mycotoxin content of less than approximately 10 ppm, or less than approximately 9 ppm, or less than approximately 8 ppm, or less than approximately 7 ppm, or less than approximately 6 ppm, or less than approximately 5 ppm, or less than approximately 4 ppm, or less than approximately 3 ppm, or less than approximately 2 ppm, or less than approximately 1 ppm, or less than approximately 0.9 ppm, or less than approximately 0.8 ppm, or less than approximately 0.7 ppm, or less than approximately 0.6 ppm, or equal to 1 / 10 ppm of any of these, or a total mycotoxin content of less than 10 ppm.

[0018] In one embodiment, the filamentous fungus may contain more than approximately 15 wt.% of branched-chain amino acids.

[0019] In one embodiment, the particles of filamentous fungi may be in the form of a powder. The powder may have a particle size of 30-400 microns, but this is not essential. The powder may be approximately 400 microns, approximately 390 microns, approximately 380 microns, approximately 370 microns, approximately 360 microns, approximately 350 microns, approximately 340 microns, approximately 330 microns, approximately 320 microns, approximately 310 microns, approximately 300 microns, approximately 290 microns, approximately 280 microns, approximately 270 microns, approximately 260 microns, approximately 250 microns, approximately 240 microns, approximately 230 microns, approximately 220 microns, approximately 210 microns, approximately 200 microns, approximately 190 microns, approximately 180 microns, approximately 170 microns, approximately 160 microns, approximately 1 The particle diameter is not greater than 50 microns, not greater than approximately 140 microns, not greater than approximately 130 microns, not greater than approximately 120 microns, not greater than approximately 110 microns, not greater than approximately 100 microns, not greater than approximately 90 microns, not greater than approximately 80 microns, not greater than approximately 70 microns, not greater than approximately 60 microns, not greater than approximately 50 microns, not greater than approximately 40 microns, not greater than approximately 30 microns, not greater than approximately 20 microns, not greater than approximately 10 microns, not greater than approximately 9 microns, not greater than approximately 8 microns, not greater than approximately 7 microns, not greater than approximately 6 microns, not greater than approximately 5 microns, not greater than approximately 4 microns, not greater than approximately 3 microns, not greater than approximately 2 microns, or not greater than approximately 1 micron, or any approximately integer micron between approximately 1 micron and approximately 400 microns. 10 Particle size, D 25 Particle size, D 50 Particle size, D 75 Particle size, D 90The particle diameter or weight-average particle diameter may be 1 or more. In one embodiment, substantially all particles may have a particle diameter of at least about 30 microns and not exceeding about 400 microns.

[0020] In one embodiment, the particles may have a particle length of approximately 0.05 mm to approximately 500 mm, a particle width of approximately 0.03 mm to approximately 7 mm, and a particle height of approximately 0.03 mm to approximately 1.0 mm. All particles may, but are not required, have a particle length, particle width, and particle height exceeding approximately 0.02 mm, approximately 0.03 mm, approximately 0.04 mm, approximately 0.05 mm, approximately 0.06 mm, approximately 0.07 mm, approximately 0.08 mm, approximately 0.09 mm, approximately 0.10 mm, approximately 0.11 mm, approximately 0.12 mm, approximately 0.13 mm, approximately 0.14 mm, approximately 0.15 mm, approximately 0.16 mm, approximately 0.17 mm, approximately 0.18 mm, approximately 0.19 mm, or approximately 0.20 mm, or any nearly integer micron that is at least approximately 20 microns.

[0021] In one embodiment, the food ingredient may be a liquid dispersion of filamentous fungal particles. The liquid dispersion may be produced under nitrogen, but this is not essential. The liquid dispersion of filamentous fungal particles may be stable for at least about one day, but this is not essential.

[0022] In one embodiment, the ingredients can be vegan.

[0023] In one embodiment, fungal particles may be the only protein component present in the food.

[0024] In one embodiment, the fungal particles may contain all essential amino acids. The fungal particles may, but may not contain, at least one branched-chain amino acid selected from the group consisting of leucine, isoleucine, and valine.

[0025] In one embodiment, the filamentous fungal particles may be unsustainable.

[0026] Another aspect of the present invention is to provide a yogurt-like food containing particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than about 40 wt.% and an RNA content of less than about 8 wt.%.

[0027] In one embodiment, filamentous fungi include the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, and Pleurotaceae. It may belong to a family selected from the group consisting of aceae, Tuberaceae, Morchellaceae, Sparassidaceae, Physalacriaceae, Omphalotaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

[0028] In one embodiment, the filamentous fungi include Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.It may belong to a group of species selected from the genus Leucoagaricus, which includes PTA-10698, Disciotis venosa, Clonostachys rosea, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and Leucoagaricus spp.

[0029] In one embodiment, the filamentous fungus may be a Fusarium species.

[0030] In one embodiment, the filamentous fungus may be Fusarium venenatum.

[0031] In one embodiment, the filamentous fungus may be strain MK7 (ATCC Accession Deposit No. PTA-10698).

[0032] In one embodiment, the filamentous fungus may have characteristics selected from the group consisting of (a) having a protein content of more than approximately 45 wt.%; (b) having a protein content of more than approximately 50 wt.%; (c) having a protein content of more than approximately 55 wt.%; (d) having a protein content of more than approximately 60 wt.%; (e) having an RNA content of less than approximately 5 wt.%; (f) having an RNA content of less than approximately 4 wt.%; (g) having an RNA content of less than approximately 3 wt.%; (g) having an RNA content of less than approximately 2 wt.%; and a combination of one of a to d and one of e to h.

[0033] In one embodiment, the ratio of filamentous fungal particles to water may be in the range of about 1:10 to about 10:1.

[0034] In one embodiment, the ratio of filamentous fungal particles to water can be selected from the group consisting of about 1:3, about 1:2, about 1:1, and about 2:1.

[0035] In one embodiment, the yogurt-like food may further contain invert sugar.

[0036] In one embodiment, the yogurt-like food may further contain a thickening agent.

[0037] In one embodiment, the cells of a filamentous fungus can be lysed.

[0038] In one embodiment, the yogurt-like food may further contain Lactobacillus bulgaricus and Streptococcus thermophilus.

[0039] In one embodiment, the product may be vegan.

[0040] In one embodiment, the filamentous fungal particles may contain all essential amino acids.

[0041] In one embodiment, the filamentous fungal particles may be the sole protein component.

[0042] In one embodiment, the filamentous fungal particles may be unsustainable.

[0043] In one embodiment, the yogurt-like food may further contain rennet. The rennet may be of a source selected from the group consisting of animal, plant, and microbial sources, but this is not required. The rennet may be of a source selected from the group consisting of plant and microbial origins, but this is not required.

[0044] In one embodiment, the product does not need to contain milk solids.

[0045] In one embodiment, the yogurt-like food may further contain probiotics.

[0046] In one embodiment, the yogurt-like food may further contain enzyme water.

[0047] Another aspect of the present invention is the provision of a foaming material comprising filamentous fungal biomat particles and a liquid phase, wherein the solid component of the foaming material is about 5% to about 30%, and the foam is stable. The liquid phase may be an aqueous phase, i.e., it may contain water, but this is not essential.

[0048] In one embodiment, the foamed material may not spontaneously collapse immediately after the foaming process is stopped during its production.

[0049] In one embodiment, the foaming material may be stable for at least about 7 days.

[0050] In one embodiment, the foam material may have an overrun of at least about 10%.

[0051] In one embodiment, the filamentous fungal biomat may contain Fusarium species.

[0052] In one embodiment, the foaming material does not need to contain milk solids.

[0053] Another aspect of the present invention is the provision of a food product containing the foaming material.

[0054] Another aspect of the present invention provides a bioreactor comprising a container; at least one membrane disposed inside or on the surface of the container, including a first surface and a second surface; a feed material for the growth of filamentous fungi in contact with the first surface of at least one membrane; and a filamentous fungal inoculant material disposed on the first or second surface of at least one membrane, wherein, by culturing the inoculant material in the bioreactor, a biomat of the filamentous fungi is formed on the second surface of at least one membrane after the biomat growth phase. The term “membrane” as used herein means any flexible or semi-flexible enclosing or separating portion that forms a plane or film and has pores that separate two environments and allow for the exchange of at least a portion of the fluid between the two environments.

[0055] In one embodiment, the container is a bag, where the first and second surfaces of at least one membrane are the first and second surfaces of at least a portion of the bag.

[0056] In one embodiment, the supply material may be subjected to positive or negative pressure applied to at least one of the first and second surfaces opposite each other of at least one of the membranes.

[0057] In one embodiment, the bioreactor may further include cyanobacteria, which provide at least one of oxygen gas and carbon to promote the growth of the biomat.

[0058] In one embodiment, at least one of the following may be true: i) the density of the biomat is at least about 0.05 grams / cubic centimeter; and ii) the density of the biomat after drying is at least about 0.01 grams / cubic centimeter.

[0059] In one embodiment, the biomat may include at least one layer.

[0060] In one embodiment, the biomat may have a tensile strength of at least about 3 kilopascals or at least about 30 grams per square centimeter. The biomat may have a tensile strength of at least about 100 kilopascals or at least about 1,020 grams per square centimeter, but this is not required.

[0061] In one embodiment, the film may comprise at least one polymer selected from the group consisting of polypropylene, polytetrafluoroethylene, polycarbonate, polyamide, cellulose acetate, polyvinylidene fluoride, mixed cellulose esters, polyethersulfone, polyethylene, and polypyrrole.

[0062] In one embodiment, at least one membrane may comprise at least one material selected from the group consisting of polypropylene cloth, polytetrafluoroethylene cloth, and nylon net filter.

[0063] In one embodiment, at least one of the films may include at least one of a glass fiber material and a porous ceramic material.

[0064] In one embodiment, the average pore size of at least one membrane may be approximately 0.2 μm to approximately 25 μm. The average pore size of at least one membrane may be approximately 5 μm to approximately 11 μm, but is not required.

[0065] In one embodiment, the container may be sealed and substantially airtight, and the container encloses a gas head space in which the biomat grows.

[0066] In one embodiment, the biomat can spontaneously separate from at least one membrane.

[0067] In one embodiment, when the biomat is removed from at least one membrane, new inoculum of filamentous fungi may remain on at least one membrane.

[0068] In one embodiment, filamentous fungi may belong to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

[0069] In one embodiment, the filamentous fungi may belong to a family selected from the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae.

[0070] In one embodiment, the filamentous fungi include strain MK7 (ATCC Accession Deposit No. PTA-10698), Fusarium venenatum, Rhizopus oligosporus, Ustilago esculenta, Heliculum erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, and Agaricus bisporus. bisporus), Stropharia rugosoannulata, Hypholoma lateritium, Pluerotus eryngii, Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa, Ophiocordyceps sinensis and Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina vertipes velutipes), Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, Leucoagaricus holosericeus, Calvatia fragilisThe group may be selected from the species *Pholiota fragilis*, *Handkea utriformis*, and *Pholiota adiposa*.

[0071] In one embodiment, the feedstock may include at least one of animal feces and animal urine. The animal may be human, but is not required.

[0072] In one embodiment, at least one film may be a single composite film, where the first surface comprises a first material and the second surface comprises a second material.

[0073] In one embodiment, at least one film may include at least a first film and a second film, where the first surface is the surface of the first film and the second surface is the surface of the second film. The first and second films may, but are not required to, be in physical contact with each other.

[0074] In one embodiment, the bioreactor may further include a selective gas permeable membrane, where a first gas produced during the growth of the biomat is selectively separated into the gas headspace on the first side of the selective gas permeable membrane. A second gas produced during the growth of the biomat may be selectively separated into the gas headspace on the second side of the membrane, but this is not mandatory. In one embodiment, a gas dissolved or dispersed in the liquid feed material or otherwise positioned on the feed material side of the membrane may be selectively separated from the feed material and pass through to the other side of the membrane.

[0075] Another aspect of the present invention provides a method for producing a biomat of filamentous fungi, comprising inoculating a bioreactor with filamentous fungi, wherein the bioreactor comprises a container; at least one membrane disposed inside or on the surface of the container, comprising a first surface and a second surface, either or both of which are suitable for receiving the inoculation material of the filamentous fungi; and a feed material for the growth of the filamentous fungi, which is in contact with the first surface of the at least one membrane.

[0076] In one embodiment, the container may be a bag, where the first and second surfaces of at least one membrane are the first and second surfaces of at least a portion of the bag.

[0077] In one embodiment, the feed material may be subjected to positive or negative pressure applied to the feed material side opposite the first surface of at least one membrane. Positive or negative pressure may, but not necessarily, facilitate the inoculation process.

[0078] In one embodiment, the method further comprises providing cyanobacteria to a bioreactor, where the cyanobacteria provide at least one of oxygen gas and carbon to promote the growth of the biomat.

[0079] In one embodiment, at least one of the following may be true: i) the density of the biomat after harvesting is at least about 0.6 grams / cubic centimeter; and ii) the density of the biomat after harvesting and drying is at least about 0.1 grams / cubic centimeter.

[0080] In one embodiment, the biomat may include at least one layer.

[0081] In one embodiment, during or after the harvesting process, the biomat may have a tensile strength of at least about 3 kilopascals or at least about 30 grams per square centimeter. During or after the harvesting process, the biomat may have a tensile strength of at least about 100 kilopascals or at least about 1,020 grams per square centimeter, but this is not required.

[0082] In one embodiment, the film may comprise at least one polymer selected from the group consisting of polypropylene, polytetrafluoroethylene, polycarbonate, polyamide, cellulose acetate, polyvinylidene fluoride, mixed cellulose esters, polyethersulfone, polyethylene, and polypyrrole.

[0083] In one embodiment, at least one membrane may comprise at least one material selected from the group consisting of polypropylene cloth, polytetrafluoroethylene cloth, and nylon net filter.

[0084] In one embodiment, the film may include at least one of a glass fiber material and a porous ceramic material.

[0085] In one embodiment, the average pore size of at least one membrane may be approximately 0.2 μm to approximately 25 μm. The average pore size of at least one membrane may be approximately 5 μm to approximately 11 μm, but this is not required.

[0086] In one embodiment, the container may be sealed and substantially airtight, and the container encloses a gas head space in which the biomat grows.

[0087] In one embodiment, the biomat can spontaneously separate from at least one membrane.

[0088] In one embodiment, the method may further include collecting a biomat, where, when the biomat is removed from at least one membrane, a fresh inoculum of filamentous fungi remains on at least one membrane.

[0089] In one embodiment, filamentous fungi may belong to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

[0090] In one embodiment, the filamentous fungi may belong to a family selected from the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae.

[0091] In one embodiment, the filamentous fungi include strain MK7 (ATCC Accession Deposit No. PTA-10698), Fusarium venenatum, Rhizopus oligosporus, Ustilago esculenta, Heliculum erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, and Agaricus bisporus. The group may be selected from the following species: bisporus, Stropharia rugosoannulata, Hypholoma lateritium, Pluerotus eryngii, Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa, Ophiocordyceps sinensis, and Cordyceps militaris.

[0092] In one embodiment, the feedstock may include at least one of animal feces and animal urine. The animal may be human, but is not required.

[0093] In one embodiment, at least one film may be a single composite film, where the first surface comprises a first material and the second surface comprises a second material.

[0094] In one embodiment, the film may include at least one film, at least a first film and a second film, where the first surface is the surface of the first film and the second surface is the surface of the second film. The first and second films may, but may not, be in physical contact with each other.

[0095] In one embodiment, the bioreactor may further include a selective gas permeable membrane, where a first gas produced during the growth of the biomat is selectively separated into the gas headspace on the first side of the selective gas permeable membrane. A second gas produced during the growth of the biomat may be selectively separated into the gas headspace on the second side of the membrane, but this is not essential.

[0096] Another aspect of the present invention provides a method for producing fresh water, comprising: inoculating a bioreactor containing a container and a feed material for the growth of filamentous fungi with filamentous fungi; culturing the filamentous fungi to form a biomat on at least one of the surface of the feed material and the surface of the bioreactor membrane, wherein the filamentous fungi produce water as a byproduct during the formation or growth of the biomat; and collecting the water produced by the formation or growth of the biomat.

[0097] In one embodiment, the feedstock may include at least one of animal feces and animal urine. The animal may be human, but is not required.

[0098] In one embodiment, the method may further include recirculating the collected water to a bioreactor.

[0099] In one embodiment, the method may further include preparing a feedstock containing the collected water. The method may further include, but is not required, recirculating the prepared feedstock containing the collected water to a bioreactor.

[0100] Another aspect of the present invention provides a method for producing gas, comprising: inoculating a bioreactor containing a container and a feed material for the growth of filamentous fungi; culturing the filamentous fungi to form a biomat on at least one of the surface of the feed material and the surface of the bioreactor membrane, wherein the filamentous fungi produce gas as a metabolic byproduct during the growth of the biomat; and collecting the gas produced by the growth of the biomat.

[0101] In one embodiment, the gas may be selected from the group consisting of ammonia, ammonium species, hydrogen gas, and volatile esters.

[0102] One aspect of the present invention provides a method for producing a filamentous fungal biomat, comprising: (a) inoculating an effective amount of cells of at least one filamentous fungus into a first aliquot of a growth medium to produce an inoculated growth medium; (b) incubating the inoculated growth medium for a first time to produce an initial biomat; (c) removing at least a portion of the first aliquot of the growth medium and adding a second aliquot of the growth medium to provide a refreshed growth medium; and (d) incubating the refreshed growth medium for a second time to produce a final biomat.

[0103] In one embodiment, the dry mass density of the final biomat may be at least about 75 grams / liter.

[0104] In one embodiment, the biomat may contain more than approximately 40 wt% protein and less than approximately 8 wt% RNA.

[0105] In one embodiment, at least one filamentous fungus may belong to an order selected from the group consisting of Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

[0106] In one embodiment, at least one filamentous fungus may belong to a family selected from the group consisting of Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae.

[0107] In one embodiment, at least one filamentous fungus is strain MK7 (ATCC Accession Deposit No. PTA-10698), Fusarium venenatum, Ustilago esculenta, Heliculum erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoanulata The group is selected from the following species: rugosoannulata, Hypholoma lateritium, Pluerotus eryngii, Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa, Ophiocordyceps sinensis, and Cordyceps militaris.

[0108] In one embodiment, the final biomat may have the following characteristics selected from the group consisting of (a) a protein content greater than approximately 45 wt.%; (b) a protein content greater than approximately 50 wt.%; (c) a protein content greater than approximately 55 wt.%; (d) a protein content greater than approximately 60 wt.%; (e) an RNA content of less than approximately 5 wt.%; (f) an RNA content of less than approximately 4 wt.%; (g) an RNA content of less than approximately 3 wt.%; (h) an RNA content of less than approximately 2 wt.%; and (i) a combination of one of a to d and one of e to h.

[0109] In one embodiment, the final biomat may contain less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zeararenone, beuvericin, fusarin C, fusaric acid, and any combination thereof.

[0110] In one embodiment, the final biomat may contain a total mycotoxin content of less than approximately 10 ppm.

[0111] In one embodiment, the final biomat may contain a total mycotoxin content of less than approximately 5 ppm.

[0112] In one embodiment, the final biomat may contain more than approximately 15 wt.% of branched-chain amino acids.

[0113] Another aspect of the present invention provides a biomat of at least one filamentous fungus having a dry mass density of at least about 75 grams / liter.

[0114] In one embodiment, the biomat may contain more than approximately 40 wt% protein and less than approximately 8 wt% RNA.

[0115] In one embodiment, at least one filamentous fungus may belong to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

[0116] In one embodiment, at least one filamentous fungus belongs to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, and Pleurotaceae. It may belong to a family selected from the group consisting of eae, Physalacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

[0117] In one embodiment, at least one filamentous fungus is Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus bisporus), Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum venenatum), strain MK7 (ATCC Accession Deposit No.The group is selected from PTA-10698), Disciotis venosa and Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and the genus Leucoagaricus spp.

[0118] In one embodiment, the biomat may have the following characteristics selected from the group consisting of (a) a protein content greater than approximately 45 wt.%; (b) a protein content greater than approximately 50 wt.%; (c) a protein content greater than approximately 55 wt.%; (d) a protein content greater than approximately 60 wt.%; (e) an RNA content of less than approximately 5 wt.%; (f) an RNA content of less than approximately 4 wt.%; (g) an RNA content of less than approximately 3 wt.%; (h) an RNA content of less than approximately 2 wt.%; and (i) a combination of one of a to d and one of e to h.

[0119] In one embodiment, the biomat may contain less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zeararenone, fusarin C, fusaric acid, and any combination thereof.

[0120] In one embodiment, the biomat may contain a total mycotoxin content of less than approximately 10 ppm.

[0121] In one embodiment, the biomat may contain a total mycotoxin content of less than approximately 5 ppm.

[0122] In one embodiment, the biomat may contain more than approximately 15 wt.% of branched-chain amino acids.

[0123] In one embodiment, the biomat may be produced by the method described herein.

[0124] Another aspect of the present invention provides a method for producing a biomat of filamentous fungi, comprising inoculating a bioreactor with filamentous fungi, wherein the bioreactor comprises a container; at least one mesh scaffold disposed inside or on the surface of the container, comprising a first surface and a second surface, either or both of which are suitable for receiving the inoculation material of the filamentous fungi; and a feed material for the growth of the filamentous fungi in contact with the first surface of the mesh scaffold.

[0125] In one embodiment, the mesh scaffolding may include nylon material.

[0126] Another aspect of the present invention provides a cultured food comprising filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, with particles of filamentous fungi constituting a protein content of more than about 40 wt.% and an RNA content of less than about 8 wt.%; and a microbial food culture.

[0127] In one embodiment, the microbial food culture may contain lactic acid bacteria.

[0128] Another aspect of the present invention provides a method for producing a cultured food comprising inoculating a microbial food culture with particles of filamentous fungi, wherein the filamentous fungi belong to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales and Hypocreales, and the filamentous fungi contain a protein content of more than about 40 wt.% and an RNA content of less than about 8 wt.%.

[0129] In one embodiment, the microbial food culture may contain lactic acid bacteria.

[0130] In one embodiment, the cultured food described herein may be produced by the method for producing the cultured food described herein. [Brief explanation of the drawing]

[0131] [Figure 1] Growth of Fusarium strain MK7 biomats in nutrient medium that was refreshed daily after the initial 4-day biomat growth stage.

[0132] [Figure 2] (From day 4 onwards) A 3 cm thick biomat of Fusarium strain MK7 formed on a daily refreshed liquid nutrient medium. A nylon mesh screen beneath the biomat is shown, used to lift and move the biomat to keep the medium fresh.

[0133] [Figure 3] A continuous flow system designed to continuously supply Fusarium strain MK7 biomats for growth and remove nutrients from the culture medium. The biomat is shown in grooves after 7 days of growth from the time of inoculation.

[0134] [Figure 4] Bio-mat proliferation after 10 days of growth from the time of inoculation (6 days of continuous flow + 4 days of rest / static conditions).

[0135] [Figure 5] (A) Semi-continuous production of biomat showing the removal of the majority of the mature biomat on day 12. After harvesting one-third of the majority of the mature biomat at the bottom of the tray, the remaining biomat is physically moved downward in the direction of the arrow until the edge of the biomat touches the edge of the tray (B). Moving the biomat creates a fresh, open space on the tray where new biomat is formed.

[0136] [Figure 6] Cumulative biomass production over time using a semi-continuous production method. The dotted line is the linear regression line from day 5 to day 19 (y=0.57x-1.52, r²=0.973). Error bars are the standard deviation of the mean of the three replicated trays. Error bars are not visible when they are smaller than the data point symbols.

[0137] [Figure 7] Continuous production of biomat, with the removal of the majority of the mature biomat shown on the right. The majority of the mature biomat is continuously harvested on the right side of the tray, while simultaneously creating a fresh, open space at the left end of the tray, allowing for the formation of new biomat. The liquid culture medium in the tray can be replenished and / or increased as needed or continuously.

[0138] [Figure 8] The Fusarium MK7 biomat after 4 hours of UVB light irradiation shows orange discoloration (the two cut discs on the right). The two cut discs from a non-irradiated control biomat are shown on the left.

[0139] [Figure 9]Field emission scanning electron microscopy examination of a 4-day-old Fusarium strain MK7 (ATCC Accession Deposit No. PTA-10698) biomat produced using MK7-1 medium supplemented with glycerol, corn starch, and corn steep liquor (as described in PCT / US2017 / 020050). Images A, B, and C show the biomat with extracellular matrix (ECM) removed by ethanol washing. A) Top view of the biomat with aerial mycelium. B) Cross-section of the dense bottom layer with arrows indicating the layers. The cross-section was made by cutting the biomat with a razor. The bottom of the biomat is shown in the lower left corner of the image, and the less adherent transition layer on the dense bottom layer is shown in the upper right corner. C) Bottom surface view of the biomat. D) Bottom surface view of the biomat with in-situ ECM (i.e., ECM not removed by ethanol washing).

[0140] [Figure 10] Transmitted light microscope images (100x magnification) of biomats grown in glycerol, starch, and corn steep liquor. The left image of the aerial mycelial layer confirms the predominantly fibrous, nearly vertical orientation. The right image shows the dense subsomal layer and adjacent transitional layer.

[0141] [Figure 11] Biomat produced using the disclosed method. A: Reishi mushroom; B: Marbled oyster mushroom; C: Oyster mushroom; D: Sparassis crispa; E: White oyster mushroom; F: Giant puffball.

[0142] [Figure 12] Biomat growth in a sealed reactor begins when cells attach to a gas-permeable membrane where oxygen is readily available. Over time, the biomat grows downwards, eventually filling the reactor space and consuming all the fluid and nutrients.

[0143] [Figure 13](A)-(C) Fusarium strain MK7 biomats grown for 5 days under static conditions in petri dishes covered with semipermeable membranes constructed of polypropylene and (D) polycarbonate. Essentially, no free liquid remained in the petri dishes, and all nutrients were absorbed into the biomats. The reactor space / liquid volume was essentially filled with the biomats.

[0144] [Figure 14] A connected bag, separated from the liquid culture medium by a gas-permeable membrane, is used for gas supply and capture. The integrated multi-functional membrane allows for oxygen entry and release of CO2 and other produced gases. The fungal biomass (yellow) growing in the lower liquid compartment converts the supplied material and nutrients into a biomat, which fills the compartment as it grows. The dense, integrated biomat can be easily collected and removed from the bag by opening the reactor sealing system (e.g., Ziploc® type).

