Methods for producing food using microbial growth and products thereof

Abstract

A method for preparing a food product is described, the method comprising the steps of providing a scaffold substrate; inoculating the scaffold substrate with at least two microbial species, wherein the at least two microbial species comprise at least one filamentous fungal species, preferably a first filamentous fungal species and a second filamentous fungal species; and incubating the scaffold substrate to grow the at least two microbial species inside, outside, or inside and outside the scaffold substrate, wherein the at least two microbial species allow significant growth of the at least one filamentous fungal species.

Description

[Technical Field]

[0001] Technical Field The present application relates to a method of providing a food product by combining pieces of a textured vegetable protein substrate that are attached to one another by growth of two or more microbial species by at least one filamentous fungus, thus forming a fungal-containing food product. [Background technology]

[0002] background Currently, various approaches are applied to produce meat substitute products, resulting in large differences in texture and nutritional value.In fact, there are few meat substitutes that not only have an attractive texture but also provide the protein recommended for daily intake.It is also possible to add some ingredients to bind the pieces together or to give the product or part of the product a specific texture, so as to obtain a food product with a texture similar to meat and / or meat-derived foods.

[0003] Tempeh is a well-known vegan product made from soybeans fermented by a filamentous fungus, usually Rhizopus oligosporus. Recently, the term tempeh has been used more broadly to include the fermentation and binding of grains or food processing by-products in addition to soybeans. One drawback of currently available tempeh products is that they lack flavor and texture compared to real meat and / or meat-derived foods. Another drawback is that the product is neither very juicy nor flavorful on its own, and the most common cooking method requires extensive marinating and, usually, deep frying.

[0004] More recent advances have been made by linking textured vegetable protein with fungi, as presented in various patent applications (US2021045410, US2022132893). These applications use a textured substrate that can be made from several pieces of textured vegetable protein (TVP) that are bound together using at least one fungus. One of the limiting parameters in the production of mycelium-based foods is the density of the fungal hyphae within the substrate. Fungal growth can be limited by available nutrients, lack of oxygen, and / or