[0145] [Figure 15] Basic reactor (1). Multiple grooves (4) sharing a wall / baffle (9), a front valve (6) and a back valve (8), and a gas permeable membrane (2) are shown.

[0146] [Figure 16] A basic sealed reactor (1) having a single gas collection chamber (14).

[0147] [Figure 17] A basic sealed reactor (1) having grooved gas collection chambers (15, 20).

[0148] [Figure 18] A basic sealed reactor (1) having a grooved gas collection chamber (15) having gas-specific grooves (30, 40) equipped with gas-specific permeable membranes (2, 50).

[0149] [Figure 19]A basic sealed reactor (1) having a cylindrical groove (4), walls / baffles (9), a front valve (6) and a back valve (8) and a gas permeable membrane (2).

[0150] [Figure 20] Refractive index, density, and particle size analysis of 10- to 1000-fold dilutions of vegan milk produced using filamentous fungi.

[0151] [Figure 21] Structure of vegan milk under an optical microscope.

[0152] [Figure 22] Plot of viscosity of vegan milk samples against shear rate.

[0153] [Figure 23] A generalized schematic diagram of various bioreactor configurations.

[0154] [Figure 24] An illustration of a sealed embodiment of bioreactor configuration "1".

[0155] [Figure 25] An illustration of a sealed embodiment of bioreactor configuration "4".

[0156] [Figure 26] The process of biomat growth in a bag reactor.

[0157] [Figure 27] An illustration of a sealed embodiment of bioreactor configuration "4".

[0158] [Figure 28] An illustration of a bioreactor that utilizes "biomembrane."

[0159] [Figure 29] An illustrative schematic diagram of a bioreactor containing photosynthetic cyanobacteria.

[0160] [Figure 30] An illustration of the biomat produced in a raw material comparison test.

[0161] [Figure 31] Diagram of a continuous supply, back pressure relief bioreactor.

[0162] [Figure 32] Illustration of biomat growth in a membrane-bag biofilm reactor.

[0163] [Figure 33] An illustration of a nylon net filter membrane.

[0164] [Figure 34] Comparison of biomats grown under light and dark conditions.

[0165] [Figure 35] Figure 35A: Diagram of a membrane envelope bioreactor (MEBR).

[0166] Figure 35B: An inset of Figure 35A showing details of the membrane envelope bioreactor.

[0167] [Figure 36] Biomat growth rate in trays with and without refreshing the growth medium. [Modes for carrying out the invention]

[0168] Detailed description As used herein, the term "biomat" refers to a sticky mass of filamentous fungal tissue, including a network of interwoven hyphal fibers, unless otherwise specified. A biomat may, but is not required, be characterized by a density of approximately 50 to 200 grams / liter, approximately 5 wt% to 20 wt% solid components, and a tensile strength sufficient to lift it substantially intact from the surface of the growing medium. As used herein, the term "extracellular matrix" refers, unless otherwise specified, to the extracellular material that at least partially surrounds the filamentous fungal structure of a biomat, protecting, supporting, and / or isolating the fungal mycelium of the biomat from the surrounding environment. The extracellular matrix, as the term is used herein, generally includes, but is not limited to, a variety of macromolecules including proteoglycans (e.g., heparin sulfate, chondroitin sulfate, keratan sulfate), non-proteoglycan polysaccharides (e.g., hyaluronic acid), and proteins (e.g., collagen, elastin).

[0169] Edible filamentous fungi can be used alone or incorporated into food as a source of nutrients such as protein.

[0170] While the fruiting bodies of Basidiomycota and Ascomycota fungi are used in food, there are few products that primarily contain plant mycelium of Basidiomycota or Ascomycota fungi. This is partly because the mycelium is generally located underground or largely inseparable from the material that supports its growth.

[0171] Furthermore, under specific conditions, filamentous fungi can form fungal biomats via surface fermentation under anaerobic, microaerophilic, or aerobic conditions, or a combination thereof. Here, the filamentous fungal biomat mainly consists of mycelium, fragments of mycelium, hyphae, fragments of hyphae, and to a lesser extent conidia, microconidia, macroconidia, or any and all combinations thereof, and may also include, in some cases, conidiophores, chlamydospores, and parts of the extracellular matrix.

[0172] Generally, filamentous fungal biomats consist mainly of mycelium, i.e., a complex network of interwoven plant mycelial fibers. The average length of non-destructive fibers within a biomat is generally at least 0.1 mm, and is defined by any two integers between 0.1 mm and 100 cm, for example, between 0.1 mm and 100 cm or between 1 mm and 100 cm. In one embodiment, the average length may be at least 0.1 mm, 0.25 mm, 0.5 mm, 1.0 mm, 1.4 mm, 1.6 mm, 1.7 mm, 1.8 mm, 2 mm, 2.5 mm, 5 mm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 85 cm, or 100 cm or any value in between.

[0173] The food items listed here include edible filamentous fungi, particularly those that grow as biomats before being processed into particles.

[0174] Filamentous fungi suitable for use in the present invention (as particles in biomats or food ingredients) may be selected from the phyla Zygomycota, Glomermycota, Chytridiomycota, Basidiomycota, or Ascomycota. Basidiomycota includes, among other things, the Agaricales, Russulales, Polyporales, and Ustilaginales; Ascomycota includes, among other things, the Pezizales and Hypocreales; and Zygomycota includes, among other things, the Mucorales. The edible filamentous fungal particles of the present invention belong to orders selected from Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, Hypocreales, and Mucorales.

[0175] In one embodiment, filamentous fungi of the order Ustilaginales are selected from the family Ustilaginaceae. In one embodiment, filamentous fungi of the order Russulales are selected from the family Hericiaceae. In one embodiment, filamentous fungi of the order Polyporales are selected from the family Polyporaceae or Grifolaceae. In one embodiment, filamentous fungi of the order Agaricales are selected from the families Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae or Omphalotaceae. In one embodiment, the filamentous fungi of the order Pezizales are selected from the family Tuberaceae or Morchellaceae. In another embodiment, the filamentous fungi of the order Mucorals are selected from the family Mucoraceae.

[0176] In one embodiment, the filamentous fungi may be selected from the genera Fusarium, Aspergillus, Trichoderma, and Rhizopus.

[0177] Examples of filamentous fungal species include Ustilago esculenta, Hericululm erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmariuos (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.This includes, but is not limited to, PTA-10698, Disciotis venosa, Cordyceps militaris, Ganoderma lucidum (Reishi mushroom), Flammulina velutipes, Lentinula edodes, and Ophiocordyceps sinensis. Further examples include, but are not limited to, Trametes versicolor, Ceriporia lacerate, Pholiota gigantea, Leucoagaricus holosericeus, Pleurotus djamor, Calvatia fragilis, Handkea utriformis, and Rhizopus oligosporus.

[0178] In one embodiment, the filamentous fungus is a Fusarium species. In one embodiment, the filamentous fungus is Fusarium strain MK7 (ATCC PTA-10698 deposited in the American Type Culture Collection, 1081 University Boulevard, Manassas, Virginia, USA). Strain MK7 was previously reported as Fusarium oxysporum strain. However, it has since been identified as not being an oxysporum strain. In one embodiment, the filamentous fungus is Fusarium strain venenatum.

[0179] As detailed herein, the filamentous fungi of the present invention have a remarkably high protein content. It is noteworthy that filamentous fungi grown naturally, in the wild, or by prior art methods do not have such a high protein content, whereas filamentous fungi grown or cultured as disclosed herein have a high protein content, and in particular a higher protein content than that achieved in nature or by the prior fermentation methods for certain fungi. For example, the protein content of filamentous fungi described herein refers to the protein content of filamentous fungi grown in the biomat according to this disclosure. Consequently, the ingredients of the present invention have a high protein content based on the filamentous fungal component of the material, without the need for and / or in the absence of protein content from non-filamentous fungal sources. Therefore, in various embodiments, the ingredients of the present invention do not contain or lack protein content from non-filamentous fungal sources.

[0180] In one embodiment, the filamentous fungi contain at least approximately 30 wt.% protein. Unless otherwise specified, the percentages of components such as proteins, RNA, and lipids in the biomat or filamentous fungal particles are expressed on a dry weight percentage basis. For example, the biomat can be dried at 99°C for two days and then air-dried for several more days, at which point the biomat is expected to contain less than approximately 5 wt.% moisture, e.g., less than 4 wt.%, less than 3 wt.%, less than 2 wt.%, less than 1 wt.%, less than 0.5 wt.%, or less than 0.1 wt.%. The total protein content of a dried biomat sample can be measured using a total nitrogen analysis method for protein estimation.

[0181] In one campaign, filamentous fungi accounted for at least approximately 30%, at least approximately 31 wt.%, at least approximately 32 wt.%, at least approximately 33 wt.%, at least approximately 34 wt.%, at least approximately 35 wt.%, at least approximately 36 wt.%, at least approximately 37 wt.%, at least approximately 38 wt.%, at least approximately 39 wt.%, at least approximately 40 wt.%, at least approximately 41 wt.%, at least approximately 42 wt.%, at least approximately 43 wt.%, at least approximately 44 wt.%, at least approximately 45 wt.%, at least approximately 46 wt.%, at least approximately 47 wt.%, at least approximately 48 wt.%, at least approximately 49 wt.%, at least approximately 50 wt.%, at least approximately 51 wt.%, at least approximately 52 wt.%, at least approximately 53 wt.%, at least approximately 54 wt.%, at least approximately 55 wt.%, and a small amount. It contains at least approximately 56 wt.%, at least approximately 57 wt.%, at least approximately 58 wt.%, at least approximately 59 wt.%, at least approximately 60 wt.%, at least approximately 61 wt.%, at least approximately 62 wt.%, at least approximately 63 wt.%, at least approximately 64 wt.%, at least approximately 65 wt.%, at least approximately 66 wt.%, at least approximately 67 wt.%, at least approximately 68 wt.%, at least approximately 69 wt.%, at least approximately 70 wt.%, at least approximately 71 wt.%, at least approximately 72 wt.%, at least approximately 73 wt.%, at least approximately 74 wt.%, at least approximately 77 wt.%, at least approximately 76 wt.%, at least approximately 77 wt.%, at least approximately 78 wt.%, at least approximately 79 wt.%, or at least approximately 80 wt.% of protein. Alternatively, in embodiments of the present invention, the filamentous fungus may contain an integer percentage of protein in the range of 30 wt.% to 80 wt.% or 30 wt.% to 80 wt.%. See Examples 21-23.

[0182] The filamentous fungi of the present invention also have a remarkably low RNA content. High levels of RNA in food have been shown to have harmful health or physiological effects. For example, a diet high in purines (present in RNA) is associated with the development of gout. The filamentous fungi grown or cultured as disclosed herein have an inherently low RNA content, and no additional or supplementary treatment is required to modify or reduce the RNA content. Therefore, in various embodiments, the food ingredients of the present invention do not contain filamentous fungal components that have a substantial level of RNA and / or have been treated to modify or reduce the RNA content of the component or food ingredient. In other embodiments, it is understood that the food ingredients of the present invention, which necessarily have a low amount of RNA filamentous fungal components, may be treated, for example, by heating or steam treatment to inactivate the fungi, or by other treatments that similarly reduce or degrade the RNA, if present. Such materials are characterized as having a filamentous fungal component with the low RNA content described herein, either before or without such reduction or degradation of any RNA.

[0183] In one embodiment, the filamentous fungus has an RNA content of less than approximately 8 wt.%. The wt.% RNA content is expressed on a dry weight basis. For example, the total RNA content in a dried biomat sample can be measured using purine analysis. See Example 24.

[0184] In one embodiment, the RNA content in the filamentous fungus is less than approximately 8.0 wt.%, less than approximately 7 wt.%, less than approximately 6 wt.%, less than approximately 5.0 wt.%, less than approximately 4 wt.%, less than approximately 3 wt.%, less than approximately 2 wt.%, or less than approximately 1 wt.%, or less than any increment of 0.1 wt.% of less than approximately 8.0 wt%. Alternatively, in an embodiment of the present invention, the filamentous fungus contains RNA in the range of 0.5 wt.% to 8 wt.% or any sub-range.

[0185] In one embodiment, the filamentous fungus contains a high protein content combined with a low RNA content as described above. For example, in one embodiment, the filamentous fungus may contain more than 45 wt.% protein, more than 50 wt.% protein, more than 55 wt.% protein, or more than 60 wt.% protein and an RNA content of less than 8 wt.%, less than 5 wt.% RNA, less than 4 wt.% RNA, less than 3 wt.% RNA, or less than 2 wt.% RNA. In another embodiment, the filamentous fungus may have any of the above protein content combined with any of the above RNA content.

[0186] The filamentous fungi and related food ingredients of the present invention can also be characterized as having a remarkably low mycotoxin content. Known mycotoxins include aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, beuvericin, fusarin C, fusaric acid, and zearalenone. In some embodiments, the total amount of mycotoxins and / or the total amount of any one or subset of the above mycotoxins in the filamentous fungi, biomat, or food ingredients of the present invention is less than about 10 ppm. In other embodiments, the total amount of mycotoxins and / or the total amount of any one or subset of the above mycotoxins is less than about 9 ppm, less than about 8 ppm, less than about 7 ppm, less than about 6 ppm, less than about 5 ppm, less than about 4 ppm, less than about 3 ppm, less than about 2 ppm, less than about 1 ppm, less than about 0.9 ppm, less than about 0.8 ppm, less than about 0.7 ppm, or less than about 0.6 ppm. See Example 25.

[0187] The filamentous fungi of the present invention also have a surprisingly high branched amino acid content. Branched amino acids refer to leucine, isoleucine, and valine. In one embodiment, the total amount of branched amino acids is greater than approximately 10 wt.%, greater than approximately 11 wt.%, greater than approximately 12 wt.%, greater than approximately 13 wt.%, greater than approximately 14 wt.%, greater than approximately 15 wt.%, greater than approximately 16 wt.%, greater than approximately 17 wt.%, greater than approximately 18 wt.%, greater than approximately 19 wt.%, greater than approximately 20 wt.%, greater than approximately 21 wt.%, greater than approximately 22 wt.%, greater than approximately 23 wt.%, greater than approximately 24 wt.%, greater than approximately 25 wt.%, greater than approximately 26 wt.%, greater than approximately 27 wt.%, greater than approximately 28 wt.%, greater than approximately 29 wt.%, and greater than approximately 30 wt.%. See Example 23 for branched amino acid content in strains MK7 and Fusarium venenatum biomats. Example 23 also shows the fatty acid profiles of these two fungi.

[0188] Growth and harvesting of filamentous fungal biomats The growth of filamentous fungal biomats can be achieved via surface fermentation. This involves inoculating filamentous fungal cells into a liquid medium containing a carbon source and a nitrogen source. Suitable carbon sources include sugars (e.g., sucrose, maltose, glucose, fructose, Japanese rare sugars, etc.), sugar alcohols (e.g., glycerol, polyols, etc.), starch (e.g., corn starch, etc.), starch derivatives (e.g., maltodextrin, cyclodextrin, glucose syrup, hydrolysates, and modified starch), starch hydrolysates, hydrogenated starch hydrolysates (HSH; e.g., hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, etc.), lignocellulose pulp or lignocellulose raw materials (e.g., beet pulp, agricultural pulp, wood pulp, dried grains for brewing, brewing wastewater, etc.), corn steep liquor, acid whey, sweet whey, whey, wheat steep liquor, carbohydrates, food waste, olive oil processing waste, hydrolysates from lignocellulose materials, and / or combinations thereof. Filamentous fungi produce biomats located on the surface of the growth medium.

[0189] The liquid growth medium of the present invention may be characterized by a desirable or pre-selected mass ratio of carbon to nitrogen ("C:N ratio"). Generally, the C:N ratio of the liquid growth medium of the present invention may be any ratio between about 1:1 and about 50:1, or about 2.5:1 and about 30:1, or about 5:1 and about 10:1, or about 1:1 and about 50:1, and any other ratio between about 1:1 and about 50:1. As a non-limiting example, the growth medium of the present invention may have a C:N ratio of about 2.5:1, about 5:1, about 7.5:1, about 10:1, about 12.5:1, about 15:1, about 17.5:1, about 20:1, about 22.5:1, about 25:1, about 27.5:1, about 30:1, about 32.5:1, about 35:1, about 37.5:1, about 40:1, about 42.5:1, about 45:1, about 47.5:1 or about 50:1, or any ratio of about about X:2 (where X is an integer from about 2 to about 100).

[0190] Inoculation can be performed using an inoculum containing planktonic filamentous fungal cells, conidia, microconidia or macroconidia, or spores or fruiting bodies. In many cases, particularly for Ascomycota fungi, an inoculum containing planktonic filamentous fungal cells, conidia, microconidia or macroconidia is inoculated into the growth medium. Ideally, the cells in the inoculum, such as cells with a high lipid content, will float on the surface of the growth medium and increase their growth rate. Cells or cell clumps that settle in the growth medium can negatively affect the cells floating on the surface and the biomat they form. Specifically, biomats derived from growth medium containing a significant number of settled cell clumps tend to be discolored and do not grow into a uniformly dense mat.

[0191] In one embodiment, the inoculum may include spores. For example, in one embodiment, approximately 2 cc of sterile Basidiomycota spores suspended in deionized water from a spore syringe (e.g., MycoDirect, Huntley, IL) was used to inoculate approximately 75 mL of growth medium in a small Pyrex tray. Alternatively, 1 cc of spores suspended in deionized water from a spore syringe was seeded under standard sterile conditions in a container having malt extract agar medium + CF (30 g dried malt extract, 20 g agar, 1000 mL water + 0.01% chloramphenicol). The container was sealed with Parafilm and incubated at room temperature until the mycelium completely covered the agar surface. Mycelial sections from the wedge-shaped agar preparation, approximately 2 cm wide, were then cut into the smallest possible size and transferred to tubes containing growth medium. The liquid culture tubes were sealed and incubated at room temperature, shaking by hand or mechanical means (i.e., continuous shaking or continuous stirring tank reactor) for about 1 minute at least 5 times per day to disrupt the mycelium as much as possible. The liquid cultures were incubated for generally 3 days or more until they became visibly cloudy. The liquid cultures were then used to inoculate trays of growth medium at 10% or 15% of the total growth medium volume.

[0192] In one embodiment, the inoculum may include fruiting bodies. For example, in one embodiment, Basidiomycota fruiting bodies were used to produce inoculum for initiating a filamentous biomat. In one example, the inoculum was prepared by (a) surface sterilizing the fruiting bodies with, for example, a 5% bleach solution, (b) rinsing them in a sterile medium, (c) grinding them under sterile conditions into aggregates less than 5 mm in length or larger than 5 mm, depending on the end use, and (d) surface sterilizing the ground mushroom biomass with, for example, a 5% bleach solution and rinsing it again in a sterile medium. 5 grams of ground, surface-sterilized fruiting body biomass was used directly as the inoculum. In another example, a pure culture derived from fruiting bodies was used. In this case, the fruiting bodies were approximately 3 mm in length. 3The mycelium was seeded onto agar medium containing 0.01% chloramphenicol and incubated at room temperature. After 2–5 days of growth, the mycelium was transferred to fresh agar + chloramphenicol medium and grown for a further 3–7 days. Culture purity was confirmed by extracting and purifying DNA (FastDNA Spin Kit, MP Biomedicals), sequencing the 18S rRNA sequence and / or ITS region, and performing phylogenetic classification of the sequences using Blast (NCBI database). After confirmation, the mycelium was used to inoculate 50 mL of sterile liquid medium, stirred / rotated at 185 rpm for approximately 5 days, and then used as inoculum in a ratio of approximately 7.5% inoculum to 92.5% liquid medium.

[0193] Numerous different media may be used, but certain media may work better than others for the growth of filamentous fungal biomats; as a non-limiting example, Hansen's medium (1.0 g peptone, 0.3 g KH2PO4·7H2O, 2.0 g MgSO4·7H2O, 5.0 g glucose per liter with a C:N ratio of 26.9) did not produce a complete, sticky, clumpy biomat, but media that worked very well included MK7A, MK7-1, MK7-3 (all described in WO2017 / 151684) and the media listed below. These are also described in Example 12.

[0194] [Table 1]

[0195] [Table 2] [Table 3]

[0196] [Table 4]

[0197] The osmolality, expressed as osmolality, can be determined by measuring the culture medium with an osmometer capable of measuring up to 5000 mOsm / kg (e.g., Model 3250 SN: 17060594). Three values ​​were read for several culture media, and the following results were obtained: Hansen = 39, 39, 38; Malt 001 = 169, 168, 169; MK-7 SF = 1389, 1386, 1387; Malt 001 + NH4NO3 = 288, 287, 286 。

[0198] As previously described, the method of the present invention may result in enhancement of the protein content, amino acid profile (e.g., branched-chain amino acid content), and / or nutritional content of filamentous fungi. While not bound by theory, this result is thought to depend on the culture medium used for the growth of certain fungi.

[0199] For example, the natural protein content of the fruiting body of Pleurotus ostreatus var. Columbinus has been reported to be approximately 16.32% (Ulziijargal and Mau (2011) Int J Medicinal Mushrooms, 13(4):343-49) or 24.65% (Stamets (2005) Int J Medicinal Mushrooms 7:103-110). However, as shown in Example 12, the Pleurotus biomat grown in malt 001 medium according to the present invention has a high water-corrected protein content of 29.82%, which is an increase of 13.6% or 5.71% in protein content.

[0200] The protein content of the fruiting body of Pleurotus ostreatus (also known as marbled oyster mushroom) has been reported to be approximately 23.85% (Ulziijargal and Mau (2011) Int J Medicinal Mushrooms, 13(4):343-49) or 27.25% (Stamets (2005) Int J Medicinal Mushrooms 7:103-110); however, the marbled oyster mushroom biomat propagated by the present invention has a high water-corrected protein content of 39.77%, representing an increase of at least 46% to a maximum of 67% in protein content.

[0201] The protein content of the fruiting body of Sparassis crispa is reported to be approximately 13.4% (Kimura (2013) BioMed Research International); however, the Sparassis crispa biomat grown using the present invention has a high water-corrected protein content of 32.21% to 46.24%, representing a minimum increase of 140% to a maximum increase of 245% in protein content.

[0202] Other characteristics of the medium considered important for the growth of biomats on the surface of the fermentation medium are the osmotic pressure and ionic strength of the medium. In one embodiment, the osmotic pressure of the medium for biomat growth may be greater than about 3 atm, greater than about 10 atm, greater than about 20 atm, greater than about 30 atm, greater than about 40 atm, greater than about 50 atm, greater than about 60 atm, greater than about 70 atm, greater than about 80 atm, greater than about 90 atm, greater than about 100 atm, greater than about 110 atm, greater than about 120 atm, greater than about 125 atm, or greater than any integer from greater than 3 atm to greater than 125 atm. In another embodiment, the osmotic pressure may be in the range of about 3 atm to about 125 atm, about 20 atm to about 100 atm, or between any two integer atm values ​​from 3 to 125.

[0203] In certain embodiments, the ionic strength of the medium that can be used for the growth of the biomats can exceed about 0.02 M, exceed about 0.05 M, exceed about 0.10 M, exceed about 0.20 M, exceed about 0.30 M, exceed about 0.40 M, exceed about 0.50 M, exceed about 0.60 M, exceed about 0.70 M, exceed about 0.80 M, exceed about 0.90 M, exceed about 1.0 M or exceed any 1 / 100 M value in the range from exceeding 0.02 M to exceeding 1.00 M. In another embodiment, the ionic strength can be in the range of about 0.02 M to about 1.0 M, about 0.10 M to about 0.50 M or any range of two molar concentration values from 0.01 to 1.0.

[0204] Harvesting of the biomats can be performed at any point when a sufficiently thick biomats has formed. Harvesting is generally performed after growth for 2 - 3 days, but a longer growth period may be desirable in certain instances, such as when a thicker or higher density biomats is desired / required. For example, harvesting can be performed after growth for 2 days to 60 days or after any period of days or part of a day (e.g., hours) between 2 days and 60 days. For example, such growth periods can be 3.5 - 4 days, 3 - 5 days, 4 - 6 days, 5 - 7 days, 6 - 9 days, 7 - 10 days or 19 - 21 days, or approximately integer days up to and including about 21 days. As used herein, the term "harvesting" refers to any process or step that stops the growth of the biomats (e.g., separation from the nutrient source or change in temperature conditions) and / or modifies the physical properties of the biomats (e.g., conversion to particles or strips of the biomats).

[0205] Due to the adhesive mass structure of the filamentous biomats grown under the surface fermentation conditions described in PCT / US2017 / 020050 and herein, the filamentous biomats have a tensile strength sufficient to lift them essentially intact from the surface of the medium at the end of the growth period. In various embodiments, the biomats of the present invention have at least about 30 g / cm 2 , at least about 40 g / cm 2 , at least about 50 g / cm 2 , at least about 60 g / cm [[ID=第十五]] 2at least approximately 70 g / cm³ 2 at least approximately 80 g / cm³ 2 at least approximately 90 g / cm³ 2 at least approximately 100 g / cm³ 2 at least approximately 150 g / cm³ 2 at least approximately 200 g / cm³ 2 at least approximately 250 g / cm³ 2 at least approximately 300 g / cm³ 2 at least approximately 350 g / cm³ 2 , at least about 400g / cm 2 at least approximately 450 g / cm³ 2 , at least about 500g / cm 2 at least approximately 550 g / cm³ 2 Or at least about 600g / cm³ 2 Or at least about 650 g / cm³ 2 Or at least about 700g / cm³ 2 Or at least about 750 g / cm³ 2 Or at least about 800 g / cm³ 2 Or at least about 850 g / cm³ 2 Or at least about 900 g / cm³ 2 Or at least about 950 g / cm³ 2 Or at least about 1000 g / cm³ 2 Or at least about 1500g / cm³ 2 Or at least about 2000 g / cm³ 2 Or at least about 2500 g / cm³ 2 Or at least about 3000 g / cm³ 2 Or at least about 3500 g / cm³ 2 Or at least about 4000 g / cm³ 2 It may have a tensile strength of 30 g / cm². In another embodiment, the biomat of the present invention has a tensile strength of 30 g / cm². 2 It may have a tensile strength greater than some integer exceeding . Alternatively, the tensile strength of the biomat of the present invention is approximately 30 g / cm². 2 ~About 4000g / cm 2 The range is approximately 30 g / cm³. 2 ~About 4000g / cm 2The range can be any integer range. A suitable method for measuring tensile strength is described in Example 41.

[0206] Table 1A shows some examples of tensile strength and other physical properties measured in various filamentous fungi. [Table 5]

[0207] Table 1B shows further examples of tensile strength and other physical properties measured in various filamentous fungi obtained using other culture media. [Table 6]

[0208] In various embodiments, the biomat of the present invention may have a thickness ranging from about 0.05 cm to at least about 2 cm.

[0209] In various embodiments, the biomat of the present invention may have a width ranging from about 0.6 cm to about 3 m. Generally, the biomat produced by the present invention may have a width approximately the same as the width of the container in which the biomat was grown.

[0210] Surface fermentation can be carried out under a variety of conditions, including static medium conditions (described in PCT Publication WO2017 / 151684, which is incorporated herein by reference as a whole), semi-static medium conditions, and continuous medium flow conditions. Some embodiments are described in Examples 1 to 4.

[0211] Growth under semi-static media conditions means that at least a portion of the media is replaced before the filamentous fungal biomat is harvested. These conditions enable long-term linear dry biomass production and demonstrate the suitability of this system to operate as a continuous production system. For example, in one experiment, linear dry biomass production was achieved from day 4 to day 18 (r 2(=0.995), and then the biomass weight is approximately 2.5 kg dry / m². 2 It stabilized.

[0212] Biomats can also be produced under continuous medium flow conditions, and biomat growth is limited to the surface of the growth medium, so that the medium beneath the mat is continuously or semi-continuously refreshed.

[0213] In some cases, however, it is preferable to harvest the growing biomat on a semi-continuous basis. Here, a portion of the biomat is removed, and the remaining portion is then physically moved into the empty area of ​​the culture medium created by the removal of that portion of the biomat. This can be achieved by physically grasping the biomat and pulling it until it reaches the edge of the surface fermentation vessel, or by other mechanical means. The resulting empty area is then available for mat growth without another or additional inoculation step, as the culture medium already contains viable fungal cells. This process can be repeated periodically, which can be useful, especially when the culture medium is refreshed or when depleted nutrients are reintroduced.

[0214] Biomat harvesting can also be carried out on a continuous basis. Continuous harvesting can be facilitated by a number of mechanisms. One such example is a roller wheel connected to the mature end of the biomat (see Figure 7). The roller wheel rotates slowly, harvesting the mature biomat and simultaneously creating an open medium for the growth of new biomat at the other end of the surface fermentation vessel. In various embodiments, sampling can be carried out at a rate of at least about 0.1 cm / day, 0.2 cm / day, 0.3 cm / day, 0.4 cm / day, 0.5 cm / day, 0.6 cm / day, 0.7 cm / day, 0.8 cm / day, 0.9 cm / day, 1.0 cm / day, 1.1 cm / day, 1.2 cm / day, 1.3 cm / day, 1.4 cm / day, 1.5 cm / day, 1.6 cm / day, 1.7 cm / day, 1.8 cm / day, 1.9 cm / day, 2.0 cm / day, 2.1 cm / day, 2.2 cm / day, 2.3 cm / day, 2.4 cm / day, or 2.5 cm / day. A typical sampling rate is 1.56 cm / day, but this can be varied depending on the specific application or user preference.

[0215] In some cases, UVB light (290-320 nm) can induce pigment production by filamentous fungi such as Fusarium strain MK7 (ATCC Accession Deposit No. PTA-10698), which produces colored biomats. In addition to color changes that may be useful for creating various food effects, UVB treatment converts ergosterol present in the fungal cell membrane into vitamin D2 and increases the production of carotenoids such as beta-carotene and astaxanthin. Consequently, UVB irradiation of filamentous fungi such as Fusarium strain MK7 increases vitamin D2 and carotenoids in the resulting biomats.

[0216] In some cases, the formed filamentous fungal biomat consists of multiple layers with a uniform appearance, where one surface of the filamentous biomat is in contact with air and the other surface is in contact with the synthetic medium. In other cases, there are at least two distinct layers: an aerial mycelial layer on the top surface and a dense multicellular bottom layer in contact with the synthetic medium. More often, there are three distinct layers: (a) an aerial mycelial layer on the top surface, (b) a dense bottom layer, and (c) a transitional layer between the upper and bottom layers. The transitional layer may only be loosely attached to the dense bottom layer, in which case it allows for easy separation of the bottom layer from the rest of the biomat. The fiber density of the transitional layer ranges from slightly lower than the bottom layer in the zone in contact with the bottom layer to a density similar to the aerial mycelium near the top of the biomat.

[0217] In one embodiment, the biomat contains strain MK7 (ATCC Accession Deposit No. PTA-10698), Fusarium venenatum, Rhizopus oligosporus, Morchella esculenta (morel), Morchella conica (morel), Morchella importuna (morel), Calvatia gigantea (giant puffball), Pleurotus ostreatus (marbled oyster mushroom), and Pleurotus ostreatus var. columbinus. It may include *Pleurotus columbinus* (oyster mushroom), *Sparassis crispa* (flower mushroom), *Ganoderma lucidum* (reishi mushroom), and *Hypsizygus ulmarius* (white oyster mushroom).

[0218] Inactivation of filamentous fungal biomats The biomat can be rinsed to remove excess growth medium, but rinsing is not necessary; however, in some cases, removal of growth medium or excess growth medium is preferable. Similarly, the biomat can be squeezed to remove excess growth medium, which is also not necessary, but may be preferable in certain applications.

[0219] Eliminating cell viability and the possibility of further biomat growth is desirable in certain cases, such as for the use of biomats as a single protein source or protein component in food. This can be achieved by heating, irradiation, ethanol, and / or steam treatment.

[0220] Regarding the heating process, the filamentous fungal biomat can be treated, for example, in accordance with WO95 / 23843 or British Patent 1,440,642, or incubated at a temperature that destroys the vast majority of the organism's RNA without adversely affecting the organism's protein composition.

[0221] During irradiation, the filamentous fungal biomat was used. 60 Co (or rarely) 137 Exposure to ionization energies such as those produced by radioactive isotopes of Cs, X-rays produced by machines operating at nominal energies below 5 MeV, and accelerated electrons produced by machines operating at nominal energies below 10 MeV.

[0222] Steam treatment can be used to inactivate certain filamentous fungal biomats, such as those produced by Fusarium strain MK7 (ATCC Accession Deposit No. PTA-10698) and Fusarium venenatum, because steam treatment can also remove certain specific metabolites from the biomat structure, if those metabolites have been produced. Here, the biomat is positioned so that biomat effluent and condensed steam can easily drip from the biomat. Suitable biomat holding systems include porous plastic mesh and porous trays. Other biomat holding systems include, but are not limited to, systems that hold the biomat in a vertical position, such as systems with a clamping mechanism that fixes at least one end of the biomat while the other end of the biomat is suspended from the clamp, and mesh systems that fix at least two sides of the biomat.

[0223] The biomat is placed in a steamer so that heated steam, such as steam at a temperature of 85°C or 95°C or higher, comes into contact with the biomat. If multiple trays are placed in a single steamer, for example, one above the other, it is preferable to protect the biomat placed at the bottom from water droplets from the biomat placed at the top. The protection must allow the steam to come into contact with the biomat, thereby inactivating the biomat's viability, and divert the biomat discharge liquid and condensed steam produced in the upper part of the steamer from coming into contact with the biomat placed in the lower part of the steamer. In one embodiment, a cone is placed between the upper and lower trays to achieve this result. In other embodiments, the separation of the upper and lower trays also includes at least one other geometric shape, such as a cylinder, cube and / or cuboid, pyramid, sphere, torus and / or other regular polyhedron. In yet another embodiment, the tray may be separated using at least one cylindrical, cubic and / or rectangular prism, pyramidal, sphere, torus, other regular polyhedron, mesh, porous belt, or a combination thereof.

[0224] Steam-treat the biomats until the viability of the biomats is reduced to a point where further biomats growth and / or cell regeneration within the biomats can be ignored. The viability of the biomats is a function of the original substrate, biomats progression, steam / heat transfer characteristics, biomats position within the steamer and biomats orientation to the generated steam. As an example, Fusarium strain MK7 biomats grown on glycerol or acid whey substrates are non-viable after 5 minutes of steam treatment and in some cases less than 5 minutes. The steam-treated mats can be rinsed and / or pressed to remove mat excretions and condensed steam.

[0225] The inactivated edible filamentous fungus biomats can be used directly as a protein source, for example, in the preparation of foods that are comparable to, for example, just a few examples, tofu, bacon and jerky.

[0226] Filamentous fungal biomat particles The inactivated edible filamentous fungus biomats can be miniaturized for use as a protein source in foods. Miniaturization can be performed by mechanical means such as cutting, shearing, dicing, mincing, grinding, blending, etc. or by sonication and is carried out prior to mixing with other ingredients or liquids. The miniaturized particles may be of uniform size or heterogeneous.

[0227] Generally, the length of the miniaturized particles is 0.05 - 500 mm, the width is - 0.03 - 7 mm and the height is 0.03 - 1.0 mm. For example, flour-type particles are generally in the range of 0.03 mm - 0.4 mm and jerky-type particles are in the range of 100 mm - 500 mm, etc. Larger particles can be produced. For example, the biomats are grown in a rubber pool (66 inches in diameter) that produces a single biomat that is 66 inches in diameter and completely spherical. Larger containers can be used to grow on larger mats.

[0228] The number of miniaturized particles produced per biomat depends on the initial biomat size and the purpose for which the biomat miniaturized particles are used. [[ID=十九]]

[0229] Large particles In one embodiment, the inactivated edible filamentous fungal biomat is reduced to particles in which at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particles have a particle length of about 0.05 mm to about 500 mm, a particle width of about 0.03 mm to about 7 mm, and a particle height of about 0.03 mm to about 1.0 mm, or any partial range within these ranges. For example, at least 50%, 60%, 70%, 80%, or 90% of the particles may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

[0230] In one embodiment, the inactivated edible filamentous fungal biomat is reduced to particles in which at least 50%, 60%, 70%, 80%, or 90% of the particle mass has a particle length of about 0.05 mm to about 500 mm, a particle width of about 0.03 mm to about 7 mm, and a particle height of about 0.03 mm to about 1.0 mm, or any sub-range within these ranges. For example, at least 50%, 60%, 70%, 80%, or 90% of the particle aggregate may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

[0231] For example, at least 50%, 60%, 70%, 80%, or 90% of the particles may have a particle length of about 0.08 mm to about 100 mm, or 10 mm to about 70 mm, or 130 mm to about 200 mm; a particle width of about 0.05 mm to about 2 mm, or about 1 mm to about 3 mm, or about 4 mm to about 6 mm; and a particle height of about 0.03 mm to about 0.06 mm, or about 0.04 mm to about 0.07 mm, or about 0.08 mm to about 1.0 mm.

[0232] Such particles mimic the texture and chewiness of meat products such as chicken nuggets or hamburgers, and are useful in the preparation of such products, such as as an excipient or filler for meat products or their vegetarian versions. When the particles of the present invention are used as an excipient or filler for meat products, the ratio of filamentous fungal particles to meat may be in the range of 10:90 to 90:10 or any ratio in between.

[0233] For example, in one embodiment, the filamentous fungal particles include particles in which at least 90% of the particles are less than about 1.5 mm in length and most are less than 1 mm in length, less than about 1 mm in width, and less than about 0.75 mm in height. Foods containing such particles are characterized as having a higher density in the mouth, being easy to chew, providing a creamy texture and a more refined eating experience, and such particles can be used in the preparation of food ingredients that mimic hamburgers found in high-end restaurants.

[0234] In one embodiment, the filamentous fungal particles include at least about 90% of particles having a length of about 4 mm to about 10 mm, a width of about 1.0 mm to about 3 mm, and a height of less than 0.75 mm. Food ingredients containing such particles lead to a more substantial meal experience, similar to the type of burger commonly found in burger restaurants or BBQs.

[0235] Fine particles (powder) In one embodiment, the inactivated edible filamentous fungal biomat is reduced to fine particles. In one embodiment, the filamentous fungal particles are in the form of a powder. In such embodiments, the particle size and particle size distribution may be the same as or similar to those conventionally used for flour-like materials such as wheat or other flours. In one embodiment, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles fall within the range of 0.03 mm to about 0.4 mm, or some sub-range within this range, for example, about 0.03 mm to 0.07 mm, about 0.07 mm to about 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about 2.0 mm, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm, or 0.2 mm to about 0.4 mm. In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particles fall within the range of 0.075 mm to approximately 0.12 mm.

[0236] In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particle aggregate falls within the range of 0.03 mm to about 0.4 mm, or any sub-range within this range, for example, about 0.03 mm to 0.07 mm, about 0.07 mm to about 0.12 mm, about 0.12 mm to about 0.15 mm, about 0.15 mm to about 2.0 mm, about 0.04 mm to about 0.2 mm, or 0.06 mm to about 0.120 mm, or 0.2 mm to about 0.4 mm. In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particle aggregate falls within the range of 0.075 mm to about 0.12 mm.

[0237] Miniaturization can be carried out using a milling machine, grinder, or other conventional apparatus for miniaturization.

[0238] In one embodiment, the moisture content of the particulate material of the present invention is about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or less than about 1%. Low moisture levels help prevent particle aggregation.

[0239] Such particles are useful in preparing ingredients such as baked goods, including but not limited to bread, rolls, muffins, cakes, cookies, and pies, or can be sprinkled on other foods.

[0240] Liquid dispersion (milk) One method of introducing protein into food is the use of a liquid dispersion made from filamentous fungal biomats as a substitute for milk or milk-like substances. The liquid dispersion (also referred to here as "milk") contains particles of filamentous fungal biomats dispersed in an aqueous medium.

[0241] The particle size of filamentous fungal biomats suitable for use in liquid dispersions is generally smaller than about 10 microns. In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particles in the liquid dispersion fall in the range of about 1 micron to about 10 microns, or any sub-range within this range. In one embodiment, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the particles are less than 10 microns, less than 9 microns, less than 8 microns, less than 7 microns, less than 6 microns, less than 5 microns, less than 4 microns, less than 3 microns, less than 2 microns, or less than 1 micron. In one embodiment, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the particles may have a particle size of less than about 1 micron.

[0242] Liquid dispersions or milks can be prepared by combining and mixing a filamentous fungal biomat with an aqueous phase such as water. The mixed mixture can be gradually heated, for example, to a boiling point. The heated mixture is then allowed to cool. In one embodiment, the liquid dispersion can be produced under nitrogen. This process results in a liquid dispersion with less fungal odor and a creamier viscosity. Production under nitrogen can be achieved by bubbling with nitrogen in a sealed container so that most of the available oxygen is replaced with nitrogen during mixing or heating cycles, such as in a Vitamix or high-energy miniaturization or milling process. An example of the method is described in Example 27.

[0243] The biomat-to-water ratio can be adjusted to produce a liquid dispersion of appropriate viscosity and density. The biomat-to-water ratio may be in the range of about 1:10 to about 10:1 or any ratio in between. In one embodiment, the biomat-to-water ratio may be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0244] In various embodiments, the liquid dispersion of the present invention is stable such that the filamentous fungal particles do not easily separate from the liquid medium in which they are dispersed. For example, upon dispersion formation, the resulting liquid appears uniform in appearance and does not visibly separate into different phases. For example, no visibly identifiable or significant deposits are formed at the bottom of the container holding the dispersion. In some embodiments, the liquid dispersion may remain stable for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours, or for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 1 week, 2 weeks, 3 weeks, or 4 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In these embodiments, the dispersion may be kept at room temperature or refrigerated temperature, for example, about 35°F (1.6°C).

[0245] Example 26 illustrates the stability of the liquid dispersions of the present invention, i.e., milk. Milk that was left standing in a refrigerator for 15 and 30 days without visible separation was obtained in each sample. The milk also showed no alteration in flavor or aroma.

[0246] In certain embodiments, the dispersion comprises at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% solids. In other embodiments, the liquid dispersions of the present invention comprise a solid component of from about 4% to about 30% or any sub-range of 4% to 30%.

[0247] The liquid dispersions can be used as beverages or drinks, including alternatives to dairy products such as dairy milk, almond milk, rice milk, soy milk, etc. They can be used in a number of recipes including soups, ice cream, yogurt, smoothies, fudges, and confections such as caramel and truffles. In some cases, the filamentous fungal biomass produced from different feedstocks / carbon sources can result in liquid dispersions with different flavors. For example, when the feedstock / carbon source is glycerol, the resulting liquid dispersion produced from Fusarium strain MK7 is sweet, while the liquid dispersion obtained from Fusarium strain MK7 grown on acid whey feedstock / carbon source tends to be sour. The natural sweetness or sourness of the filamentous fungus, e.g., Fusarium strain MK7, transfers to the final food product. For example, the acid whey liquid dispersion is suitable for yogurt, while the glycerol liquid dispersion is suitable for mousse, caramel, or fudge.

[0248] In one embodiment, a liquid dispersion can be used to form stable foam that does not spontaneously collapse immediately after the foaming process is stopped. The foaming process may include whisking, incorporation of compressed gas, or other conventional foaming processes. The foam is smooth and creamy in appearance and exhibits the presence of bubbles of distributed size. Larger bubbles tend to burst after standing or pouring, while smaller bubbles remain in the suspension for a longer period, forming a stable foam product. The foam product of the present invention has the compositional characteristics of a liquid dispersion and further includes air or other gases incorporated into the foam in a stable manner. For example, the foaming material of the present invention may have a volume increase (i.e., overrun) of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to the starting volume of the liquid dispersion before foaming, due to air incorporation. In various embodiments, the foaming material is stable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days or at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days or at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days or at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days or at least about 30 days. In one embodiment, the liquid dispersion remains stable for at least about 1 month, at least about 2 months or at least about 3 months. As far as is used in reference to foam, stability means retaining at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, and at least approximately 95% of the initial foam volume.

[0249] An average overrun of approximately 12% is suitable for the preparation of ice cream (with more fat and emulsifiers), frozen yogurt, cheesecake batter, whipped cream, and the like. In one embodiment, the foam may incorporate nitrogen to provide different overrun characteristics.

[0250] In one embodiment, the food ingredient is a cultured food. Unless otherwise specified, the term "cultured food" as used herein refers to a microbial food culture, i.e., a food in which viable bacteria, yeast, or mold have been introduced into a filamentous fungus. As a non-limiting example, the fungal food ingredient of the present invention may be cultured with Lactobacillus spp. or other lactic acid bacteria (e.g., for the production of yogurt-like foods or other dairy-like foods), Saccharomyces cerevisiae or other yeasts used in brewing or baking (e.g., for making baked goods-like foods or alcoholic beverage-like foods), molds traditionally used in sausage production (e.g., Penicillium chyrsogenum or Penicillium nalgiovense for making sausage-like foods), or soy sauce (e.g., Aspergillus oryzae or Aspergillus sojae for making soy sauce-like foods). In one embodiment, the cultured food according to the present invention may be cultured simultaneously or sequentially with two or more microbial food cultures to produce an analogue of a food produced by the fermentation of two or more microbial cultures; as a non-limiting example, the cultured food according to the present invention may be a moderately soft aged cheese analogue (by subjecting the fungal material to a first culture of Lactobacillus spp. or other lactic acid bacteria and a second culture with cheese aging yeast), a blue cheese analogue (by subjecting the fungal material to a first culture of Lactobacillus spp. or other lactic acid bacteria and a second culture with mold such as Penicillium roqueforti), a soft aged cheese analogue (e.g., Brie or Camembert) (by subjecting the fungal material to a first culture of Lactobacillus spp. or other lactic acid bacteria and Penicillium camembert) It may contain (by subjecting it to a second culture by camemberti).

[0251] In one embodiment, the food ingredient includes a yogurt-like food containing particles of the filamentous fungal biomat of the present invention dispersed in an aqueous medium. In one embodiment of the yogurt-like food, the ratio of filamentous fungal particles to water may be in the range of about 1:10 to about 10:1. It is predicted that the higher the ratio of filamentous fungal particles to water, the more the texture of the yogurt-like food will increase and the less fluid it will be. In one embodiment, the ratio of filamentous fungal particles to water may be about 1:3, 1:2, 1:1, or 2:1.

[0252] In one embodiment, the yogurt-like food contains invert sugar or invert sugar. Invert sugar is resistant to crystallization, promotes moisture retention, and is commercially used in various foods such as baked goods, confectionery or fruit jams and beverages to enhance flavor and texture and extend shelf life. Examples include honey or a mixture of glucose and fructose (obtained by hydrolysis of sucrose and sweeter than sucrose).

[0253] In one embodiment, the yogurt-like food contains a thickening or gelling agent. Such substances are known in this field and include, but are not limited to, agar, gelatin, starch (i.e., arrowroot, tapioca, corn, potato), higher fat liquids (coconut milk), fats (i.e., deodorized or otherwise processed coconut flakes), chickpea water, flaxseed, xanthan gum, guar gum, psyllium husk, ground chia seeds, nut / seed butter, pumpkin puree, heated and mashed yam / sweet potato, apple sauce, crushed overripe banana or plantain, pureed dates or prunes, soaked and boiled figs, chopped fruit / vegetables, chopped coconut, gluten-free flours (e.g., teff flour, buckwheat flour, amaranth flour, chickpea flour, sorghum flour, almond flour), heated pureed legumes, cocoa powder, plant gum, polysaccharides, plant mucilage, seaweed derivatives, pectin, gluten, soy and egg analogues. The thickening agent is a fat that can be in liquid form, such as coconut milk, or in solid form, such as deodorized coconut flakes.

[0254] In one embodiment, filamentous fungal cells are lysed, which may release more proteins, increase enrichment, and improve nutrient bioavailability. Lysis can be performed by any method known in the art, such as sonication.

[0255] In one embodiment, the yogurt-like food contains lactic acid bacteria (LABs). These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation. Examples of LABs include the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus. In one embodiment, the bacteria Lactobacillus bulgaricus and / or Streptococcus thermophilus are included.

[0256] In one embodiment, the yogurt-like food further comprises rennet. The rennet may be of animal, plant, or microbial origin. In vegetarian or vegan foods, the rennet may be of plant and / or microbial origin.

[0257] In one embodiment, the yogurt-like food further contains enzyme water. For example, enzyme water may be produced as follows: 100 gm of whole rye or durum wheat seeds (or other suitable whole grain seeds) are combined with 1 liter of water and germinated for 2-4 hours. When the seeds have germinated and the first roots have appeared, the seeds are placed in a clean jar containing 1 liter of water. The jar may be covered with a permeable cloth (linen or cotton) and incubated at room temperature for 24 hours, at which point the color and odor of the water in the jar will change. This water is called enzyme water and can be used in the production of yogurt and cheese.

[0258] In one embodiment, the yogurt-like food further comprises probiotics. Probiotics are a mixture of viable microorganisms, such as bacteria and yeasts, that provide health benefits, including improved digestion.

[0259] In one embodiment, the yogurt-like food contains milk solids derived from livestock milk. In another embodiment, the yogurt-like food does not contain milk solids derived from livestock milk, i.e., it contains no milk solids at all.

[0260] It should be expressly understood that, in addition to yogurt-like foods, the ingredients of the present invention may include any one or more other dairy-like foods. As a first non-limiting example, the ingredients of the present invention may include cheese-like foods such as hard cheese (e.g., Parmesan)-like foods, semi-hard cheese (e.g., Gouda)-like foods, semi-soft cheese (e.g., Havarti)-like foods, soft or soft-ripened cheese (e.g., Brie)-like foods, cream cheese-like foods, acidic milk cheese-like foods, blue cheese-like foods, mascarpone cheese-like foods, pasta filata (e.g., mozzarella)-like foods, salted cheese (e.g., feta)-like foods, whey cheese (e.g., ricotta or brunost)-like foods, or fresh cheese (e.g., cottage cheese)-like foods. As a second non-limiting example, the ingredients of the present invention may include butter-like foods such as fresh cream butter-like foods, butterfat-like foods, clarified butter-like foods, whey butter-like foods, fermented butter-like foods, mild fermented butter-like foods, sweet cream butter-like foods, or traditional buttermilk-like foods. As a third non-limiting example, the ingredients of the present invention may include whey-like foods such as sour whey-like foods or sweet whey-like foods. As a fourth non-limiting example, the ingredients of the present invention may include cream-like foods such as crème fraîche-like foods, smetana-like foods, sour cream-like foods, half-and-half-like foods, table cream-like foods, whipping cream-like foods, double cream-like foods, clotted cream-like foods, sour cream-like foods, pasteurized cream-like foods, or condensed cream-like foods. As a fifth non-limiting example, the ingredients of the present invention may include acidic milk-like foods such as quark-like foods, cheese curd-like foods, sour milk-like foods, kefir-like foods, organic yogurt or mild yogurt-like foods, yogurt-like foods, cream yogurt-like foods, or cultured buttermilk-like foods.As a sixth non-limiting example, the ingredients of the present invention may include milk-like foods such as raw milk-like foods, low-fat or non-fat raw milk-like foods, pasteurized milk-like foods, fresh whole milk-like foods, low-fat milk-like foods, skim milk-like foods, extended shelf life (ESL) milk-like foods, ultra-high temperature (UHT) processed milk-like foods, pasteurized milk-like foods, concentrated or unsweetened condensed milk-like foods, partially skimmed condensed milk-like foods, or condensed skim milk-like foods. As a seventh non-limiting example, the ingredients of the present invention may include powdered dairy-like foods such as powdered whey-like foods, powdered milk-like foods, or skim milk powder-like foods. In some embodiments, the dairy-like foods of the present invention may be vegan foods, i.e., foods that do not contain animal products, and therefore may be foods that vegan observers may include in their diet.

[0261] The particles of the filamentous fungal biomat may be added as a protein or other nutrient source to enhance the nutritional content of the food, or they may be, for example, the sole protein component. For foods consisting of filamentous fungal biomat or miniaturized particles of such biomat, the particles may be optimized for specific textures, mouthfeels, and chewability. Modifications to texture, mouthfeels, and chewability allow for customization to accommodate individuals with specific dietary needs, such as those with chewing difficulties, or individuals who need / desire softer foods or those who desire foods with more texture, a stronger mouthfeel, and a greater chewiness, while still providing the same nutritional and taste experience. Because particle size is easily controlled, foods fortified with or made solely from filamentous fungal biomat have a texture very similar to the mimicked standard protein foods, as shown in Table 2. [Table 7]

[0262] The particles of the filamentous fungal biomat can be used as the sole protein component in food ingredients or to enhance the protein components of other food ingredients. Examples of foods that can be produced using only the reduced particle size of the filamentous fungal biomat, with or without flavoring, include, but are not limited to, 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 substitutes, bread, pasta, noodles, dumplings, pastries (e.g., choux pastry), cookies, cakes, pies, desserts, frozen desserts, ice cream substitutes, yogurt, candies, and candies.

[0263] The protein content of food fortified with a filamentous fungal biomat containing smaller particles can be significantly increased, which is particularly important for individuals adhering to a vegan diet. For example, soups, beverages, or smoothies can be fortified with a liquid dispersion of strain MK7.

[0264] Whether miniaturized biomat particles are used to enhance the protein content of food or as the sole protein component, in some cases, binders are useful in achieving the desired texture. Approved food binders such as egg white, gluten, chickpea flour, vegetable binders, arrowroot, gelatin, pectin, guar gum, carrageenan, xanthan gum, whey, chickpea water, ground flaxseed, egg substitutes, wheat flour, agar, chia seeds, and psyllium are suitable and can be used alone or in combination. In addition to food binders, miniaturized filamentous biomat particles may also be used with approved flavorings, spices, flavor enhancers, fats, fat substitutes, preservatives, sweeteners, colorants, nutrients, emulsifiers, stabilizers, thickeners, pH regulators, acidulants, leavening agents, anti-caking agents, wetting agents, yeast nutrients, dough strengtheners, dough improvers, stabilizers, enzyme preparations, gases, and combinations and mixtures thereof. Generally, binders, flavorings, and spices are selected to suit the needs of a particular group. For example, milk and / or milk solids are not used to suit individuals with dairy allergies / sensitivity, and wheat flour cannot be used to suit individuals with gluten allergies / sensitivity.

[0265] In some applications, a substantially unimodal particle size distribution, i.e., where all particles are approximately the same size, may be used, while in other applications, a broad or multimodal particle size distribution or a combination of distributions may be used. Similarly, miniaturized particles may originate from a single-source filamentous fungal biomat or a combination of filamentous fungal biomats from various sources; for example, strain MK7 alone, strain MK7 and Fusarium venenatum, or strain MK7 and Fusarium venenatum and giant puffball biomat.

[0266] The conventional use of filamentous fungi for commercial production generally required substantial infrastructure and / or equipment, energy supply, expensive additives, and / or considerable human resources. Filamentous fungi have the ability to produce a wide range of organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, pigments, and recombinant heteroproteins (Wiebi (2002) Myco-protein from Fusarium venenatum: a well-established product for human consumption. Appl Microbiol Biotechnol 58, 421-427; El-Enshasy (2007) Chapter 9-- Filamentous Fungal Cultures - Process Characteristics, Products, and Applications. In. Bioprocessing for Value-Added Products from Renewable Resources. Editor: Shang-Tian Yang. Elsevier; Gibbs et al (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit. Rev. Biotechnol. 20). 17-48), and it is well known that they possess the greatest metabolic diversity of all microorganisms on Earth, including the ability to decompose many types of difficult-to-handle materials such as lignocellulose and soil humic substances.

[0267] Although widely used, production by water fermentation still faces many challenges, including significant factors such as limited oxygen availability and growth limitations due to excessive shear forces caused by agitation (Gibbs et al (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit. Rev. Biotechnol. 20, 17-48). Under Earth's surface conditions, oxygen solubility in water is approximately 8 mg / L, which is quickly depleted by rapid growth in liquid culture. Therefore, continuous aeration using complex, expensive, and energy-intensive aeration and agitation systems is necessary to maintain high growth rates. Cultivating filamentous fungi is further challenging because their filamentous morphology imposes non-Newtonian fluid behavior, which further inhibits oxygen transfer to the solution (Norregaard et al. (2014) Filamentous Fungi Fermentation. In Industrial Scale Suspension Culture of Living Cells, H.-P. Meyer, and DR Schmidhalter, eds. (Wiley-VCH Verlag GmbH & Co. kGaA), pp. 130-162). As culture density increases, the amount of energy required to aerate and mix the culture increases nonlinearly, and similarly, the energy requirement for aerating dense cultures is extremely high. For many filamentous species, vigorous stirring and aeration of the culture is detrimental to mycelial growth, resulting in a dramatically slower growth rate. These and other challenges of aquatic fermentation of filamentous microorganisms necessitate innovative solutions for the effective utilization of these organisms in spacecraft and space stations where available resources are limited.

[0268] The disclosed reactor system (1) addresses these challenges and has the following advantages: - Active aeration or stirring of the liquid culture is unnecessary; - In-situ aggregation of biomass into a single mat with sufficient tensile strength (>0.1 kg / cm of biomat width) to facilitate collection; - Textured biomats can be used in a wide range of applications as essential products (i.e., as expression platforms for food, bioplastics, biofuels, nutritional supplements, and various pharmaceuticals); - High biomass production (80-120g / m³) 2 While maintaining a concentration of 0.55 g / L / hour (or 0.55 g / day), water usage is minimized, and residual wastewater or nutrients from the treatment are minimal and / or none; - The growth rate can result in the production of a fully formed biomat in as little as two days, or it can be extended beyond 10 days, and in some embodiments, up to at least about 21 days; - High biomass density (biomat is generally 0.684 g / cm³) 3 Wet weight or 0.123 g / cm³ 3 dry weight); - High yield (total 6.9kg / m 2 Wet weight or 1.23 kg / m² 2 Dry weight and, in some embodiments, at least approximately 206 g / m² 2 dry weight / day); - A variety of filamentous fungi (including extremophilic microorganisms and known edible and commercially viable mushrooms) with specific advantages for various processing methods can be cultivated; - It is relatively easy to scale up or down, at least about 150cm. 2 Do not reduce productivity to the extent of the growth area; - The process can utilize a wide variety of C and N-rich waste substrates generated by natural disasters and / or space missions.

[0269] The disclosed reactor system provides a self-sufficient biomat reactor including a container, in which a feedstock, fungal inoculant, at least a semipermeable membrane (e.g., gas-permeable, liquid-permeable, gas-semipermeable, and / or liquid-semipermeable) and optionally a liquid nutrient medium are arranged. Depending on the circumstances, the reactor may be a disposable or reusable reactor.

[0270] Generally, containers can be sealed and may include a container cover in addition to a seal. Some examples of containers are coated trays, coated petri dishes, and other types of coated containers or bags. In certain uses or environments, containers may have multiple growth chambers, for example, utilizing a manifold design and / or a flow rectifier system.

[0271] The feedstock is inoculated with filamentous fungal strains as described above. Examples of Ascomycetes strains include strain MK7 (ATCC PTA-10698 deposited at the American Type Culture Collection, 1081 University Boulevard, Manassas, Virginia, USA), Fusarium venenatum, Fusarium avenaceum, and / or combinations thereof. Inoculation of the feedstock may be performed when the feedstock is placed in the container or at some point after the feedstock has been placed. That is, the reactor (1) may be induced by freeze-dried filamentous fungal inoculum that is regenerated upon contact with the feedstock, or the feedstock may be inoculated directly after being placed in the reactor channel, or the feedstock may be inoculated and then placed in the reactor channel (4).

[0272] The feedstock used in the reactor may be as described above. For example, it may be waste (excrement) such as urine and / or feces that inevitably occur, food waste, plant material, industrial waste and waste by-products such as glycerol, starch and / or starch hydrolysis by-products, acid whey, sugar alcohols and / or combinations thereof. Synthetic or manufactured excrement surrogates such as surrogate human urine may also be used. Plant material feedstock is generally lignocellulose. Some examples of lignocellulose feedstocks include agricultural crop residues (e.g., wheat straw, barley straw, rice straw, small grain straw, corn stalks and leaves), corn fiber (e.g., corn fiber gum (CFG), brewing dry grain (DDG), corn gluten mean (CGM), switchgrass, sugar beet pulp, waste streams from palm oil production, alfalfa hay, sugarcane bagasse), non-agricultural biomass (e.g., algal biomass, cyanobacteria biomass, urban tree debris), forest products and industrial residues (e.g., first / second offcuts of coniferous timber, first / second offcuts of hardwood, recycled paper pulp sludge), lignocellulose-containing waste (e.g., newspaper, waste paper, brewing grain, used rubber tires (URT), municipal organic waste and by-products, garden waste and by-products, clinical organic waste and by-products, as well as waste and by-products produced during biofuel production (e.g., processed algal biomass, glycerol) and combinations thereof).

[0273] In some embodiments, the permeable membrane may, as non-limiting examples, include polymer materials such as polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, polyamide (e.g., nylon), polypyrrolone, poly(amideamine)dendrimer composites, polyvinylidene fluoride, polyethersulfone, cellulose acetate, cellulose ester mixtures, and / or butadiene-acrylonitrile. In addition to and / or separately from these, the permeable membrane may, as non-limiting examples, include glass fiber materials, porous ceramic materials, and / or fabrics, such as polypropylene cloth, polytetrafluoroethylene cloth, and / or nylon net filters. The pore size of the permeable membrane (2) may generally be any 0.01 μm increment value between approximately 0.2 μm and approximately 25 μm, or a range of values, including, as non-limiting examples, 0.2 μm, 0.22 μm, 0.45 μm, 1.0 μm, 5 μm, 10 μm, and 11 μm, and the membrane (2) may be in the form of a sterile cloth-like material or a paper-like material.

[0274] The permeable membrane allows for the optimization of the system in several different ways, as shown in Figures 15-18. The reactor system shown in the figures has a total of nine channels (4), but those skilled in the art will recognize that any number of channels (4), from one to many, can be present, depending on the space available for placement in the reactor (1). Similarly, the shape of the channels (4) is not limited to right-angle prisms or cylindrical bodies, but can take any shape suitable for fitting into the available space of the reactor (1).

[0275] In some cases, the membrane (2) is positioned in direct contact with the surface of the feed material present in the container, optionally with the liquid culture medium and inoculant, as shown in Figure 12. The membrane can also be in close contact with the surface of the feed material by being bonded, for example, to a rubber gasket integrated with a plastic frame.

[0276] In other examples, the membrane is suspended in a baffle system located between the membrane and the feedstock, allowing the membrane to fall down the mat or the aerial mycelium to grow as the fungi grow and oxygen is consumed. Such a system is shown in Figure 15. Here, the reactor (1) has multiple channels (4) that begin at an inlet valve (6) at the front (7) of the reactor and end at an outlet valve (8) at the back (5) of the reactor, separated by a baffle / wall (9). A gas permeable membrane (2) forms the top of the reactor. The bottom (3) of the reactor can be formed from any suitable material, including but not limited to rigid and flexible plastics such as polyethylene terephthalate, high-density polyethylene, polyvinyl chloride, polylactic acid, polycarbonate, acrylic acid, acetal, nylon, acrylonitrile butadiene styrene, metals such as glass, aluminum, titanium, stainless steel, etc., and / or combinations thereof. The baffle / wall (9) can be made from similar materials. Suitable front (6) and back (8) valves include, but are not limited to, check valves, two-way valves, ball valves, butterfly valves, gate valves, plug valves, globe valves, pinch valves, disc check valves, accessory valves, separator valves and / or combinations thereof. The inlet valve (6) serves as access to the chamber (4) for the delivery of feedstock / culture medium, while the outlet valve (8) allows for the removal of consumed feedstock and / or filamentous biomat. The permeable membrane (2) may, in non-limiting examples, include polymer materials such as polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, polyamide (e.g., nylon), polypyrrolone, poly(amideamine)dendrimer complexes, polyvinylidene fluoride, polyethersulfone, cellulose acetate, cellulose ester mixtures and / or butadiene-acrylonitrile. The permeable membrane may, in addition to and / or separately therefrom, include glass fiber materials, porous ceramic materials and / or, in non-limiting examples, fabrics such as polypropylene cloth, polytetrafluoroethylene cloth and / or nylon net filters.The pore size of the permeable membrane (2) can generally be any 0.01 μm increment value between approximately 0.2 μm and approximately 25 μm, or a value of this value, including, in non-limiting examples, 0.2 μm, 0.22 μm, 0.45 μm, 1.0 μm, 5 μm, 10 μm, and 11 μm, and the membrane (2) can be in the form of a sterile cloth-like material or a paper-like material. For some use, the membrane surface may have a smooth texture, and for others, the surface may have a rough texture. Furthermore, the pathways for gas diffusion may vary from essentially straight to more meandering pathways.

[0277] In other situations, the membrane facilitates the entry of oxygen and the release of other gases produced during fungal growth (Figure 14). In this situation, the sealed reactor (1) has a gas collection chamber (14) located directly above the gas permeable membrane (2) (see Figure 16). The gas collection chamber (14) may be made of materials similar to those used for the reactor walls / baffles (9) or bottom (3); i.e., both rigid and flexible plastics such as polyethylene terephthalate, high-density polyethylene, polyvinyl chloride, polylactic acid, polycarbonate, acrylic acid, acetal, nylon, acrylonitrile butadiene styrene, glass, aluminum, titanium, stainless steel, and / or combinations thereof. Alternatively, the gas collection chamber may consist of channels (15) that closely resemble the channels (4) of the sealed reactor (1) or include one or more sealed reactor channels (20) (see Figure 17).

[0278] In other systems, separate gas permeable membranes are used for gas entry and exit. Figure 18 illustrates such a system. In this case, two different gas permeable membranes (2, 50) are provided to separate gas sampling trenches (30, 40) and are located across a single reactor trench (4). This type of system allows for the entry, exit, and / or sampling and / or separation of multiple different and useful gases. For example, one membrane is prepared for oxygen passage, and the second membrane is prepared for carbon dioxide or hydrogen passage or other related gas systems.

[0279] As a non-limiting example, filamentous fungi can be inoculated and a biomat can grow on any or any side of the membrane, including the top, bottom, side facing the atmosphere and / or the side facing the gas headspace or the side facing the feed material. Biomat growth characteristics, including but not limited to the density on which the biomat grows and the side of the membrane, can be controlled by controlling various parameters such as the bioreactor, e.g., the side of the membrane on which the fungi are inoculated, the membrane material, membrane pore size and thickness, temperature, humidity, pressure, wavelength of light, or light intensity. It has generally been found that the highest quality biomat is achieved when the feed material is constantly interacting (i.e., in physical contact) with the permeable membrane and the biomat grows on the opposite side of the membrane (i.e., facing the atmosphere or gas headspace). In particular, in some embodiments, it may be advantageous to seal the biomat or otherwise isolate it from the ambient atmosphere and grow it in a gas headspace that allows the biomat to become moist as it grows and produces water. It is also advantageous to configure the bioreactor so that the biomat and, in particular, the hyphae of filamentous fungi on the biomat remain substantially dry during biomat growth (i.e., not in contact with liquid growth media or other free liquid moisture).

[0280] As the biomat grows, water is produced by the respiration of the growing fungi and is stored within the bioreactor (generally on the gas phase side of the membrane). In addition to providing a humid environment suitable for further biomat growth, this stored water can be collected as a valuable product in itself and used for some suitable purpose. Thus, one advantage of the present invention is that a portion of the feedstock, which may include waste (excrement) such as feces or animal urine, can be converted into tap water. Other feedstocks from which tap water can be properly produced include sugars (e.g., sucrose, maltose, glucose, fructose, rare sugars, etc.), sugar alcohols (e.g., glycerol, polyols, etc.), starch (e.g., corn starch, etc.), starch derivatives, starch hydrolysates, hydrogenated starch hydrolysates, lignocellulose pulp or feedstocks (e.g., sugar beet pulp, agricultural pulp, wood pulp, brewing dry grains, brewing wastewater, etc.), corn steep liquor, acid whey, sweet whey, whey, wheat steep liquor, industrial liquor, food processing product / waste streams, agricultural crop residues (e.g., wheat straw, barley straw, rice straw, peas, oats, small grain straw, corn stalks and leaves), corn fiber (e.g., corn fiber gum (CFG), brewing dry grain (DDG),CFG), brewing dry grain (DDG), corn fiber This may include, but is not limited to, one or more of the following: ruten coarse grain (CGM), switchgrass, alfalfa hay, sugarcane bagasse, non-agricultural biomass (e.g., algal biomass, cyanobacteria biomass, urban tree debris), plants (e.g., carrots, broccoli, garlic, potatoes, beets, Sparassis crispa), forest products and industrial residues (e.g., first / second offcuts of coniferous timber, first / second offcuts of hardwood, recycled paper pulp sludge, anaerobic fermentation residues), lignocellulose-containing waste (e.g., newspaper, waste paper, brewing grains, used rubber tires (URT), municipal organic waste, garden waste, clinical organic waste, sugars, starches, waste oil, olive oil, olive oil processing waste, cricket waste, and waste produced during biofuel production (e.g., processed algal biomass, glycerol).

[0281] As the biomat grows, the respiration of the growing fungi may produce one or more gases, such as ammonia, hydrogen gas, and / or volatile esters. In one embodiment, the membrane may be a selective gas permeable membrane that allows for the separation and / or isolation of at least one gas produced by fungal respiration.

[0282] The pressure difference between one side of the membrane and the other can be utilized to influence various properties of the fungal biomat. For example, the present invention intends non-atmospheric pressure on one or both sides of the membrane, e.g., one side being ultra-atmospheric or sub-atmospheric and the other atmospheric pressure, or both sides being ultra-atmospheric or sub-atmospheric, or one side being ultra-atmospheric and the other sub-atmospheric. In non-limiting examples, the growth rate of the biomat may be affected; for example, positive pressure on the feeding side of the membrane is similar to "supplying" the biomass, promoting biomat production and / or increasing yield, while positive pressure on the "dry" side of the membrane may result in a slowdown in biomass production, leading to alterations in the composition or physical properties of the fungal biomass. Conversely, in some embodiments, it may be advantageous for the biomat to remain dry during growth, and therefore the opposite effect may be observed: positive pressure results in a "wetter," slower-growing mat, while negative pressure results in a "drier," faster-growing mat. The selective application of pressure may also be suitable for determining the membrane type that is most advantageous for a particular application of the bioreactor, for example, pressure fluctuations may be more or less advantageous for membranes of a certain pore size. Changes in gas headspace and / or ambient atmospheric pressure may affect biomat growth characteristics, such as aerial mycelial production, hyphae, mycelium and / or fiber production and biomass density, and may also promote filamentous fungal inoculation into the membrane.

[0283] In one embodiment, cyanobacteria may be provided to either side of the membrane within the bioreactor, either naturally or by inoculation. Whether alive or dead, the cyanobacteria advantageously contribute to the nutritional characteristics of the feedstock as carbon and nitrogen sources. Furthermore, living cyanobacteria can act as oxygen sources that promote biomass growth as products of respiration. One or both of these advantages may make the bioreactor of the present invention particularly suitable for certain applications where other solutions are unavailable, such as long-duration manned space missions. An exemplary schematic diagram of an embodiment of a bioreactor including respiratory cyanobacteria is shown in Figure 29.

[0284] In most embodiments, a gentle force must be applied for biomat collection (i.e., removal from the membrane surface), but in some embodiments, the biomat can be "self-collecting," i.e., it can spontaneously separate from the membrane. The inventors have observed this phenomenon particularly when, for example, the biomat has a high moisture content, oleic acid is present in the feedstock, and / or the membrane is characterized by being relatively thin (e.g., less than 0.2 mm thick), smooth, and low inflexibility. In some embodiments, self-collection can be achieved by applying pressure. The adhesion strength of the biomat to the membrane depends on one or more bioreactor parameters, such as the filamentous fungal species, the feedstock composition, membrane characteristics (e.g., material, pore size, geometry / roughness, etc.), the pressure on either side of the membrane, and / or the pressure difference across the membrane, and therefore can be selected by control. Providing a self-collecting mat by spontaneously separating from the membrane surface is one aspect of the present invention.

[0285] Reactor (1) produces a biomat that serves as a food source, such as a protein source and / or an oil source. However, the biomat may also serve as a source of leather analogs, bioplastics, biofuel precursors, biofuels, and / or combinations thereof. In yet another embodiment, the biomat is useful for producing organic products such as organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, pigments, and recombinant heterologous proteins.

[0286] The disclosed biomat reactor fermentation technology enables growth using standard and specialized feedstocks and media such as human excrement (urine / feces), producing highly integrated and textured products without the need for separation or concentration processes.Relatively high biomass production rate - i.e., at least about 0.05 g / L / hour dry biomass (i.e., grams of dry biomass produced per liter of feed per hour) or at least about 0.10 g / L / hour dry biomass or at least about 0.15 g / L / hour dry biomass or at least about 0.20 g / L / hour dry biomass or at least about 0.25 g / L / hour dry biomass or at least about 0.30 g / L / hour dry biomass or at least about 0.35 g / L / hour dry biomass or at least about 0.40 g / L / hour dry biomass or at least about 0.45 g / L / hour dry biomass or less At least approximately 0.50 g / L / hour of dry biomass or at least approximately 0.55 g / L / hour of dry biomass or at least approximately 0.60 g / L / hour of dry biomass or at least approximately 0.65 g / L / hour of dry biomass or at least approximately 0.70 g / L / hour of dry biomass or at least approximately 0.75 g / L / hour of dry biomass or at least approximately 0.80 g / L / hour of dry biomass or at least approximately 0.85 g / L / hour of dry biomass or at least approximately 0.90 g / L / hour of dry biomass or at least approximately 0.95 g / L / hour of dry biomass or at least approximately 1.00 g / L / hour of dry biomass - and / or, for example, in the core of a batch process, high production (i.e., biomattgrams produced per liter of feedstock) of at least about 10 g / L, at least about 20 g / L, at least about 30 g / L, at least about 40 g / L, at least about 50 g / L, at least about 60 g / L, at least about 70 g / L, at least about 80 g / L, at least about 90 g / L, at least about 100 g / L, at least about 110 g / L, at least about 120 g / L, at least about 130 g / L, at least about 140 g / L, at least about 150 g / L, at least about 160 g / L, at least about 170 g / L, at least about 180 g / L, at least about 190 g / L, or at least about 200 g / L is achieved without active aeration or stirring.Scaling up the system vertically, horizontally, and / or in more than two dimensions is straightforward and does not result in a decrease in productivity. The produced biomats are typically 0.2–2.5 cm thick with a dry matter content of 10–30% and can be readily used for critical needs such as meat substitutes, countless other appetizing foods, and building materials.

[0287] The fungal biomats grown in the disclosed reactor system can be described as a thick outer layer, which in many ways resembles a microbial biofilm grown on a surface, but corresponds to the biomat thickness described herein and is located at the gas-liquid interface. For example, bacterial cells within the biofilm can withstand extreme disinfection treatments with sodium hypochlorite (bleach) and sodium hydroxide (Corcoran, 2013). The disclosed reactor system leverages the advantages of biofilm structure to enable growth in unpleasant human waste, industrial waste, and by-products that may be produced under extreme conditions, such as those caused by space missions or other undesirable conditions resulting from natural disasters.

[0288] The disclosed reactor designs include a permeable membrane that is either directly installed or suspended just above the liquid surface of the feedstock. In one embodiment, a sealed reactor design allows gas exchange with the external atmosphere but is sealed to prevent contamination or leakage due to gas / liquid ingress. The sealed reactor design may also allow separation of consumable gases from generated gases by means of a gas permeable membrane. To achieve this, in one example, valves and / or further porous membranes having the same or different properties are used to form different layers between one or more feedstocks and various embodiments of the liquid culture medium, which is optionally present.

[0289] Rapid biomat growth using the disclosed reactor design has been demonstrated with a variety of permeable membrane materials. Figure 13 shows a biomat approximately 7 mm thick grown in a reactor where the container placed directly on the surface of the feedstock / liquid medium is a Petri dish covered with a propylene membrane. The initial biomat was formed by direct binding to the membrane and grew downward into the liquid medium over time (see Figure 12). At the end of the 5-day growth period, essentially all of the feedstock / liquid medium was consumed, and dense biomass completely filled the volume beneath the membrane.

[0290] The biomat was produced with only light adhesion to the membrane and was easily harvested by simply peeling the biomat from the membrane (see Figures 13A-13D). Further experiments with polycarbonate membranes yielded similar results (data not shown). Therefore, the total reactor volume can be efficiently utilized for the production of dense, easily harvestable biomass. In a non-limiting example, the bioreactor of the present invention may take the form of a sealed "envelope" including four membranes: an outer upper membrane, an inner upper membrane, an inner lower membrane, and an outer lower membrane. In such an embodiment, the feedstock may be provided between the inner upper membrane and the inner lower membrane, thereby "sandwiching" the feedstock between either of the vertical gas headspaces (i.e., between the outer and inner upper membranes and between the outer and inner lower membranes). Thus, the biomat grows from the outer surface of the inner membrane into the gas headspace on either side of the feedstock, while simultaneously being at least partially isolated from the ambient atmosphere by the outer membrane (which may be a gas-permeable or gas-semipermeable membrane embodiment). An "envelope" bioreactor could be a highly volumetrically efficient bioreactor that can be used in situations where space is scarce, such as on a spacecraft or in emergency or rescue situations.

[0291] The biomats generally produced in the disclosed reactors are highly dense, as described herein, and exhibit a fibrous texture depending on the fungi and growth conditions. The production of fibrous biomass is essential for certain products, such as foods requiring a meat-like texture, as well as fibrous materials that mimic leather and wood. The dense nature of the biomass allows for easy harvesting without the need for concentration processes (e.g., centrifugation, filtration). The density of the biomats is approximately 0.01 g dry biomat weight / cm³. 3 ~Approx. 1g / cm 3 and any lower range within this range. In one embodiment, the density is 1 cm 3 It may be more than approximately 0.01, more than approximately 0.02, more than approximately 0.03, more than approximately 0.04, more than approximately 0.05, more than approximately 0.06, more than approximately 0.07, more than approximately 0.08, more than approximately 0.09, more than approximately 0.1, more than approximately 0.2, more than approximately 0.3, more than approximately 0.4, more than approximately 0.5, more than approximately 0.6, more than approximately 0.7, more than approximately 0.8, more than approximately 0.9, or more than approximately 1g.

[0292] Herein, Figure 23 illustrates several different bioreactor configurations, each within the scope of the present invention. In configuration 1, the feedstock is placed beneath the membrane and in physical contact, and the biomat grows upward from the top surface of the membrane (however, in some embodiments, the fungal material may also grow from the bottom of the membrane, i.e., the feedstock-side surface). In configuration 2, the biomat grows directly from the feedstock surface (i.e., there is no membrane between the biomat and the feedstock) into the gas headspace, and the feedstock, biomat and headspace are separated from the surrounding environment by a membrane; the membrane in this embodiment may be similar to or different from the membrane placed between the feedstock and the biomat in other embodiments. In configuration 3, two membranes are provided: a lower membrane that separates the feedstock from the biomat (as in configuration 1) and an upper membrane that separates the biomat and the gas headspace on the biomat from the surrounding environment (as in configuration 2). In the configuration shown in 4, an "inverted" bioreactor scheme is provided, in which the feedstock is supplied to the upper surface of the membrane and the biomat grows downward from the lower surface of the membrane into the gas headspace and / or the surrounding environment (however, in one embodiment, the fungal material may grow upward from the upper surface of the membrane, i.e., the feedstock side surface).

[0293] Next, Figures 24A and 24B show the "airtight" (tightly sealed to the environment) configuration of the bioreactor in the arrangement indicated as "1" in Figure 23, before (Figure 24A) and after (Figure 24B) the production of the biomat. As shown in Figure 24B, the fresh water produced by the respiration of the filamentous fungi condenses and accumulates on the gas phase side of the membrane.

[0294] Next, Figure 33 shows a nylon net filter membrane. In this embodiment, the nylon net filter consists of two nylon membranes (pore size 11 μm) that are "laminated" (i.e., arranged in physical contact such that the second surface of the first membrane is adjacent to the first surface of the second membrane) in order to reduce the effective porosity of the membrane and leave the membrane on the surface of the feed material. This type of membrane is suitable for the growing fungal biomat according to the present invention.

[0295] Next, Figures 35A and 35B illustrate a membrane envelope bioreactor (MEBR). In this embodiment, the MEBR includes a sealed chamber or rocker that is in fluid communication with a culture medium reservoir and surrounds a plurality of membrane envelopes. As shown in Figure 35A, the growth medium is brought from the culture medium reservoir to the MEBR and flows into each of the plurality of membrane envelopes. As shown in Figure 35B, each membrane envelope includes a porous inner core material surrounded on each side by a semipermeable membrane. As the growth medium flows through the membrane envelope, it comes into contact with the porous inner core material and flows, allowing the filamentous fungal biomat to approach the growth medium through the semipermeable membrane and thus grow on the outer surface of the semipermeable membrane.

[0296] It should be explicitly understood that various other configurations of the bioreactor are intended and within the scope of the present invention. As a non-limiting example, one such configuration is a “trough” bioreactor, in which an upward-opening, arc-shaped hydrophilic membrane having a relatively small pore size (e.g., 0.2 μm) is provided as a trough, and a hose having holes passing through it is provided parallel to the longitudinal direction of the membrane. The feedstock can be sprayed, for example, through the holes in the hose, ensuring that the inner / upper arc-shaped surface of the trough is uniformly saturated with growth medium at all times. The biomass can then grow on the opposite side of the trough membrane, i.e., the outer / lower surface, which in one embodiment may allow it to fall down from the membrane by its own weight when it reaches a certain weight.

[0297] Use of biomat reactors in zero gravity The primary physical force controlling the formation and growth of biomats in the disclosed reactor is adhesion to the membrane. While not constrained by theory, it is believed that biomats growing in the disclosed reactor would not be affected by the microgravity conditions experienced during spaceflight. Growth controlled by gravity-driven directional growth or physical mixing or flow is not a critical factor of the system, as is the tendency that occurs in a gravity environment. Previous experiments in space have successfully demonstrated fungal growth (European Space Agency, Expeditions 25-28, Growth and Survival of Colored Fungi in Space (CFS-A)), providing further confidence that the disclosed reactor system will function in a space environment.

[0298] For ease of space operations and development, the reactor can be pre-filled with freeze-dried inoculum and essential components (if necessary) that support the growth of specific feedstocks. Astronauts and space travelers can then prepare the feedstocks, inoculum, and any culture medium components. Incubation time depends on the feedstocks, microbial strains, and other growth parameters such as pH, temperature, and moisture content. Incubation conditions are simple in that fermentation is carried out under static conditions that allow the reactor to simply be incubated in the correct position. A dense, integrated biomat is harvested simply by opening the reactor closure (e.g., a Ziploc® type) and taking the mat.

[0299] Various embodiments of the present invention are described in detail, but it will be apparent that modifications and adaptations of these embodiments can be put into practice by those skilled in the art. However, it should be expressly understood that such modifications and adaptations are within the scope of the present invention, as set forth in the appended claims. Examples and figures are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Each publication or other reference disclosed herein is incorporated herein by reference in its entirety, to the extent that it does not conflict with this disclosure. [Examples]

[0300] Example 1: Growth of Fusarium strain MK7 and other fungi in a static tray reactor. Filamentous acidophilic Fusarium strain MK7, Ganoderma lucidum (Reishi mushroom; Figure 1A), Pleurotus ostreatus (marbled oyster mushroom; Figure 1B and oyster mushroom; Figure 1C), Sparassis crispa (flower mushroom; Figure 1D), Hypsizygus ulmarius (white oyster mushroom; Figure 1E), Calvatia gigantea (giant puffball; Figure 1F), and Fusarium venenatum biomats were propagated in shallow static tray reactors as described in PCT / US2017 / 020050.

[0301] Example 2: Growth of Fusarium strain MK7 biomat in a nutrient medium refreshed daily (semi-static conditions). A dense Fusarium strain MK7 biomat, approximately 3 cm thick, was grown for 21 days in nutrient medium refreshed daily. The biomat was grown in a 12.7 × 17.8 cm Pyrex® glass tray in sterile MK7-1 liquid medium (described in PCT / US2017 / 020050) containing 7.5% glycerol at pH 3.0. To begin the experiment, 200 mL of nutrient medium was inoculated with a 5% (volume / volume) late-stage exponential growth Fusarium strain MK7 culture, as previously described in PCT / US2017 / 020050. 200 mL of inoculum was added to each of three sterile trays lined with sterile coarse nylon mesh screens. The cultures were incubated gently at room temperature (approximately 22°C) for 4 days to allow the growth of the initial biomat layer formed on the surface of the liquid. After 4 days of growth, the biomat was gently lifted from the tray using a nylon mesh screen and tilted at a 45° angle to drain the liquid from the mat. The biomat was drained in this position until the dripping liquid was less than one drop every 5 seconds. On average, it took approximately 3 minutes to drain completely. The screen-dried biomat was placed in a fresh, pre-weighed 12.7 × 17.8 cm Pyrex® tray containing 200 mL of fresh MK7-glycerol medium (as described in PCT / US2017 / 020050). The tray containing the biomat was weighed again. The process of transferring the biomat to other trays containing fresh medium was repeated on an almost daily basis for another 17 days. Sampling of one biomat was performed on days 12, 15, and 21 to determine the moisture content of these biomats. The average moisture content of the biomat was 17.3% (standard deviation = 0.7), and this value was used to calculate the dry biomass production over the experimental period. Dry biomass production is linear from day 4 to day 18 (r 2 (=0.995), and the biomass weight is then approximately 2.5 kg / m² dry. 2 Stable (Figure 1, m 2 The y-axis is standardized by the base, and growth is generally exponential from day 0 to day 4. The average growth rate during this period for linear growth is 6.04 g / m². 2 The time was / hour. Figure 2 shows the approximately 3cm thick biomat that developed after a total of 21 days of growth using this method.

[0302] To confirm this finding, the same experimental protocol was repeated with a control tray where the biomat growth medium was not refreshed. The results of this comparative experiment are shown in Figure 36. As Figure 36 shows, the tray with refreshed medium showed biomat growth approximately three times faster (10⁹ vs. 34 dry biomass grams per square meter per day) and for approximately twice as long (21 vs. 10 days). Surprisingly, refreshing the growth medium not only maintained the same growth rate as the control, but actually accelerated growth; the refreshed tray and control showed similar amounts of growth until day 3 (the period before the medium was first refreshed), but a rapid increase in the growth rate in the refreshed tray was observed on days 3-4, and this increase persisted for almost the entire duration of the experiment.

[0303] Example 3: Growth of biomats under continuous flow conditions. A continuous flow bioreactor system was constructed to demonstrate the growth of biomats on the surface of a flowing liquid culture medium. The system was constructed from a 2.44 m long transparent plastic roof panel with a series of corrugated sections used as flow channels (Figure 3). The ends of each groove were sealed with silicone (100% silicone, DAP Products Inc., Baltimore, MD) to allow the liquid to remain within the channels. A peristaltic pump facilitated the flow of the liquid culture medium from one end of the channel through the channel, and at the other end of the channel, the liquid flowed out through a hole at the bottom of the channel. The entire plastic roof panel system was tilted at an angle of 1 cm per 1 m, so that approximately 500 mL of liquid was held in each channel, ensuring that the constant flow was a function of the liquid volume and the tilt angle.

[0304] The panel system was disinfected, wrapped in Saran®-like plastic wrap, and isolated from the surrounding room environment. Sterile air was pumped through the plastic wrap at a rate of 400 mL / min to create positive pressure in the system. To initiate biomat development before starting flow, 500 mL of nutrient medium inoculated with the desired filamentous fungus was added to each channel and incubated under static conditions for 4 days. After 4 days, the peristaltic pump "supplied" the biomat with a continuous pulsed flow of 400 mL / day (ON for 49 minutes, 39 seconds, at 2.016 mL / min; OFF for 5 hours, 10 minutes, and 21 seconds). Two independent experiments were conducted, each using two different flow channels as replication (Figure 3).

[0305] The integrated biomats were harvested after 10 days of growth in nutrient medium (4 days under static conditions, followed by 6 days of continuous flow; Figure 4). The average dry weight of the biomass produced was 2.38 g per replicated flow channel. During the continuous flow period (days 4-10), the average removal rates of C and N from the flowing liquid medium by the growing biomats were 11.9 mg and 1.2 mg / L / hour, respectively. The C and N removal rates from the liquid medium were determined by measuring the liquid volume and total C and N input and output from the bioreactor system using a Costech total C and N analyzer (ECS 4010, Costech Analytical Technologies, Valencia, CA). Therefore, the continuous flow system supported biomat growth on the surface. The experiment helped to provide laboratory-scale proof of continuous-feed Fusarium strain MK7 biomat growth and integrated biomat production. It should be noted that other feedstocks, flow rates, and resulting growth rates can be achieved with this type of system. For example, with 10% glycerol in MK7-1 medium at pH 2.8 (as described in PCT / US2017 / 020050), the predicted yield is 40 grams of dry biomass / day / m². 2 Larger.

[0306] Example 4: Semi-continuous and continuous production of Fusarium strain MK7 biomats. Dense Fusarium strain MK7 biomats were grown and harvested on a semi-continuous basis over 19 days. The biomats were supplemented with 1 / 2 volume of MK7-1 medium salt adjusted to pH 4.0 (as described in PCT / US2017 / 020050), and acid whey was used as the feedstock / carbon source. To begin the experiment, 200 mL of nutrient medium inoculated with Fusarium strain MK7 (5% vol / vol) in the late exponential growth stage was added to a sterilized 12.7 × 17.8 cm Pyrex® glass tray, then wrapped in Saran® plastic wrap and incubated at room temperature. After 5 days of growth, 1 / 3 of the biomat was removed by cutting from one end of the tray, and a 5.9 × 12.7 cm section of the biomat was removed (Figure 5A). The remaining 2 / 3 of the biomat was then physically moved into the open area created by the removal of the 1 / 3 portion of the biomat. The biomat was moved by physically grasping it with a hand wearing sterile gloves, and the biomat was lifted up until it reached the edge of the tray to open the culture medium, with no biomat formed at the other end of the tray (Figure 5B). A 1 / 3 section of the majority of the mature biomat was harvested and subsequently moved to the remaining 2 / 3 of the biomat into the empty area, a process repeated periodically. 50 mL of culture medium was aseptically removed from the tray every 4 days and replaced with 50 mL of fresh sterile culture medium (acid whey and 1 / 2 volume MK7-1) to replenish the nutrients removed from the liquid medium by the biomat removal. Dry biomass production using this method was 0.57 g / day / tray or 25.2 g / day / m² from days 5 to 19. 2 This produced (Figure 6). Therefore, the mature ends of most of the biomats were harvested at an average rate of 1.56 cm / day, demonstrating a semi-continuous production system in which fresh biomat growth begins in the empty area of ​​the culture medium at the other end of the tray.

[0307] The system is also suitable for continuous harvesting and propagation of biomats, where continuous harvesting is facilitated by a roller wheel attached to the mature end of the biomat (Figure 7). The roller wheel rotates slowly, harvesting the mature biomat and simultaneously creating empty medium at the other end of the tray for the propagation of new biomats. To replicate the semi-continuous system described above, the roller wheel rotates at a speed of 1.56 cm / day to harvest the biomats. It is desirable to replenish the nutrients in the liquid medium at a rate equal to the nutrient depletion rate by the biomat.

[0308] Example 5: Membrane-sealed bioreactor. Dense Fusarium strain MK7 biomats were grown in liquid growth medium surrounded by a bioreactor system without gas headspace. A sterile Petri dish bottom (55 mm diameter) was filled to the brim with 57 mL of inoculated MK7-1 medium containing 8% glycerol (as described in PCT / US2017 / 020050). Gas-permeable / semipermeable polypropylene and polycarbonate membranes were placed directly on the liquid surface medium and sealed with rubber bands. No gas headspace was provided at the start of the growth phase.

[0309] After inoculation with culture medium and membrane sealing, the bioreactor was left undisturbed until harvesting. Figure 8 shows biomats of Fusarium strain MK7 approximately 5 mm and 1 mm thick, grown directly under polypropylene (Figures 13A-13C) and polycarbonate (Figure 13D) membranes, respectively, for 5 days. The biomats adhered loosely to the membrane and were easily harvested by simply peeling them off the membrane (Figure 13).

[0310] Example 6: Production of pigments and vitamin D2 by irradiation of Fusarium MK7 biomat with UVB. Pigment production was induced in Fusarium strain MK7 biomats using UVB light (290-320 nm). Fusarium strain MK7 biomats produced for 3 days in 7.5% glycerol MK7-1 medium (described in PCT / US2017 / 020050) were irradiated with UVB light for 4 hours. The UVB light was emitted from a 50W bulb (Slimline Desert 50 UVB T8 fluorescent bulb, 46 cm; Zilla, Franklin, WI) placed 10 cm above the biomats. Orange coloration was visible after 0.5 hours of irradiation and was significant after 4 hours (Figure 9). Furthermore, biomats not exposed to UVB light had a vitamin D2 content of less than 50 IU / 100g biomat, and after approximately 12 hours of UVB light exposure, the vitamin D2 content increased to approximately 1.2 million IU / 100g biomat.

[0311] Example 7: Fusarium strain MK7 biomat grown in a mixture of glycerol, starch, and corn steep liquor. Fusarium strain MK7 biomats were produced in just 4 days from a mixture of glycerol, starch, corn steep liquor, and MK7-1 salt (described in PCT / US2017 / 020050). Glycerol was purchased from Duda Energy LLC (Decatur, AL; 99.7% purity; USP grade; Lot# 466135376340); 100% Argo Corn Starch manufactured by Argo Food Companies, Inc (Memphis, TN) was purchased from Albertsons Supermarket in Bozeman, MT; and corn steep liquor was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX; Lot# B0116). The growth medium was a mixture of 7.5% glycerol (wt / wt), 2.5% starch, and 2.5% corn steep liquor with MK7-1 salt. The mixture was adjusted to pH 3.3 by adding an appropriate amount of HCl and boiled in a suitable container for 15 minutes. After cooling to room temperature, the pH of the mixture was readjusted to 3.3, and then inoculated with 5% Fusarium strain MK7 inoculum (vol / vol) prepared as in PCT / US2017 / 020050. 1.5 L of inoculum was divided into three 0.25 m disinfection chambers. 2 In addition to polypropylene trays, the trays were placed in a disinfection tray rack system, completely covered with aluminum foil to create dark conditions, and incubated at 23°±1°C. The filamentous fungal biomats that had grown on the surface of the culture medium were collected after 4 days by simply lifting the biomats from the trays.

[0312] The average final pH of the residual liquid in the three trays was 4.45 (standard deviation = 0.14). Three 56.7 cm² locations on the biomat were randomly selected. 2 The circular portions were cut out and dried at 50°C for 48 hours to obtain the dry weight. The average dry biomass weight (standard deviation) was 124.6 g / 0.25 m 2 (43.4) or 498.4 g / m 2 (173.6) The average thickness of the wet biomat was 7.5 mm, and the average density on a dry weight basis was 0.66 g / cm³. 3 That was the case.

[0313] To expose the biomatt fibers and enable testing with a field emission scanning electron microscope (FE-SEM), the extracellular matrix (ECM) between the fibers was removed by washing with ethanol. To achieve this, 1 cm of the biomatt was used. 2 A portion (1 cm x 1 cm) was cut out with a razor immediately before collection, and the excised portion was subjected to an ethanol washing / dehydration series by continuously immersing the sample in 40 mL of ethanol mixture the following number of times: 25% ethanol, 75% deionized H2O for 20 minutes; 50% ethanol, 50% deionized H2O for 20 minutes; 75% ethanol, 25% deionized H2O for 20 minutes; 95% ethanol, 5% deionized H2O for 20 minutes; 100% ethanol, 0% deionized H2O for 60 minutes. The 100% ethanol treatment was repeated two more times, after which the sample was stored in 100% ethanol.

[0314] To maintain the microstructural integrity of the biomatt for FE-SEM, it was washed / dehydrated with ethanol, followed by critical drying using a Tousimis Samdri-795 critical point dryer as instructed by the manufacturer (Tousimis Samdri-795 Operations Manual; Tousimis, Rockville, MD). After critical drying, the sample was either placed directly onto an aluminum stub or cut into <0.3 mm thick sections with a razor before mounting. The sample was then coated with iridium (20 μm, EMITECH K575X, Electron Microscopy Sciences, Hatfield, PA) and tested on a JEOL 6100 FE-SEM using a 1 keV incident beam energy (JEOL USA, Inc., Peabody, MA).

[0315] FE-SEM images revealed a complex network of interwoven mycelial fibers (Figure 10) that closely resembled the structure observed by optical microscopy of a biomat grown on glycerol, as reported in PCT / US2017 / 020050. Three distinct layers were observed: (a) an aerial mycelial layer on the upper surface, (b) a dense bottom layer, and (c) a transitional layer between the upper and bottom layers. The transitional layer was only loosely attached to the dense bottom layer, thus allowing for easy separation of the bottom layer from the rest of the biomat. The fiber density of the transitional layer ranged from slightly lower than the bottom layer at the point where the two layers met to a density comparable to the aerial mycelium near the top of the biomat.

[0316] The excised samples were also prepared for use with a light microscope by slowly immersing them in the following solutions in the order and for the duration described below:

[0317] Xylene, 3 minutes; Xylene, 3 minutes; 100% ethanol, 3 minutes; 100% ethanol, 3 minutes; 95% ethanol, 3 minutes; 95% ethanol, 3 minutes; 70% ethanol, 3 minutes; Deionized water, 3 minutes; Hematoxylin 1, 1.5 minutes; Rinse with tap water, 1 minute; Cleaning solution, 1 minute; Rinse with tap water, 1 minute; Bluing solution, 1 minute; Rinse with tap water, 1 minute; 70% ethanol, 30 dips; 95% ethanol, 30 dips; 95% ethanol, 30 dips; 100% ethanol, 30 dips; 100% ethanol, 30 dips; Xylene, 30 dips; Xylene, 30 dips; Xylene, 30 dips; Apply cover glass.

[0318] Approximately 2cm 2Biomat sections of a specific size were cut from fresh biomats using a razor blade immediately before collection. These sections were then immersed in 35 mL of deionized water in 50 mL conical bottom centrifuge tubes. The tubes were subjected to ultrasonic treatment (CP200T Ultrasonic Cleaner, Crest Ultrasonics, Ewing, NJ) for 0, 40, 90, or 150 seconds to disperse the fibers in the liquid and enable microscopic observation. A certain amount of liquid (approximately 100 μL) from these tubes was placed on a glass slide, covered with a coverslip, and observed under a 100x optical microscope (B400B, Amscope, Irvine, CA). The average length (standard deviation) of the non-destructive fibers was measured and determined to be 1.1(0.6) mm, 1.2(0.4) mm, 1.0(0.4) mm, and 1.2(0.2) mm for 0, 40, 90, and 150 seconds of ultrasonic treatment, respectively. The maximum fiber lengths observed in each treatment were 2.5 mm, 1.4 mm, 1.8 mm, and 1.4 mm, respectively. These fiber lengths were significantly longer compared to the growth of Fusarium strain MK7 in water-shaking flask culture, where the average length was 0.02 mm.

[0319] Example 8: Production of chicken nuggets using Fusarium strain MK7 biomat grown in a mixture of glycerol, starch, and corn steep liquor. Chicken nuggets were prepared using the Fusarium strain MK7 biomat produced as described above. The moist biomat was steamed in a pot steamer at 97°C for 0.5 hours, cooled to room temperature, and used as the base for making the chicken nuggets. Steam-treated moist biomat (200g) was chopped into pieces less than 0.5mm long and homogenized with 4% (by weight; 8g) chicken base and 4% egg white protein (8g). The resulting mixture consisted of over 90% Fusarium strain MK7 biomat. A portion of this biomat mixture (approximately 30g) was shaped into nuggets and steamed in a pot steamer. The prepared nuggets were baked by coating them with egg white, then mixed with breadcrumbs to adhere to the surface, and then fried. The prepared nuggets had a chicken-like texture and a typical chicken aroma. In a taste test by five people, the nuggets were judged to closely mimic actual chicken nuggets containing chicken meat in terms of taste and texture.

[0320] Example 9: Production of Fusarium strain MK7 biomat extract. A highly concentrated and viscous extract was produced from Fusarium strain MK7 biomats. After culturing for 4-16 days as previously described, the biomats were rinsed, steam-treated, drip-dried for 5 minutes through a porous plastic mesh, placed in a plastic bag, and sealed. The sealed bag was frozen at -20°C or -80°C for 24 hours, and then the pH of the remaining culture medium liquid was adjusted to pH 4-6. After that, the original sealed bag was incubated in a 60°C incubator for 48 hours. After heat treatment, the biomats were sieved through a <1.5 mm pore size filter, and the resulting liquid was collected. The collected liquid was boiled in a non-reactive container for 10 minutes, and then dried at 60°C until the moisture content was approximately 6-8%, forming a viscous paste extract. The nutritional value of the extract was similar to that of the steam-treated biomats and the powder made from the steam-treated biomats.

[0321] Example 10: Production of yogurt from Fusarium strain MK7 biomat grown in acid whey. The Fusarium strain MK7 biomat was used directly in yogurt production. The biomat was grown in trays using acid whey, a by-product of Greek yogurt production, as a raw material / carbon source. After 6 days, it was harvested and steamed within 20 minutes of harvesting. 200g of cooled, moist biomass was mixed with 600g of drinking water to produce a milky suspension called "MK7 liquid dispersion." The MK7 liquid dispersion was used as a standalone component and in combination with milk in yogurt production.

[0322] Three mixtures with different ratios of MK7 liquid dispersion to whole milk were prepared: 1) 25% MK7 liquid dispersion: 75% whole milk, 2) 50% MK7 liquid dispersion: 50% whole milk, and 3) 100% MK7 liquid dispersion. Using these mixtures, three batches of yogurt were produced by heating each mixture to 83°C and holding it at that temperature for 14 minutes with constant stirring. The mixtures were cooled to 43°C, and then live yogurt cultures were added as inoculants. The resulting mixtures were incubated at 44°C in a yogurt maker (model YM80; EuroCuisine, Los Angeles, CA) for 8 hours. All of the resulting mixtures had the appearance and texture of yogurt, as well as an odor and taste similar to typical yogurt.

[0323] Example 11: Propagation of mushroom biomats in glycerol. A biomass biomat consisting of Baby Bella brown mushrooms (Agaricus bisporus) and white mushrooms was produced in just 10 days using glycerol as the primary carbon source (supply material). These common edible mushrooms were purchased from Albertson Supermarket in Bozeman, Montana, and stored at 4°C. The culture medium used for mushroom propagation consisted of 1 L of 7.5% glycerol and MK7-1 salt (as described in PCT / US2017 / 020050), which was boiled for 10 minutes and then cooled to room temperature (approximately 23°C). The pH of the mixture was adjusted to 2.7, and 200 mL of the pH-adjusted mixture was poured into two sterile 12.7 × 17.8 cm Pyrex® trays. The inoculant consisted of 5 g of mixed, surface-sterilized brown or white mushrooms, which was added to the culture medium in each tray. Mushroom inoculation material was prepared as follows: 1) 10 g of moist brown or white mushrooms were added to 200 mL of 5% bleach solution, and the suspension was stirred for 2 minutes to sterilize the surface of the mushrooms; 2) The mushrooms were then rinsed by transferring them to 200 mL of sterile glycerol / MK7-1 salt medium (as described in PCT / US2017 / 020050) and stirred for 2 minutes; 3) The surface-sterilized mushrooms were mixed in a coffee grinder sterilized by rinsing with 70% ethanol for 30 seconds; 4) The crushed mushroom biomass (<5 mm long aggregates) was surface-sterilized again by repeating steps 1 and 2 with the crushed biomass; 5) 5 grams of crushed mushroom biomass were added to the liquid medium in a Pyrex® tray (final pH = 4.0~4.1 after adding the mushrooms); and 6) the tray was covered and incubated in the dark at room temperature (22±2℃).

[0324] The biomats were observed to grow on the surface of the culture medium after 3 days of incubation, and the integrated biomats were harvested after 10 days of growth. The brown mushroom biomats covered the entire surface of the liquid culture medium in the tray, while the white mushroom biomat growths covered approximately half of the liquid culture medium as five floating biomat islands. The average thickness of the biomats was 1.5 mm for brown mushrooms and 1.7 mm for white mushrooms. The biomass biomats were dried at 50°C for 48 hours, and the dry weight produced per tray was 1.14 g and 2.12 g for brown and white mushrooms, respectively. The dry weight-based density of the dried biomass biomats was 0.033 g / cm³ for brown and white mushrooms, respectively. 3 and 0.111 g / cm³ 3 That was the case.

[0325] Microscopic images were used to confirm the mycelial properties of the biomats. The average mycelial thickness was 25.2 μm (standard deviation = 6.2) and 18.7 μm (standard deviation = 4.0) for brown and white mushroom biomats, respectively.

[0326] Chicken nuggets were made using the produced brown mushroom biomat. The biomat was steamed at 97°C for 0.5 hours, cooled to room temperature, and used as the base for making the chicken nuggets. Steam-treated moist biomass (2.5g) was mixed with 3% (wt / wt; 75mg) Better Than Bouillon Chicken Base (Southeastern Mills, Inc. Rome, GA) and 3% egg white protein (75mg; Now Foods, Bloomingdale, IL), and chopped into pieces less than 2mm long using a razor. The mixture was formed into nuggets and steamed for 0.5 hours. The prepared nuggets had a typical chicken aroma with a slight mushroom flavor. When tasted, the nuggets had a chicken flavor or an indistinct taste.

[0327] Example 12: Growth of mushroom biomats in malt and glycerol media. A biomass biomat consisting of Calvatia gigantea (Giant Puffball), Pleurotus ostreatus (Marbled Oyster Mushroom), Pleurotus ostreatus var. columbinus (Oyster Mushroom), Hypsizygus ulmarius (White Pyrophytum), Sparassis crispa (Flower Oyster Mushroom), and Ganoderma lucidum (Reishi Mushroom) was produced in just 5 days using malt extract medium 001, glycerol medium 002, Hansen medium, MK7-SF medium, and malt extract + NH4NO3 medium 003 (Table 3). The final culture medium contained 0.01% chloramphenicol.

[0328] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0329] Using the recipes in Table 3, the culture medium was prepared in a 2L Pyrex® bottle or an 8L stainless steel pot by mixing the specified ingredients with a specific volume of water corresponding to the desired volume of medium. The ingredients were added to the water while continuously stirring the liquid with a stirring rod or spoon. Each ingredient of the medium was thoroughly mixed into the liquid, and then the next ingredient was added to adjust the pH of the MK7-SF medium to 5.0, and the solution was autoclaved. All other pH levels were achieved simply by mixing the ingredients. The medium and container were autoclaved at 20 psi and 121°C for at least 20 minutes. The osmotic pressure (as osmolality) of the liquid was measured using an Advanced Instruments, Inc. osmometer Model 3250 (Two Technology Way, Norwood, MA).

[0330] After autoclaving, the culture medium was cooled to room temperature, and the mushroom species shown in Table 4 were inoculated into individual containers.

[0331] [Table 14]

[0332] Inoculation of the growth medium was performed using the following method, employing aseptic techniques. All aseptic work in these experiments was carried out in a Class II biohazard handling cabinet. Using a spore syringe, approximately 75 mL of growth medium was directly inoculated into a pre-autoclaved 12.7 × 17.8 cm Pyrex® glass tray. This was achieved by aseptically transferring the liquid medium to the autoclaved Pyrex® tray, allowing the medium to cool to room temperature and enabling inoculation with a 2 cc suspension contained in the spore syringe. The tray was covered with sterile aluminum foil and then gently swirled to mix the inoculum.

[0333] Malt extract agar (MEA; Table 5) plates were prepared aseptically by autoclaving the MEA, cooled to 50°C, and approximately 25 mL was poured into a 100 × 15 mm sterile petri dish.

[0334] TIFF2026048715000016.tif49160

[0335] MEA plates were inoculated by taking 1 cc of liquid from the suspension contained in a spore syringe. The agar plates were then sealed with Parafilm® and kept at room temperature in a clean, dark drawer.

[0336] After the mycelium covered the entire surface of the MEA plate, it was used to inoculate 1.5 L of medium in a 2 L baffled shaking flask. Approximately 2 cm of agar plate with mycelium on the surface. 2 Cut out the portion from the plate with a sterile razor, approximately 2mm. 2 The culture medium was cut into sections and then added to two flasks containing 1.5 L of malt extract 001 medium. The medium was incubated at room temperature (23±1°C) for 3 days with intermittent hand shaking (the flasks were shaken vigorously by hand for 1 minute at least 5 times a day).

[0337] Next, the cultures in the shaker flask were used as inoculants for 6 L of malt extract medium 001 and 6 L of malt extract + NH4NO3003 medium. 15% (vol:vol) of the inoculant culture was inoculated into the medium and thoroughly mixed. 2 liters of inoculant medium were divided into three 0.25 m³ containers. 2 The mixture was poured into individual plastic trays and placed in a tray rack. The rack was wrapped in Saran® and incubated for 6 days. Relatively dense biomats were covered completely for 4 days, and the biomats were harvested after 6 days.

[0338] 12.7 x 17.8 cm Pyrex® glass tray and 0.25 m 2Biomat was collected from the plastic tray by lifting it from the tray and gently squeezed by hand. A portion of the biomat (3-50g) was passed over boiling water (approximately 5cm above the water surface) in a pot steamer placed on a kitchen oven burner for 20 minutes. After steam treatment, the biomass was cooled to room temperature, immediately placed in a Ziploc® bag, and sent to Eurofins (Des Moines, IA) for protein analysis (N is due to combustion, Test Code QD252).

[0339] [Table 15] [Table 16]

[0340] Example 13. Fusarium strain MK7 chicken nuggets Chicken-flavored Fusarium strain MK7 is a basic ingredient as a chicken substitute in numerous recipes, including breaded or unbreaded chicken nuggets, Asian chicken, or other chicken dishes. Fusarium strain MK7 biomats produced from different feedstock / carbon sources yield slightly different chicken flavors. Glycerol chicken tends to be sweeter, while acid whey chicken tends to be slightly sour.

[0341] The texture of chicken nuggets varies depending on the amount of food processor processing and blades used (i.e., sharp metal blades, blunt metal blades, plastic blades). Furthermore, acceptable chicken nuggets can be produced from a wide range of biomass sizes. That is, biomass can be cut with a knife, lightly processed in a food processor, or heavily processed in a food processor, and still yield an acceptable chicken-like product.

[0342] A 50-20:1:1 ratio of Fusarium strain MK7:chicken stock:binder was used, with or without fat, at a ratio of approximately 66.6% Fusarium strain MK7:fat. Suitable fats include duck fat, coconut butter, and cocoa butter. After mixing, the mixture was steamed for about 30 minutes to set the binder; however, the time required for some binders may be longer or shorter. The nuggets were then breaded and processed according to the conventional treatments for such foods.

[0343] Example 14: Breakfast sausages and / or hot dogs and / or burgers Add the appropriate spice mix to the miniaturized Fusarium strain MK7 biomat, with or without additional ingredients such as onions, binders, and fats such as cocoa butter, to impart the desired flavor. This can range from 10 wt.% of the spice mix to 20% of the Fusarium strain MK7, often in a ratio of 10:1. The mixture is then fried to remove the appropriate amount of moisture. It is then shaped into the desired form and, before cooking, additional ingredients such as bulgur, vegetable broth, and potatoes may be added.

[0344] Example 15: Ice cream and mousse Fusarium strain MK7 biomats with a particle size ratio of approximately 1:3 and an average fiber length of less than 900 microns: produce water. Gently heat this mixture until it no longer smells of fungus, then use it with cashews in a ratio of approximately 4:1 along with an appropriate amount of xanthan gum and / or flavoring as desired to make a mixture, which is heated as desired and then cooled to make a mousse. For a frozen dessert, then put the mixture into an ice cream churner, churn, and freeze to make a non-melting frozen dessert.

[0345] Example 16: Production of truffle oil from truffle biomat. Oil extracts can be prepared from truffle (Tuber sp.) biomats grown as described above. As an example, truffle biomats were grown in trays for just 7 days using malt extract, glucose, and peptone as the primary carbon sources (supply materials). Edible truffle mushrooms were purchased from IGB Trading LLC on Amazon Marketplace and stored at 4°C. Pure cultures of the Tuber sp. fungi were prepared using truffles approximately 3 mm in size. 3 A preparation was made from purchased truffles by placing portions (cut with a sterile razor) in malt extract agar + 0.01% chloramphenicol (used to inhibit bacterial growth). The malt extract agar was prepared by mixing 20g of malt extract, 20g of glucose, 1g of peptone, and 20g of agar in 1L of deionized water, then autoclaved for 30 minutes, cooled to 50°C, and then 0.01% chloramphenicol was added. The sterile mixture was then poured into a 9cm diameter Petri plate and allowed to cool and solidify.

[0346] The fungi were observed to grow in the tray after 3 days. After 4 days of growth, the hyphae were isolated using a sterile microbiological loop and traced onto a new malt extract agar + chloramphenicol plate. The fungi were grown on this plate for 5 days, after which the hyphae were isolated using a microbiological loop and used for confirmation of culture purity by DNA sequencing. The DNA was extracted and purified (FastDNA Spin Kit, MP Biomedicals), the metagenomic ITS region was sequenced, and the sequence was subsequently phylogenetically classified using Blast (NCBI database) to achieve confirmation.

[0347] Malt extract broth was prepared by mixing 20 g of malt extract, 20 g of glucose, and 1 g of peptone in 1 L of deionized water, and then sterilizing the mixture. Mycelial trimmings from the microbiological loop were also used in 50 mL of sterile malt extract broth in a sterile baffled shaking flask and capped with sterile gauze. The sterile gauze was used to allow gas exchange inside and outside the shaker flask. The shaker flask was then shaken at 185 rpm for 5 days. The rotated culture was then inoculated into 350 mL of sterile malt extract broth in a sterile 12.7 × 17.8 cm Pyrex® glass tray. The inoculum density of this culture medium was 7.5% inoculum to 92.5% broth. After 7 days of growth in the tray, the filamentous biomat formed on the surface was collected by lifting the biomat from the liquid medium. The collected biomat was dried at 40°C for 48 hours. Lipids / oils were extracted from these collected biomats by mechanical pressing or solvent extraction using hexane, but other extraction methods can also be used.

[0348] Example 17: MK7 powder. Using the Fusarium strain MK7 biomat produced as described above, a dry powder with particle size and particle size distribution similar to standard bread flour was prepared. Here, the wet biomat was steamed in a pot steamer at 97°C for 0.5 hours, cooled to room temperature, and dehydrated in a Cuisinart dehydrator (model DHR-20) for 2–8 hours, with an average dehydration time of 4 hours. The dehydration time is a function of the amount of biomass placed in the dehydrator, the distribution of the biomat within the dehydrator (which affects airflow), the moisture content of the biomat (average moisture content approximately 75%), and room temperature. The moisture content after dehydration varied from 4–14%, with an average of less than 12%. The dehydrated biomass was pulverized using a coffee mill (KRUPS, electric coffee and spice mill, stainless steel blade F2034251) until it was ground. The average particle size of the ground biomat powder ranged from 75 microns to 120 microns. A small portion of the large particles, approximately 5 wt%, had a particle size exceeding 180 microns. A small portion of the small particles, approximately 5 wt%, had a particle size less than 75 microns. These small particles were of a size that allowed them to remain suspended for extended periods. Particle size was determined by sieving a 100-gram sample of the miniaturized biomat through screens with 180 μm, 120 μm, and 75 μm openings for 5 minutes. The moisture content after dehydration and miniaturization was preferably less than 6%, as a high moisture content can lead to drying and coagulation of the milled biomass.

[0349] Next, BioMat powder was used as an additive to other standard wheat flours (King Arthur flour, Bob's Red Mill Flour, and Bob's Red Mill Wheat Flour) to produce a variety of baked goods. Adding BioMat powder at 5wt%, 10wt%, 20wt%, and 30wt% concentrations did not adversely affect the taste, fermentation, texture, appearance, or smell of the final baked goods. Tested products included bread (7 grain, refined & wheat), pastry (choux pastry), cookies, pasta, and dumplings. The resulting products tasted good, and the presence of strain MK7 flour was undetectable to tasters.

[0350] Example 18: MK7 bulking agent Using the Fusarium strain MK7 biomat produced as described above, biomass particles were created for use as a meat and fish additive as a bulking agent (i.e., increasing the total amount of food by adding strain MK7 to other finished foods). The wet biomat was steamed in a pot steamer at 97°C for 0.5 hours and cooled to room temperature. The biomat was then granulated to the desired particle size distribution (i.e., cut with a knife or processed in a food processor). The granulated biomass was then added to various foods to increase the amount of meat in the case of a meat bulking agent or fish in the case of a fish bulking agent. As an example of meat bulking, 10%, 20%, 30%, 40%, and 50% of the granulated biomass were added to hamburger meat. The granulation of the biomass was evaluated in numerous different size distributions. Smaller particle sizes tended to result in a denser and creamier texture. Larger particles tended to produce products that were more textured, had a stronger mouthfeel, and required more chewing before swallowing. Next, the increased meat volume was processed as if no biomass had been added. In the case of increased hamburger volume, spices or binders may be added as desired, the increased meat volume was formed into patties or meatballs, and the meat was cooked at the temperature desired by the consumer. Cooking methods included on the stovetop, in the oven, frying, and grilling. Taste tests showed that acceptable food products were produced at all levels and size distributions of added biomass. Expansion to chicken and pork was also attempted at similar levels of added biomass, with similar cooking and taste test results.

[0351] The process was expanded to fish at addition levels of 10%, 20%, 30%, and 40%. Fish fillets and fish balls were produced by adding strain MK7 treated with a diverse range of different particle sizes, from small particles (less than 1.0 mm) to large particles (greater than 2 mm), and there were no adverse effects on taste, color, smell, or overall eating experience. With the addition of small particle sizes, the resulting food had a creamier texture. With the addition of large particle sizes, the resulting food had a harder texture, characterized by the need for more chewing before swallowing as the particle size increased. Taste tests showed that acceptable food was produced at all addition levels and size distributions of the added biomass.

[0352] Example 19: MK7 Jerky Using the Fusarium strain MK7 biomat produced as described above, we created bacterial jerky that resembled meat jerky (i.e., beef jerky, buffalo jerky, pork jerky, chicken jerky, turkey jerky, etc.) in appearance and taste. The moist biomat was steamed in a pot steamer at 97°C for 0.5 hours and then cooled to room temperature. The biomat was miniaturized to a size consistent with that typically found in jerky products. The miniaturized biomat pieces were, in some cases, flavored and dehydrated in a Cuisinart dehydrator (model DHR-20) for 20–200 minutes, with an average dehydration time of 40–120 minutes. The dehydration time is a function of the amount of biomass placed in the dehydrator, the distribution of the biomat in the dehydrator affecting the airflow, the moisture content of the biomat (average moisture content approximately 75%), room temperature, and the desired moisture content in the final product. The moisture content after dehydration varied from 8% to 12% depending on the desired product characteristics. In some cases, the biomass was perforated and then dehydrated to make the product easier to break into smaller pieces, thereby facilitating consumption. Perforation of the biomass was carried out using a fork, knife, or meat tenderizer, which resulted in both perforation and destruction of the fibrous network of the biomass, making it easier to tear. A wide variety of spice mixtures (i.e., Cajun, cheese, soy, vinegar, herbs, sour cream and onion, liquid smoke, vegan meat flavor, etc.) were evaluated. The spice mixtures were evaluated both before and after dehydration. Samples spiced before dehydration resulted in a stronger flavor and better adhesion of the biomass than those spiced after dehydration. All of the resulting jerky passed taste tests well.

[0353] Example 20: Bacterial chip Using the Fusarium strain MK7 biomat produced as described above, chips similar in appearance and taste to potato or corn chips were created. The moist biomat was steamed in a pot steamer at 97°C for 0.5 hours and then cooled to room temperature. The biomat was miniaturized to a size consistent with that typically found in chip products, highly processed into a paste, and molded into chip-like forms. The microbial chips were then placed in a frying pan with hot oil (temperature approximately 380°F) until browned. Cooking time varied as a function of the biomass form, but was usually cooked very quickly, in less than 15 seconds. The produced fried chips were shown to be very palatable and offered a wide range of flavor experiences due to the spices added to or coated on the biomass before frying. [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26]

[0354] Example 22: Protein content data from MK7 biomats grown in two different culture media. The biomat was dried in an oven for 2-3 days (99°C) and then placed in a drying oven for several days. The sample was ground and prepared for total nitrogen analysis. The H2O content of the dried strain MK7 sample was estimated to be approximately 5%.

[0355] Total protein MK7 membrane-grown strain in MK102 medium - 41.2% Membrane-grown strain MK7 in AUM medium - 43.8% The calculation was performed assuming this. [Table 27] * For maximum yield, use a membrane, 8.5 mL of medium, and a 35 mm Petri dish to cultivate strain MK7 biomat for 5-7 days. ** Lignocellulose biomass - derived from hay, treated with sulfuric acid. [Table 28] [Table 29] [Table 30]

[0356] Example 23: This example shows comparative nutritional data from two Fusarium filamentous fungi, strain MK7 and Fusarium venenatum.

[0357] Comparative nutritional data obtained from strain MK7 and Fusarium venenatum. [Table 31] * The product contains proteins from non-fungal sources (eggs, egg whites, yeast, wheat gluten). [Table 32] [Table 33] [Table 34]

[0358] Example 24. Measurement of RNA content from various filamentous fungi. Purine Analysis Experimental Method: Preparation of 1000 μg / ml Purine Standard: 100 mg of each purine base (adenine, guanine, xanthine, and hypoxanthine) was added separately to four 100 ml volume flasks. 90 ml of ultrapurified water was added to each flask, and the flasks were shaken to separate the purine solids. 10 M NaOH prepared with ultrapurified water was added dropwise until the purine bases began to dissolve. The flasks were shaken repeatedly until all solids were completely dissolved. Additional base was added as needed to ensure complete dissolution. The flasks were capped and stored in a refrigerator.

[0359] Preparation of 200 μg / ml mixed purine standard: 20 ml each of 1000 μg / ml purine standards was placed in a 100 ml volumetric flask, and water was added to make a total volume of 100 ml. The flask was stored in the refrigerator.

[0360] Preparation of pH 2.5-2.8, 150 mM phosphate buffer: A 150 mmol sodium phosphate buffer was prepared by dissolving 74.88 g of NaH2PO4 in 2 liters of ultrapurified water. The solution was filtered under reduced pressure through a 45 μm pore size filter. 7.88 ml of 80% phosphoric acid was added to 2 liters of ultrapurified water, and the resulting solutions were combined. If the buffer solution is outside the pH range of 2.5-2.8, the pH may be adjusted by adding 80% phosphoric acid dropwise until the endpoint is approximately pH 2.6.

[0361] Sample preparation: Lyophilized mushroom samples were processed as obtained. Wet samples were transferred to 15 ml Falcon tubes in tare bags. The samples were then frozen at -80°F for lyophilization preparation. Pre-freezing samples were lyophilized in a Labconco lyophilizer for 24–48 hours until dry.

[0362] The weight of the dried sample was measured and recorded. The dried material was ground into a fine powder using a mortar and pestle, and weighed into a 50 ml Ellenmeyer flask to obtain approximately 500 mg of sample. 15 ml of 70% perchloric acid was added to the reaction flask. The reaction mixture was then heated to 95°C in a water bath for 1 hour while stirring with a magnetic stirring rod. The flask was removed from the water bath, and 5 ml of sample was taken from each reaction mixture and transferred to a 15 ml Falcon tube. The pH was raised by adding 10 M KOH solution dropwise until the solution reached pH 4. The volume of the neutralized sample was recorded, and the solution was centrifuged at 4000 rpm for 30 minutes. After centrifugation, the pH was checked and adjusted as necessary, and the final volume was recorded. 3 ml of the supernatant was withdrawn with a syringe and filtered through a 45 μm syringe filter for HPLC analysis.

[0363] HPLC conditions: HPLC:Waters e2695 Separations Module Column: Shodex Asahipak GS-320HQ 7.5mm x 300mm Solvent: 150 mM sodium phosphate buffer (pH 2.5-2.8) Injection volume: 10 uL Flow rate: .6mL / min Column temperature: 35℃ Detection wavelength: 260nm [Table 35]

[0364] Example 25. Toxicity data from biomats of the filamentous fungi Fusarium venenatum and Morchella conica (black morel).

[0365] Toxicity / growth test in Daphnia magna Fusarium venenatum biomats did not cause acute toxicity in Daphnia magna, a highly sensitive giant invertebrate commonly used in toxicity assays (EPA Publication, 1987; Guilhermino et al., 2000).

[0366] Live Daphnia magna were purchased from Carolina Biological Supply (Cat # 142330, Burlington, NC). Immediately after receiving the culture from the supplier, 100 mL of liquid medium containing live Daphnia magna was mixed with 800 mL of Arrowhead Spring Water (Nestle Waters North America, Inc., Stamford, CT) in a disinfected glass bowl (rinsed with 70% isopropanol and dried). The culture was gently mixed by stirring with a disinfected plastic spoon, and 400 mL of this liquid, which did not contain Daphnia magna, was removed and stored in an Arrowhead Spring Water bottle at 4°C until later use. One-quarter of a Daphnia magna food pellet (Carolina Biological Supply, Cat # 14-2316) was added to the remaining 500 mL of liquid in the bowl containing the Daphnia magna. The bowl was loosely wrapped in plastic wrap, and the culture was incubated at room temperature (21±2°C) as indicated in the manual provided by the supplier. After 48 hours of growth and observation, the surviving Daphnia magna were used for growth / toxicity experiments.

[0367] Sterile petri dishes (Fisherbrand 100×15 mm, Cat# 08-757-13, Thermo-Fisher) were filled with 35 mL of liquid growth medium (spring water / Carolina Biological Supply medium mixture) stored at 4°C (as described above). The liquid medium was equilibrated to room temperature. Four active Daphnia magna, each approximately 1-1.2 mm in length, were captured using an eyedropper (Carolina Biological Supply, capture method described in the supplier's manual) and added to each of the eight petri dishes. Additionally, four of the petri dishes were filled with 0.15 g of moist Fusarium venenatum biomat (18% solid) and four of the petri dishes with 0.03 g of dried Daphnia magna feed. Biomass was produced after 5 days of growth in MK7-102 medium, collected from the tray, gently pressed by hand to remove excess medium, and steam-treated for 30 minutes to kill the cells. Steam-treated biomass was pressed using a Francesco Palumbo grape press until a solid moisture content of approximately 18% (82% liquid) was obtained. The pH of both the control and Fusarium venenatum biomass in the petri dishes was 7.4–7.8 during the toxicity experiment. The number of surviving Daphnia magna was counted every 24 hours in each petri dish, as shown in the table below.

[0368] After 4 days, the control group had an average of 3.50 surviving Daphnia per dish (standard deviation = 1.29), while the Fusarium venenatum treatment group had an average of 3.75 surviving Daphnia per dish (standard deviation = 0.50). At this point, the control group had an average of 0.75 dead organisms per dish (standard deviation = 0.96), while the Fusarium venenatum treatment group had an average of 0.25 dead organisms per dish (standard deviation = 0.5).

[0369] In summary, these data indicate that steam-treated Fusarium venenatum biomass, when evaluated using the described method, does not cause acute toxicity to Daphnia magna.

[0370] Study on the toxicity of Fusarium venenatum to Daphnia magna [Table 36]

[0371] Mycotoxin content Method Reference US-Multitoxin LCMSMS 45-2-LWI [Table 37]

[0372] Example 26. Characterization of a liquid dispersion containing MK7 biomat particles.

[0373] Ingredients: MK7 Vegan Milk Sample: Approximately 8.25% solid in water

[0374] Unless otherwise specified, all measurements were taken at room temperature (25°C).

[0375] Appearance and Color: The appearance is slightly grayish-white with a slight beige tint. There is a very faint woody smell.

[0376] Figure 20 shows the refractive index, density, and particle size analysis of 10 to 1000-fold diluted samples.

[0377] The particle size in the solution was analyzed using dynamic light scattering (DLS).

[0378] The milk structure under an optical microscope is shown in Figures 21A (10x magnification) and 21B (100x magnification).

[0379] The fat content of the sample (8.25%) was found to be 0.6g / 100g.

[0380] The pH components were found to be as follows: [Table 38]

[0381] Viscosity: The viscosity of the vegan milk sample at its original concentration (8.25%) was tested. Figure 22 shows the average of three repeated measurements. The sample viscosity decreased with increasing shear rate, indicating it is a non-Newtonian fluid with shear deviscation behavior. This also means that the particles are much easier to deform under high shear rates. At a certain shear rate, the viscosity of the sample can be determined by a power law equation. This provides an opportunity to compare this vegan milk sample with other milks or smoothies.

[0382] No visible separation of milk was observed after 2 or 4 weeks.

[0383] Example 27. Method for producing a liquid dispersion using the filamentous fungus strain MK7. In a high-speed blender (Vitamix), combine 200 grams of strain MK7, 600 grams of water, and 1 teaspoon of vanilla extract and blend until smooth (for at least 90 seconds). Heat the blended mixture over low heat until the fungal aroma diminishes, preferably until it is no longer present (about 20-30 minutes). Keep the heat low to achieve a maximum low-speed boil, which was started when the MK7 milk reached about 80°C. Bring the MK7 milk to a gentle boil at a temperature of 90-92°C, without stirring or frothing, and without disturbing. Let the heated blended mixture cool and use.

[0384] After the heating cycle was complete and the milk was removed from the pot, a line was observed remaining inside the container at the interface between the MK7 milk and air. The residue was hard and consisted of a strong, adherent fusion of dehydrated / cooked strain MK7.

[0385] Example 28: Crepes component: King Arthur Medium-Strength Flour - 11.7% gluten content. C&H granulated sugar. · MK7 Biomass Strain - QCB-249 - 10% Glycerol - 09 / 03 / 2018. MK7 Milk - QCB-249 - 10% Glycerol - 09 / 03 / 2018 (25% Biomass before cooking) Nielsen vanilla paste. Plugra unsalted butter, 82% fat. Equipment: De Buyer Teflon crepe pan. [Table 39]

[0386] The original raw milk component of the crepe butter was replaced with MK7 milk. The milk was produced as follows: 200 grams of MK7 strain, 600 grams of water, and 2 grams of vanilla. The mixture was blended in a Vitamix and heat-treated at low boil for 20 minutes without nitrogen treatment. The crepe butter was too thick.

[0387] [Table 40]

[0388] The consistency of the butter was adjusted using a combination of liquids (whole egg, raw milk, and water). This adjustment resulted in the appropriate texture and flavor. No sugar was needed for the savory crepes; instead, savory flavorings such as onion and garlic powder and herbs were added. [Table 41]

[0389] To increase the protein content, the wheat flour portion was replaced with MK7 flour. The MK7 flour crepe butter was found to require more liquid because the MK7 strain has 150% higher hydration capacity (compared to 60-70% for wheat flour). Without the added water, the crepe texture resembled a tortilla.

[0390] Example 29: Pasta dough component: • MK7 Biomass Strain - QCB-249 - 10% Glycerol - 09 / 03 / 2018 MK7 Powder - QCB-249 - 10% Glycerol - 09 / 03 / 2018 Bob's Red Mill Semolina. Delallo 100% Organic Double 00 Wheat Flour. Bottled water - pH: 6.5. Equipment: KitchenAid food processor and pasta roller.

[0391] 1. Pasta dough made from semolina, MK7 flour, and water. [Table 42] The dough was mixed by hand, and no MK7 flavor was detected. The MK7 flour required an increase in liquid hydration from 50% of regular pasta dough to approximately 66.66% of dough made with 2 / 3 semolina and 1 / 3 MK7 flour.

[0392] 2. Pasta dough made with semolina, MK7 flour, and eggs. [Table 43] The dough was mixed by hand. No MK7 flavor was detected. The egg-based dough had a richer flavor. The texture was similar to test number 1, but with slightly less gluten development due to the egg fat (egg yolk contains 1 / 3 fat, so about 13% of the total weight of test number 2). The egg-based dough was too stiff to mix by hand due to interference between fat and gluten. A food processor may be used to obtain a smoother texture similar to test number 1. A 20% increase in salt in the pasta dough yielded good results.

[0393] 3. MK7 powder hydration [Table 44] The dough was mixed by hand. MK7 flour requires approximately 150% water to hydrate to the same level as standard wheat flour and starch-based dough. For reference: white wheat flour requires 60-70% hydration, and starch requires 70-90%.

[0394] 4. Pasta dough was made with double 00 wheat flour, MK7 flour, and water for comparison with test number 1. [Table 45] The dough was mixed by hand. No MK7 flavor was detected. The texture was smoother than test number 1 of double 00 wheat, confirming the performance of the much finer ground MK7 flour.

[0395] 5. Pasta dough was made with double 00 wheat flour, MK7 flour, and eggs for comparison with test number 2. [Table 46] The dough was mixed in a food processor. No MK7 flavor was detected. The texture was smoother than that of test number 2, which was finished in a food processor and then by hand, confirming that the food processor works better than the fat-based pasta dough.

[0396] 6. Pasta dough made with semolina, double 00 wheat flour, MK7 flour, and water for comparison with test numbers 1 and 4. [Table 47]

[0397] The dough was mixed in a food processor and finally by hand. No MK7 flavor was detected. The texture was similar to test number 4 in terms of smoothness. The combination of the two types of wheat flour and MK7 flour resulted in a reduction of semolina-like texture and a reduction of wheat flour texture.

[0398] 7. Pasta dough made with semolina, double 00 wheat flour, MK7 flour, and eggs for comparison with test numbers 2 and 5. [Table 48] The dough was mixed in a food processor and finally by hand. No MK7 flavor was detected. The texture was similar to test number 5 and was as smooth as the above combination of two types of wheat flour and MK7 flour.

[0399] Example 30: Spätzle component: ·MK7 Biomass - QCB-249 - 10% Glycerol - 09 / 03 / 2018 ·MK7 powder. King Arthur semi-strong flour, 11.7% gluten Equipment: ·none Test number 1: [Table 49] The dough was mixed by hand and spread with a dough scraper. No MK7 flavor was detected. It was determined that the Spätzle was moister inside and that it was advantageous to add cream or similar, namely heavy cream, crème fraîche, sour cream, buttermilk, or yogurt, along with nutmeg. After cooking, it was dried overnight and finished the next day.

[0400] Test number 2: [Table 50] The dough was mixed by hand. No MK7 flavor was detected. The Spätzle, which had been rested overnight, had the correct color when finished with butter. Cream was added to provide the necessary moisture. Several chefs from a French pastry school approved of this product.

[0401] Example 31: Bacon component: MK7 stock 2 teaspoons of soy 1.5 teaspoons of A1 • 1 teaspoon of liquid smoke 2 teaspoons of nutrient yeast 1 / 2 teaspoon of paprika • 1 teaspoon of honey Strain MK7 Biomat was cut into strips. A marinade / coating was prepared. Both sides of the Strain MK7 were coated with the batter and fried until browned, then shaken dry and dehydrated in a dehydrator to remove excess moisture until the desired texture was achieved. The resulting bacon-like product had a good texture (i.e., crispy) and flavor (had an appealing bacon-like flavor). A chef at a French pastry school approved of this product.

[0402] Example 32: Bread component: • 0.5 cups of unsweetened 7-grain cereal (purchased from a store) 2 cups boiling water • 1 packet of dried yeast 3 cups strong flour 1 cup MK7 powder • 1 teaspoon of olive oil • 1 teaspoon of dark brown sugar 1.5 teaspoons of salt 2 teaspoons of sesame seeds 2 teaspoons of flaxseed 2 teaspoons of poppy seeds 2 cups of water Pour 2 cups of boiling water over the 7-grain cereal and let it soak for 20 minutes to soften the grains. Add the yeast to the softened cereal. Add 1 cup of bread flour, oil, sugar and salt and mix gently until smooth. Mix the MK7 flour with the remaining bread flour and mix slowly with the above until a dough is formed. Cover and let it rest (15-20 minutes). Note: Must rest in a warm place. Knead the dough until smooth and elastic. Add more flour as needed. Knead for about 10-15 minutes. Grease a large bowl. Coat the dough with oil and place it in the greased bowl and cover. Let the dough rise in a warm place for about 1.5 hours until doubled in size. Mix in the seeds, mix in the dough and roll it out. (Turn the dough over on a lightly oiled surface. Knead lightly with about 1 / 2 the seeds.) Shape the dough into a ball. Spread about 2 teaspoons of seeds on a baking sheet. The dough was placed on top of the seeds, covered with a towel, and allowed to rise in a warm place for about 30 minutes, until it had almost doubled in size. One oven rack was placed in the middle of the oven, and the other on the lower rack. The oven was preheated to 425°F. One dough was brushed with water. The remaining seed mixture was sprinkled over the dough. A diagonal line about 1 / 8 inch deep was cut into the surface of the dough, and the baking sheet with the dough was placed in the oven. (2 cups of water was poured over the hot pan on the lower rack of the oven to steam.) The dough was baked for about 35 minutes, until golden brown, hard on the surface, and a tester inserted into the center could be removed without staining. The soft part of the resulting bread was good, and the taste was excellent. The addition of MK7 flour was not detected in the resulting dough.

[0403] Example 33: Vegan Chocolate Ice Cream component 95 grams of raw cashews 750 grams of MK7 milk 0.7 grams of xanthan gum 0.2 grams of instant Espelette 20 grams of cocoa powder 1 gram of salt 2 grams of vanilla extract • 110 grams of refined sugar (dissolved in MK7 milk) 150.5 grams of Green & Blacks 70% Dark Chocolate 20 grams of Ghirardelli sweet cocoa powder (cooking chocolate) • 15 grams of Rumford corn starch (non-GMO) 1:3 MK7 Milk Equivalent: Combine 200 grams of MK7 stock, 600 grams of water, and 1 teaspoon of vanilla extract in a high-speed blender (Vitamix) and blend until smooth (about 90 seconds). Heat the blended mixture over low heat for 20 minutes. (Other versions of the recipe also perform both the mixing and heating under nitrogen (i.e., nitrogen is passed through the MK7 milk equivalent during either or both of the miniaturization and / or heating cycles in the Vitamix). The use of nitrogen brings a sweet flavor to the thick cream-like milk equivalent.) Keep the heating low; maximum low speed until boiling. Add the sugar to the MK7 milk during the last 5 minutes of the heating process and mix until dissolved in the milk. (If the MK7 milk was pre-made, heat the milk, add the sugar, and dissolve it.) Foam is seen on top and in a small portion of the liquid at the bottom, indicating a slow boil.

[0404] All ingredients were combined in a high-speed blender and blended at high speed for 120 seconds. Standard teachings suggest that, to avoid aggregation, the mixture should be kept small while it is vigorously foaming, allowing the starch to dissolve into the mixture. If the starch mixture is prepared separately, it should be added to the master mixture gradually to avoid aggregation.

[0405] Pour the mixture into an ice cream making container and press the adhesive film onto the surface of the mixture to prevent a film from forming during cooling. Cool the mixture, not freeze it. Stir the mixture in an ice cream maker for at least 30 minutes, but not more than 1 hour (until the desired consistency is reached). Package into appropriate containers and freeze overnight.

[0406] Example 34: Chicken Nuggets component 200g of MK7 stock • 4g of chicken stock (either meat-based or plant-based chicken stock is acceptable) • 4g of binder (egg white or vegetable binder) • 3g of fat (duck fat works similarly to cocoa butter. Other fats are also used in sufficient quantities.) The biomat particles were finely chopped with a knife to obtain the desired particle size. All components were combined and processed until the desired particle size distribution was achieved. (If using a food processor, it is recommended to pulse the food processor for 5 seconds, use a spatula to remove the biomass stuck to the sides, and integrate it with the biomass near the blades, repeating this three times. More or less processing may be done as desired in the final product.) The finely chopped biomass was thoroughly mixed with a spoon / spatula to ensure that all components were densely mixed. A paste of the desired size was formed and steamed for 30 minutes to stabilize the binder. (Some binders may require more or less time.) The steamed paste was cooled. Some of the paste was coated with breadcrumbs and heated to allow the breadcrumbs to adhere to the paste.

[0407] Example 35: Burger breakfast sausage Ingredients: Spice mix 30g of molasses 30g tamari 15g sunflower oil 15g of A1 sauce 21g of ground flaxseed 21g of nutritional yeast 14g whole wheat flour 6g black pepper 6g of sage • 1g time 0.5g nutmeg Ingredients: Burger • 100g of food processing strain MK7 (starch supply raw material) • 100g TVP (Textured Vegetable Protein: Reconstructed from a dry mixture of 50 / 50 plant and mushroom broth) 20g spice mix 20g of cooked brown rice • 15g of cooked onion (yellow onion cooked in oil until soft) 4g of dried egg albumin 4g of cocoa butter • Available upon request, but recommended: Food-grade red pigment (see Pic). In a mixing bowl, combine the spice mixture and mix until a uniform mixture is obtained. Let it rest for 15 minutes to allow the liquid to be absorbed sufficiently to form a paste-like mixture. In a food processor, reduce the biomass particle size to the desired size, i.e., the size expected in a burger (or sausage). Combine the burger ingredients in a bowl and mix until uniform. Form the burger patties with moistened hands. Steam the patties for 30 minutes or bake and fry them at 350°C for 30 minutes.

[0408] Example 36: Hot Dog Bologna component 250g of MK7 stock 1g of ground cumin 1g of ground cardamom 1g Mace 1.5g of black mustard seeds 1g of ground coriander 4g black pepper 4g of salt 5g of fresh minced garlic 6g granulated sugar 4g of paprika 50g of peeled and chopped onion • 18g of vegetable oil (approximately 3 teaspoons) 9g of soy sauce (approximately 2 teaspoons) 30g of almond meal 140g of wheat gluten 2g Arrowroot All ingredients except gluten and arrowroot were placed in a food processor. The mixture was processed for at least 60 seconds until completely smooth, and the processed mixture was transferred to a large bowl with a spatula. Wheat gluten and arrowroot were mixed into the dough with a wooden spoon and kneaded in. The addition of gluten and arrowroot was shown to result in a dense dough. The amount of gluten and the amount of strain MK7 processed can be reduced as desired to achieve the desired density.

[0409] Place the dough on a clean surface and divide it into 8 equal portions. Prepare 8 pieces of paraffin paper and 8 pieces of aluminum foil, sized to allow for the appropriate size of hot dogs. Roll each portion of dough into a hot dog-like shape on a table with consistent pressure by hand. If the dough dries out or cracks, you can work with damp hands to create a smooth surface. Wrap each hot dog individually in paraffin paper and let it mature in aluminum foil. Twist the ends of the foil into a Tootsie roll shape. Steam the hot dogs in a steamer for 45 minutes. Remove the wrapping from each hot dog and let it cool.

[0410] Example 37: Film Comparison We constructed numerous simple bioreactor setups and compared the effects of membrane materials on biomat growth. Biomats were grown in each bioreactor under identical conditions (sealed and humid, 26°C, and inoculated with growth medium), and samples were taken on day 6. The results are shown in Table 19.

[0411] [Table 51]

[0412] As shown in Table 19, the hydrophilic PVDF membrane (MilliporeSigma, Burlington, MA) was the best performing membrane material under the given conditions, providing the best density for both upward (i.e., growth on the upper side of the membrane) and downward (i.e., growth on the lower side of the membrane) growth in all membrane configurations tested for the biomats. In addition to providing the best growth characteristics, after biomat harvesting, the membrane remained flexible and "transparent," i.e., suitable for reuse; subsequent experiments showed that the same membrane could be reused without a washing process, achieving approximately 80% of the initial efficiency. Other subsequent experiments showed that when fungi were inoculated on the upper side of the membrane (i.e., the opposite side of the feedstock), the results for the 5 μm PVDF membrane and the 0.2 μm PVDF membrane shown in Table 19 were comparable. Polypropylene and nylon membranes also provided consistently high yields, although the polypropylene membranes began to clog. The low-porosity nylon membranes could not maintain sufficient structural integrity and tended to tear midway through the experiment.

[0413] Example 38: "Inverted" Flask Bioreactor Next, referring to Figure 25A, an "airtight" (tightly sealed to the environment) embodiment of the bioreactor in the configuration indicated as "4" in Figure 23 is described. As shown in Figure 25A, the bioreactor is an "inverted" bioreactor formed by sealing two 125 mL Ellenmeyer flasks at the neck; the membrane (PVDF, pore size 5 μm) is placed in the neck of the flask, the feedstock is placed in the upper flask, and the biomat grows downward from the membrane in the gas head space provided in the lower flask. Similarly, condensate may be found at the bottom of the lower flask. This embodiment allows for continuous replenishment of the feedstock and semi-continuous harvesting and / or sampling of the biomat. The fungi are inoculated into the feedstock (i.e., on the membrane).

[0414] Next, referring to Figure 25B, a biomat with a thickness of approximately 1 cm was collected from the bioreactor as part of the second collection after 12 days of growth (the first collection was on day 8, and this was on day 20 of inoculation). The biomat grew at an average rate of approximately 1 mm in thickness per day, at approximately 100 g / m². 2The dry yield of the biomat up to 100% was achieved for at least the first three weeks after inoculation. Figure 25C shows the dry and wet yields of the bioreactor after 8 days (first harvest) and a further 12 days (second harvest). This bioreactor embodiment utilizes feedstock derived from food waste or human excrement to enable the production of at least approximately 0.7 kilograms of dry biomass / square meter per week, which is equivalent to approximately 0.35 kilograms of protein / square meter per week. 0.35 kilograms / week is roughly equivalent to the protein required by a 73-kilogram person; therefore, a membrane growth area of ​​1 square meter is sufficient to supply the protein requirements of one adult in manned spaceflight applications or terrestrial applications where available space is limited, for example.

[0415] After collection, the PVDF membrane in this example did not retain biomass visible to the naked eye; as shown in Figure 25B, the membrane appeared "transparent." However, enough cells remained on the membrane to be effectively reinoculated for continued rapid growth. Other similar experiments have shown that biomats can regrow on previously used membranes without the need for active reinoculation.

[0416] As with several other examples, PVDF performed particularly well as a membrane material in terms of structural integrity, cleanliness, and ease of biomat production and biomat harvesting; other materials, especially nylon, tended to detach, crack, clog, or otherwise degrade after only a few days, and therefore are more robust and suitable for use in long-term (e.g., 2-7 weeks) experiments than PVDF.

[0417] Example 39: Bag Bioreactor Two types of "bag bioreactors" were designed, fabricated, and their biomat growth performance was evaluated. The first bag bioreactor was constructed by molding a bag from Gore-Tex material, with the feed material placed inside the bag and the filamentous fungal inoculation material placed outside the bag. After 7 days, a biomat with a thickness of approximately 1 mm at the top of the bag and approximately 6 mm at the bottom of the bag was formed; this difference is thought to be due to differences in fluid pressure applied to different parts of the bag (i.e., greater fluid pressure at the bottom than at the top of the bag). The biomat yield was 498 g / m² on a wet basis. 2 (Estimated 124.5 g / m² on a dry basis) 2 ) is approximately 17.8 g / m 2 This resulted in a daily drying production rate and a carbon conversion efficiency of approximately 35.8%.

[0418] A second bag bioreactor was constructed by forming a bag from a bilayer of PVDF membrane with a pore size of 5 μm. In this embodiment, filamentous fungi were inoculated through the culture medium (i.e., from inside the bag). After 7 days, substantially all of the supplied material was consumed, and the carbon conversion efficiency was estimated to be 73.4%. The biomat growth process of this bag bioreactor is shown in Figure 26.

[0419] Example 40: "Inverted" Mason Jar Bioreactor Next, referring to Figure 27A, an "airtight" (tightly sealed to the environment) embodiment of the bioreactor in the configuration indicated as "4" in Figure 23 is described. The bioreactor is an "inverted" bioreactor, with the upper Mason jar (not shown in the illustration) placed on top of the lower Mason jar (Figure 27A) and separated by a nylon membrane with a pore size of 0.45 μm. After 10 days of growth, a biomat with a thickness of approximately 10 mm was produced. Figure 27A shows the growth container on day 10, with the feed material on one side of the membrane and the grown biomat on the other side. Figure 27B shows the biomat collected on day 10, and Figure 27C shows a thin section of it used for tensile strength testing (Example 41, below).

[0420] Table 20 provides the characteristics of the various biomats shown in Figure 27B.

[0421] [Table 52]

[0422] Example 41: Tensile strength test of a biomat grown in an "inverted" Mason jar bioreactor A 5cm × 1cm section of the biomat shown in Figure 27B was separated with a razor as shown in Figure 27C. The thickness of the section was recorded with calipers, and the 5cm long 1cm section of the sample was placed between two glass slides, taking care not to damage the sample by applying excessive pressure through the slides. The glass slides were then clipped to a tensile strength testing apparatus to prevent the sample from slipping due to the pressure of the clips. Two additional glass slides were used to press another 1cm long sample on the opposite end. A water inlet adapted for use in the tensile strength testing apparatus was attached to the slide, and water was filled at a rate of 1 mL / second until the sample dripped through. Once the sample had dripped through, the mass of the water, the inlet, the clips, and the remaining sample were weighed together; this weight was divided by the thickness of the sample to calculate the tensile strength of the sample. Determined by this method, the tensile strength of the biomat sample produced in Example 40 was 1784 grams-weight per square centimeter.

[0423] Example 42: Use of "biomembrane" Next, with reference to Figures 28A and 28B, a typical bioreactor setup is shown 3 days (Figure 28A) and 6 days (Figure 28B) after inoculation. Fungi were inoculated on the nylon membrane (pore size 0.2 μm) side opposite the supply material, and the biomat grew at a rate of approximately 1 mm / day. After the initial sampling on day 3, when significant denaturation of the nylon membrane was observed, the same membrane was reused, and a biomat of almost the same thickness grew over the next 3 days. Six days after inoculation, the nylon membrane was found to have failed, but the combination of nylon and the biomat itself continued to function as a membrane. Therefore, we unexpectedly discovered that even if the previously provided membrane fails, the biomat itself can act as a "biomembrane" in the operation of the bioreactor.

[0424] Example 43: Human feces and urine (excrement) as feedstock / growth medium One advantage of the bioreactor and its method of use according to the present invention is that animal waste (excrement), including but not limited to human urine and human feces, agricultural waste (excrement), and industrial waste products, can be used as feedstock or growth medium. In this example, artificial urine medium (AUM) and artificial fecal medium (AFM) were prepared with the compositions shown in Tables 21 and 22.

[0425] [Table 53]

[0426] [Table 54]

[0427] Biomats were used as feedstock, and prepared AUM and AFM were added to samples using lignocellulose biomass (an efficient carbon source) as feedstock, and then grown in various membranes in simple bioreactors. The bioreactor yields obtained from these feedstock / membrane combinations are shown in Table 23. [Table 55] The biomats produced in these experiments are shown in Figures 30A and 30B. Furthermore, it was observed that the biomats grown in 1:1, AFM, and AUM adhered very loosely to the PVDF membrane and simply fell off when they reached a certain thickness / weight. Therefore, an automated collection system was conceived in which the biomats would fall off the membrane when they reached a certain weight—no physical / manual removal from the membrane would be necessary.

[0428] Example 44: Continuous supply of raw materials in the absence of back pressure A bioreactor was constructed that eliminates back pressure caused by the depletion of liquid supply material, enables equilibration of the liquid supply material culture medium with the external environment via a gas-permeable filter, and allows for a continuous supply of supply material to the bioreactor; this was achieved by drilling a hole in the upper Ellenmeyer flask and installing an air filter in the resulting hole. This embodiment is shown in Figure 31A, and a more generalized bioreactor configuration is shown in Figure 31B.

[0429] The use of a nylon membrane with a pore size of 0.2 μm proved ineffective in this embodiment because the membrane was prone to leakage. In contrast, a polypropylene membrane with a pore size of 10 μm withstood the internal pressure of the bioreactor without detachment, cracking, or leakage.

[0430] Example 45: Membrane-Bag Biofilm Reactor (MBBR) System Membrane-bag biofilm reactor (MBBR) systems, including those utilizing excrement and carbon substrates, which are anticipated to be usable in manned spaceflight missions or terrestrial applications, provide a scalable and convenient means of producing dense, usable biomass. MBBRs offer, in non-limiting examples, lightweight, compact, simple, and reusable systems for culturing filamentous fungi that can be used not only as food but also as pharmaceuticals, functional foods, fuels, leather-like materials, fabrics, and / or building materials.

[0431] Three-ply Gore-Tex fabric was washed as an MBBR membrane due to its durability, water resistance, and gas exchange properties. The bags were fabricated using heat-fluidable sealing tape to provide a waterproof seam at the top of the bag and a simple roll-seal system, and were used bonded to a support. The filamentous fungal strain MK7 was inoculated onto the outer surface of the bags, filled with growth medium, then the top of the bags was sealed, and the bags were placed on a rack in a temperature and humidity controlled box (50cm × 50cm × 70cm, 25±1℃, approximately 95% relative humidity). After 3 days, each bag was completely covered with a strain MK7 biomat layer, showing hyphae, aerial mycelium, and a thick layer of mycelium. The biomat after 5 days of growth is shown in Figure 32. At the end of the 7-day growth period, the glycerol supply material in the bags was completely depleted, and the calculated carbon conversion of the glycerol supply material to the dry biomat was approximately 35%, not considering any glycerol absorbed by the membrane fabric.

[0432] Example 46: Effects of membrane material and pore size on biomat growth Tests in a simple bioreactor were conducted under identical conditions using four different membrane materials with five different pore sizes (a total of 20 different membranes). The characteristics of the membranes are shown in Table 24. [Table 56]

[0433] Table 25 shows the selected characteristics of the biomats grown using these membranes. [Table 57]

[0434] The minimum biomat density observed in these experiments was 0.22 g / cm³. 3 Moisture and 0.036 g / cm³ 3 It was dry.

[0435] Visual inspection of the samples in this example provided several insights. In PTFE and PP membranes, characterized by their large thickness and curvature, a considerable amount of biomass accumulated inside the membrane itself and was therefore unusable for harvesting; furthermore, in PTFE membranes, the fungal inoculant did not "spread" to cover the entire membrane surface and had to be applied using a spray, vacuum pump, Q-tip, paintbrush, etc., resulting in the biomat not growing across the entire membrane area. In addition, PET-backed nylon membranes did not exhibit the same structural integrity problems (cracking, detachment, etc.) shown in the pure nylon membranes in the previous examples. The poor results of hydrophobic PVDF with PET backing were hypothesized to be due to the thinner, smoother membranes used in these experiments, which may have resulted in poorer adhesion of fungal cells to the membrane (a problem that could potentially be solved by roughening the membrane), but PVDF membranes were also characterized by the formation of thick "slime," which requires further investigation. Another possibility was that the hydrophobic PVDF with PET backing exhibited significantly slower liquid flow velocity through the membrane compared to the same thin, smooth nylon membrane (see Table 24). Both the nylon and PVDF membranes were also extremely "transparent," i.e., suitable for reuse. 0.2 hydrophobic PVDF-PET was extremely transparent, while 0.45, 1, 3, or 5 exhibited slime. Hydrophilic PVDF 0.2 or 5.0 did not exhibit slime (see data in Table 19).

[0436] Another noteworthy result is that while yields tended to increase with increasing pore size in PTFE and PP membranes, the opposite trend was observed with PET-backed nylon membranes. While we do not wish to be bound by any particular theory, it was hypothesized that the hydrophilic nature of this membrane provides conditions that are too "wet" for optimal biomat growth in the large-pore embodiment, and that this trend would be different if the membrane were "reversed" in an attempt to grow the biomat on a rather hydrophobic surface.

[0437] Example 47: Tensile strength test The tensile strength of various biomat samples was tested using the method described in Example 41. The biomat characteristics and test results are shown in Table 26. [Table 58]

[0438] Example 48: Back pressure-eliminating bioreactor A bioreactor was constructed that essentially followed the generalized schematic diagram provided in Figure 31A, eliminating back pressure resulting from the consumption of feedstocks, and was used to test the performance of various membranes under fluid pressure. The results of these tests are shown in Table 27. [Table 59]

[0439] The trend in these results was that hydrophilic membranes and large-pore membranes were less resistant to hydrostatic pressure than small-pore hydrophobic membranes. While we do not wish to be bound by any particular theory, it was hypothesized that hydrophilic membranes and large-pore membranes may be more suitable for applications where feedstock is deposited beneath the membrane, although this may facilitate the passage of feedstock, while small-pore hydrophobic membranes may be more suitable for applications where feedstock is placed on top of the membrane. It was also hypothesized that fluctuations in fluid pressure would allow bioreactor operators to adjust or fine-tune the biomat growth rate or other biomat characteristics and / or facilitate the in situ removal of the biomat from the membrane.

[0440] Example 49: Effect of light conditions on biomat growth The fungal strain MK7 was inoculated into nylon and polypropylene membranes and grown in biomats under light and dark conditions. All mats appeared identical after 4 days of growth, but by day 7, the biomats grown under light conditions began to die, likely due to nutrient depletion in the feedstock. In contrast, the biomats grown under dark conditions appeared healthy after 7 days and showed gas bubbles, suggesting a different metabolic pathway than the biomats grown under light conditions. The biomats grown under dark conditions did not blacken and remained healthy until day 14. These results are shown in Figure 34 ("N" means nylon membrane, and "P" means polypropylene membrane).

[0441] Example 50: Comparative protein content Each of the filamentous fungi was propagated by at least one method selected from the surface fermentation of the present invention, water fermentation by prior art methods, and fruiting body (i.e., "natural") growth. In each case of surface fermentation, the carbon-to-nitrogen ratio of the liquid growth medium was 7.5. The average protein content of each fungus propagated by each method is shown in Table 28. [Table 60]

[0442] The results of this embodiment demonstrate that the surface fermentation method of the present invention produces filamentous fungal biomats with a higher protein content than can be achieved by water fermentation or natural propagation of fungal fruiting bodies. More specifically, the method of the present invention produces biomats with a protein content of at least about 40 wt% for a number of filamentous fungal species, which could not be achieved by any previously known method.

[0443] Example 51: Mesh Solid Support 1.75 kilograms of 1:1 sucrose:fructose carbon source in M2 medium was poured into a 10-inch x 13-inch Pyrex tray. 0.09 m of #7 polyolefin mesh (mesh size 2 mm, material assumed to be LDPE) was used. 2The slices were coated with the inoculum of strain MK7, applied to the surface of the growth medium, and then the tray was cultured at 27 °C for 72 hours. It was found that the initial biomatt formation was faster on the mesh surface than on the bulk medium in the tray.

[0444] The first slice of the biomatt was collected from the tray by cutting the outer periphery of the polyolefin mesh, and the second slice of the biomatt was collected from the tray by cutting around the tray. It was immediately revealed that the components of the accompanying liquid growth medium were much lower in the biomatt (wet mass 70 g) collected from the mesh surface than in the sample (wet mass 144 g) from the surface of the medium.

[0445] Both biomatt samples were steam-treated for 30 minutes, then immersed in water at 50 °C for 15 minutes and pressed by hand. The final (dry) mass of the biomatt collected from the mesh surface was 30 g (333.33 g / m 2 ) while the final (dry) mass of the biomatt collected from the bulk medium was 24 g (266.67 g / m 2 ) Thus, this example provides evidence that the biomatt yield can be improved by about 20% by simply providing a solid mesh as a scaffold or substrate to which the biomatt structure can adhere.

[0446] Example 52: Refresh Medium Experiment with Pleurotus eryngii and Ganoderma lucidum The experimental method of Example 2 (including control comparison) was repeated using Pleurotus eryngii (Pleurotys eryngii) and Ganoderma lucidum (Ganoderma lucidum) instead of strain MK7, using the mesh support of Example 51, and conducting the experiment for 22 days, except that the total biomatt was measured on the 5th, 10th, and 22nd days. The results are shown in Table 29 (total yield is shown as grams of dry mass per square meter).

Table 61

[0447] Example 53: Effects on culture medium characteristics and protein content Artificial urine medium ("AUM"), lignocellulose biomass medium ("LCBM"), and MK102 medium were prepared with the compositions shown in Tables 28, 29, and 30, respectively. [Table 62] [Table 63] [Table 64]

[0448] The culture medium was then acidified with hydrochloric acid (AUM and MK102) or sulfuric acid (LCBM) to achieve a pH of 3.5. The ionic strength and osmolality of the medium were determined, and the mats were grown in each medium, then desiccated and / or dried, and assayed to determine the protein content. The results of these determinations are shown in Table 31.

[0449] [Table 65]

Claims

1. A food ingredient containing particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than approximately 40 wt.% and an RNA content of less than approximately 8 wt.%.

2. Filamentous fungi belong to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, and Pleurotaceae. The food ingredient according to claim 1, belonging to a family selected from the group consisting of Physalacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

3. The filamentous fungi include Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.The food ingredient according to claim 1, belonging to a species selected from the group consisting of PTA-10698, Disciotis venosa, Clonostachys rosea, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and the genus Leucoagaricus.

4. The food ingredient according to claim 1, wherein the filamentous fungus is of the Fusarium species.

5. The food ingredient according to claim 1, wherein the filamentous fungus is Fusarium venenatum.

6. The food ingredient according to claim 1, wherein the filamentous fungus is strain MK7 (ATCC Accession Deposit No. PTA-10698).

7. Filamentous fungi a. Contains a protein content exceeding approximately 45 wt.%; b. Contains more than approximately 50 wt.% protein; c. Contains more than approximately 55 wt.% protein; d. Contains more than approximately 60 wt.% protein; e. Contains an RNA content of less than approximately 5 wt.%; f. Contains RNA content of less than approximately 4 wt.%; g. Contains RNA content of less than approximately 3 wt.%; h. Contains an RNA content of less than approximately 2 wt.%; and i. A combination of one from a to d and one from e to h The food ingredient according to claim 1, having characteristics selected from the group consisting of the following.

8. The food ingredient of claim 1, wherein the filamentous fungus contains less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zearalenone, beuvericin, fusarin C, fusaric acid, and any combination thereof.

9. The food ingredient according to claim 1, comprising filamentous fungi with a total mycotoxin content of less than approximately 10 ppm.

10. The food ingredient according to claim 1, comprising filamentous fungi with a total mycotoxin content of less than approximately 5 ppm.

11. The food ingredient according to claim 1, wherein the filamentous fungus contains more than 15 wt.% of branched-chain amino acids or more than 20 wt% of branched-chain amino acids.

12. The food ingredient according to claim 1, wherein the filamentous fungal particles are in the form of a powder.

13. The food ingredient according to claim 12, wherein the powder has a particle size of 30 to 400 microns.

14. The food ingredient according to claim 1, wherein the particles have a particle length of approximately 0.05 mm to approximately 500 mm, a particle width of approximately 0.03 mm to approximately 7 mm, and a particle height of approximately 0.03 mm to approximately 1.0 mm.

15. The food ingredient according to claim 1, wherein the food ingredient is a liquid dispersion of filamentous fungal particles.

16. The food ingredient according to claim 15, wherein the liquid dispersion is produced in a nitrogen-rich atmosphere.

17. The food ingredient according to claim 15, wherein the liquid dispersion of filamentous fungal particles is stable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, or at least about 5 days.

18. The ingredient according to claim 1, wherein the ingredient is vegan.

19. The food ingredient according to claim 1, wherein filamentous fungal particles are the only protein component present in the food ingredient.

20. The food ingredient according to claim 1, wherein the filamentous fungal particles contain all essential amino acids.

21. The food ingredient according to claim 1, wherein filamentous fungal particles cannot survive.

22. A yogurt-like food containing particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than approximately 40 wt.% and an RNA content of less than approximately 8 wt.%.

23. Filamentous fungi belong to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, and Physiophyceae. A yogurt-like food according to claim 22, belonging to a family selected from the group consisting of alacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

24. The filamentous fungi include Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.A yogurt-like food according to claim 22, belonging to a species selected from the group consisting of PTA-10698, Disciotis venosa, Clonostachys rosea, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and the genus Leucoagaricus.

25. The yogurt-like food according to claim 22, wherein the filamentous fungus is of the Fusarium species.

26. The food ingredient according to claim 22, wherein the filamentous fungus is Fusarium venenatum.

27. The food ingredient according to claim 22, wherein the filamentous fungus is strain MK7 (ATCC Accession Deposit No. PTA-10698).

28. Filamentous fungi a. Contains a protein content exceeding approximately 45 wt.%; b. Contains more than approximately 50 wt.% protein; c. Contains more than approximately 55 wt.% protein; d. Contains more than approximately 60 wt.% protein; e. Contains an RNA content of less than approximately 5 wt.%; f. Contains RNA content of less than approximately 4 wt.%; g. Contains RNA content of less than approximately 3 wt.%; h. Contains an RNA content of less than approximately 2 wt.%; and i. A combination of one from a to d and one from e to h The food ingredient of claim 22, having characteristics selected from among.

29. The yogurt-like food according to claim 22, wherein the ratio of filamentous fungal particles to water is in the range of approximately 1:10 to approximately 10:

1.

30. A yogurt-like food according to claim 22, wherein the ratio of filamentous fungal particles to water is selected from the group consisting of approximately 1:4, approximately 1:3, approximately 1:2, approximately 1:1, approximately 2:1, approximately 3:1, and approximately 4:

1.

31. Furthermore, the yogurt-like food according to claim 22 contains invert sugar.

32. Furthermore, the yogurt-like food according to claim 22, further comprising a thickening agent.

33. A yogurt-like food according to claim 22, wherein the cells of filamentous fungi are dissolved.

34. The yogurt-like food according to claim 22, further comprising Lactobacillus bulgaricus and Streptococcus thermophilus.

35. A yogurt-like food product according to claim 22, wherein the product is vegan.

36. The yogurt-like food according to claim 22, wherein the filamentous fungal particles contain all essential amino acids.

37. The yogurt-like food according to claim 22, wherein filamentous fungal particles are the sole protein component.

38. A yogurt-like food according to claim 22, wherein filamentous fungal particles cannot survive.

39. Furthermore, the yogurt-like food according to claim 22, further comprising rennet.

40. The yogurt-like food according to claim 39, wherein the rennet is derived from a source selected from the group consisting of animal sources, plant sources and microbial sources.

41. The yogurt-like food according to claim 39, wherein the rennet is derived from an origin selected from the group consisting of plant origin and microbial origin.

42. A yogurt-like food product according to claim 35, wherein the product does not contain milk solids.

43. Furthermore, the yogurt-like food according to claim 22 contains probiotics.

44. Furthermore, the yogurt-like food according to claim 22 contains enzyme water.

45. a. Particles of filamentous fungal biomats; b. Water phase; A foaming material containing a certain substance, wherein the solid component of the foaming material is approximately 5% to approximately 30%, and the foam is stable.

46. The foamed material according to claim 45, wherein the foamed material does not spontaneously collapse immediately after the foaming process is stopped during production.

47. The foaming material according to claim 45, wherein the foaming material is stable for at least about 7 days.

48. The foaming material according to claim 45, wherein the foaming material has an overrun of at least about 10%.

49. The foamed material according to claim 45, wherein the filamentous fungal biomat contains Fusarium species.

50. The foaming material according to claim 45, wherein the foaming material does not contain milk solids.

51. A food product comprising the foaming material of claim 45.

52. container; A film comprising a first surface and a second surface, disposed inside or on the surface of a container; A feedstock for the growth of filamentous fungi, which comes into contact with the first surface of at least one membrane; and A filamentous fungal inoculant placed on the first or second surface of at least one membrane, A bioreactor comprising the inoculant, wherein, by culturing the inoculant in the bioreactor, a biomat of the filamentous fungus is formed on the second surface of at least one membrane after the biomat growth phase.

53. The bioreactor according to claim 52, wherein the container is a bag, and the first and second surfaces of at least one membrane are the first and second surfaces of at least a portion of the bag.

54. The bioreactor according to claim 52, wherein the supply material is subjected to positive or negative pressure applied to the opposite side of at least one of the first and second surfaces of at least one membrane.

55. The bioreactor according to claim 52, further comprising cyanobacteria, the cyanobacteria providing at least one of oxygen gas and carbon to promote the growth of the biomat.

56. at least one of the following i) The density of the biomat is at least about 0.05 grams / cubic centimeter; and ii) The density of the biomat after drying is at least about 0.01 grams / cubic centimeter. The bioreactor according to claim 52, wherein the above applies.

57. The bioreactor according to claim 52, wherein the biomat comprises at least one layer.

58. The bioreactor according to claim 52, wherein the biomat has a tensile strength of at least about 3 kilopascals or at least about 30 grams by weight per square centimeter.

59. The bioreactor according to claim 58, wherein the biomat has a tensile strength of at least about 100 kilopascals or at least about 1,020 grams by weight per square centimeter.

60. The bioreactor according to claim 52, wherein at least one membrane comprises at least one polymer selected from the group consisting of polypropylene, polytetrafluoroethylene, polycarbonate, polyamide, cellulose acetate, polyvinylidene fluoride, mixed cellulose ester, polyethersulfone, polyethylene, and polypyrrole.

61. The bioreactor according to claim 52, wherein at least one membrane comprises at least one material selected from the group consisting of polypropylene cloth, polytetrafluoroethylene cloth, and nylon net filter.

62. The bioreactor according to claim 52, wherein at least one membrane comprises at least one of a glass fiber material and a porous ceramic material.

63. The bioreactor according to claim 52, wherein the average pore size of at least one membrane is about 0.2 μm to about 25 μm.

64. The bioreactor according to claim 63, wherein the average pore size of at least one membrane is about 5 μm to about 11 μm.

65. The bioreactor according to claim 52, wherein the container is sealed and substantially airtight, and the container encloses a gas head space in which the biomat grows.

66. The bioreactor according to claim 52, wherein the biomat is spontaneously separated from at least one membrane.

67. The bioreactor according to claim 52, wherein when the biomat is removed from at least one membrane, the new inoculation material for filamentous fungi remains on at least one membrane.

68. The bioreactor of claim 52, wherein the filamentous fungi belong to an order selected from the group consisting of Mucorals, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

69. Filamentous fungi belong to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, and Physiophyceae. A bioreactor according to claim 52, belonging to a family selected from the group consisting of alacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

70. The filamentous fungi include strains of Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white pygmy mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.PTA-10698), Disciotis venosa and Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, Leucoagaricus holosericeus, Calvatia fragilis, Handkea utriformis and Pholiota adiposa A bioreactor according to claim 52, selected from the group consisting of adiposa.

71. The bioreactor according to claim 52, wherein the raw material supplied includes at least one of animal feces and animal urine.

72. The bioreactor according to claim 71, wherein the animal is a human.

73. The bioreactor according to claim 52, wherein at least one membrane is a single composite membrane, the first surface comprising a first material and the second surface comprising a second material.

74. The bioreactor according to claim 52, wherein at least one membrane comprises at least a first membrane and a second membrane, where the first surface is the surface of the first membrane and the second surface is the surface of the second membrane.

75. The bioreactor according to claim 74, wherein the first membrane and the second membrane are in physical contact with each other.

76. The bioreactor according to claim 52, further comprising a selective gas permeable membrane, wherein the first gas produced during the growth of the biomat is selectively separated into the gas headspace on the first side of the selective gas permeable membrane.

77. The bioreactor of claim 76, wherein a secondary gas produced during the growth of the biomat is selectively separated into the gas headspace on the second side of the membrane.

78. A method for producing a biomat of filamentous fungi, This includes inoculating a bioreactor with filamentous fungi, where the bioreactor container; A membrane comprising a first surface and a second surface, disposed inside or on the surface of a container, wherein either or both of the first and second surfaces are suitable for receiving the inoculation material of the filamentous fungus; and A feedstock for the growth of filamentous fungi, which comes into contact with the first surface of at least one membrane. Methods that include...

79. The method of claim 78, wherein the container is a bag, wherein the first and second surfaces of at least one membrane are the first and second surfaces of at least a portion of the bag.

80. The method of claim 78, wherein the supply material is subjected to positive or negative pressure applied to the supply material side opposite the first surface of at least one membrane.

81. The method of claim 80, wherein positive or negative pressure facilitates the inoculation process.

82. The method of claim 78, further comprising providing cyanobacteria to a bioreactor, wherein the cyanobacteria provide at least one of oxygen gas and carbon to promote the growth of the biomat.

83. at least one of the following i) The density of the biomat after collection is at least about 0.6 grams / cubic centimeter; and ii) The density of the biomat after collection and drying is at least about 0.1 grams / cubic centimeter. The method of claim 78, which corresponds to the above.

84. The method of claim 78, wherein the biomat comprises at least one layer.

85. The method of claim 78, wherein during or after the harvesting process, the biomat has a tensile strength of at least about 3 kilopascals or at least about 30 grams by weight per square centimeter.

86. The method of claim 85, wherein during or after the harvesting process, the biomat has a tensile strength of at least about 100 kilopascals or at least about 1,020 grams by weight per square centimeter.

87. The method of claim 78, wherein at least one membrane comprises at least one polymer selected from the group consisting of polypropylene, polytetrafluoroethylene, polycarbonate, polyamide, cellulose acetate, polyvinylidene fluoride, mixed cellulose ester, polyethersulfone, polyethylene, and polypyrrole.

88. The method of claim 78, wherein at least one membrane comprises at least one material selected from the group consisting of polypropylene cloth, polytetrafluoroethylene cloth, and nylon net filter.

89. The method of claim 78, wherein the film comprises at least one of a glass fiber material and a porous ceramic material.

90. The method of claim 78, wherein the average pore size of at least one membrane is about 0.2 μm to about 25 μm.

91. The method of claim 90, wherein the average pore size of at least one membrane is about 5 μm to about 11 μm.

92. The method of claim 78, wherein the container is sealed and substantially airtight, and the container encloses a gas head space in which the biomat grows.

93. The method according to claim 78, wherein the biomatt is spontaneously separated from at least one membrane.

94. The method of claim 78, further comprising the collection of a biomat, wherein when the biomat is removed from at least one membrane, a fresh inoculant of filamentous fungi remains on at least one membrane.

95. The method of claim 78, wherein the filamentous fungus belongs to an order selected from the group consisting of Mucorals, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

96. Filamentous fungi belong to the families Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, and P. The method of claim 78, wherein the fungus belongs to a family selected from the group consisting of hysalacriaceae, Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

97. The filamentous fungi include Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, and Stropharia rugosoanulata. rugosoannulata), Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum, strain MK7 (ATCC Accession Deposit No.The method of claim 78, comprising selecting from the group consisting of PTA-10698), Disciotis venosa, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and the genus Leucoagaricus spp.

98. The method of claim 78, wherein the raw material supplied includes at least one of animal feces and animal urine.

99. The method of claim 98, wherein the animal is a human.

100. The method of claim 78, wherein at least one film is a single composite film, where the first surface comprises a first material and the second surface comprises a second material.

101. The method of claim 78, wherein at least one film comprises at least a first film and a second film, where the first surface is the surface of the first film and the second surface is the surface of the second film.

102. The method according to claim 101, wherein the first film and the second film are in physical contact with each other.

103. The method of claim 78, wherein the bioreactor further comprises a selective gas permeable membrane, and the first gas produced during the growth of the biomat is selectively separated into the gas headspace on the first side of the selective gas permeable membrane.

104. The method of claim 103, wherein a secondary gas produced during the growth of the biomat is selectively separated into the gas headspace on the second side of the membrane.

105. A method for producing fresh water, container; and Raw materials for the growth of filamentous fungi; A bioreactor containing filamentous fungi is inoculated, Filamentous fungi are cultured to form a biomat on at least one of the surface of the feed material and the surface of the bioreactor membrane, where the filamentous fungi produce water as a metabolic byproduct of biomat growth; and Collect water produced by the growth of biomats. A method that includes doing so.

106. The method of claim 105, wherein the raw material supplied includes at least one of animal feces and animal urine.

107. The method of claim 106, wherein the animal is a human.

108. The method of claim 105, further comprising recycling the collected water into a bioreactor.

109. The method of claim 105, further comprising preparing a supply material containing the collected water.

110. The method of claim 109, further comprising recycling the prepared feedstock, including the collected water, into a bioreactor.

111. A method for producing gas, container; and Raw materials for the growth of filamentous fungi; A bioreactor containing filamentous fungi is inoculated, Filamentous fungi are cultured to form a biomat on at least one of the surface of the feed material and the surface of the bioreactor membrane, where the filamentous fungi produce gas as a metabolic byproduct during the growth of the biomat; and Collecting gases produced by the growth of biomats. A method that includes doing so.

112. The method of claim 111, wherein the gas is selected from the group consisting of ammonia, ammonium species, hydrogen gas, and volatile esters.

113. A method for producing a biomat of filamentous fungi, (a) Inoculate an effective amount of cells of at least one filamentous fungus into a first aliquot of growth medium to produce an inoculated growth medium; (b) Incubate the inoculated growth medium for a first hour to produce the initial biomat; (c) Remove at least a portion of the first aliquot of the growth medium and add the second aliquot of the growth medium to provide a refreshed growth medium; and (d) Incubate the refreshed growth medium for a second time to produce the final biomat. A method comprising the following: the thickness of the final biomat and the dry mass density of the initial biomat are greater than those of the initial biomat.

114. The method of claim 113, wherein the dry mass density of the final biomat is at least about 75 grams / liter.

115. The method of claim 113, wherein the biomat contains more than approximately 40 wt% protein and less than approximately 8 wt% RNA.

116. The method of claim 113, wherein at least one filamentous fungus belongs to an order selected from the group consisting of Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales and Hypocreales.

117. The method of claim 113, wherein at least one filamentous fungus belongs to the families Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, and Cordycipitaceae.

118. At least one filamentous fungus strain is identified: MK7 (ATCC Accession Deposit No. PTA-10698), Fusarium venenatum, Ustilago esculenta, Heliculum erinaceus, Polyporus squamosus, Grifola fondrosa, Hypsizygus marmoreus, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoanulata. The method of claim 113, selected from rugosoannulata), Hypholoma lateritium, Pluerotus eryngii, Tuber borchii, Morchella esculenta, Morchella conica, Disciotis venosa, Ophiocordyceps sinensis, and Cordyceps militaris.

119. The final biomat a. Contains a protein content exceeding approximately 45 wt.%; b. Contains more than approximately 50 wt.% protein; c. Contains more than approximately 55 wt.% protein; d. Contains more than approximately 60 wt.% protein; e. Contains an RNA content of less than approximately 5 wt.%; f. Contains RNA content of less than approximately 4 wt.%; g. Contains RNA content of less than approximately 3 wt.%; h. Contains an RNA content of less than approximately 2 wt.%; and i. A combination of one from a to d and one from e to h The method of claim 113, having a feature selected from the group consisting of the following.

120. The method of claim 113, wherein the final biomat contains less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zearalenone, beuvericin, fusarin C, fusaric acid and any combination thereof.

121. The method of claim 113, wherein the final biomat contains a total mycotoxin content of less than approximately 10 ppm.

122. The method of claim 113, wherein the final biomat contains a total mycotoxin content of less than approximately 5 ppm.

123. The method of claim 113, wherein the final biomat contains more than approximately 15 wt.% of branched-chain amino acids.

124. A biomat of at least one filamentous fungus having a dry mass density of at least approximately 75 grams / liter.

125. The biomat of claim 124, comprising more than approximately 40 wt% protein and less than approximately 8 wt% RNA.

126. The biomat of claim 124, wherein at least one filamentous fungus belongs to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales.

127. At least one filamentous fungus belongs to the following families: Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, or Pleurotaceae. The biomat according to claim 124, belonging to a family selected from the group consisting of (Physalacriaceae), Ophiocordycipitaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae.

128. At least one filamentous fungus is found in Rhizopus oligosporus, Ustilago esculenta, Helicululm erinaceus, Polyporus squamosus, Grifola frondosa, Hypsizygus marmoreus, Hypsizygus ulmarius (also known as white oyster mushroom), Calocybe gambosa, Pholiota nameko, Calvatia gigantea, or Agaricus bisporus. bisporus), Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus (marbled oyster mushroom), Pleurotus ostreatus var. columbinus (oyster mushroom), Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa (flower mushroom), Fusarium venenatum venenatum), strain MK7 (ATCC Accession Deposit No.A biomat according to claim 124, selected from the group consisting of PTA-10698, Disciotis venosa, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and the genus Leucoagaricus.

129. Biomat a. Contains a protein content exceeding approximately 45 wt.%; b. Contains more than approximately 50 wt.% protein; c. Contains more than approximately 55 wt.% protein; d. Contains more than approximately 60 wt.% protein; e. Contains an RNA content of less than approximately 5 wt.%; f. Contains RNA content of less than approximately 4 wt.%; g. Contains RNA content of less than approximately 3 wt.%; h. Contains an RNA content of less than approximately 2 wt.%; and i. A combination of one from a to d and one from e to h A biomat according to claim 124, having features selected from the above.

130. A biomat according to claim 124, comprising less than 10 ppm of mycotoxins selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, nivalenol, deoxynivalenol, acetyldeoxynivalenol, fusarenone X, T-2 toxin, HT-2 toxin, neosolaniol, diacetoxysylpenol, zearalenone, fusarin C, fusaric acid, and any combination thereof.

131. The biomat according to claim 124, comprising a total mycotoxin content of less than approximately 10 ppm.

132. The biomat according to claim 124, comprising a total mycotoxin content of less than approximately 5 ppm.

133. The biomat according to claim 124, comprising more than approximately 15 wt.% of branched-chain amino acids.

134. A biomat according to claim 124, produced by the method of claim 113.

135. A method for producing a biomat of filamentous fungi, This includes inoculating a bioreactor with filamentous fungi, where the bioreactor container; At least one mesh scaffold placed inside or on a surface of a container, comprising a first surface and a second surface, wherein either or both of the first and second surfaces are suitable for receiving the inoculation material of the filamentous fungus; and Raw materials for the growth of filamentous fungi in contact with the first surface of the mesh scaffolding. Methods that include...

136. The method of claim 135, wherein the mesh scaffolding includes nylon material.

137. It is a cultured food, Particles of filamentous fungi belonging to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content of more than approximately 40 wt.% and an RNA content of less than approximately 8 wt.%; and microbial food culture Cultured foods, including those containing this ingredient.

138. The cultured food according to claim 137, wherein the microbial food culture contains lactic acid bacteria.

139. A method for producing cultured food, A method comprising inoculating granules of filamentous fungi into a microbial food culture, wherein the filamentous fungi belong to an order selected from the group consisting of Mucorales, Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales, and Hypocreales, wherein the filamentous fungi contain a protein content greater than approximately 40 wt.% and an RNA content less than approximately 8 wt.%.

140. The method of claim 139, wherein the microbial food culture contains lactic acid bacteria.

141. A cultured food according to claim 137, produced by the method of claim 139.

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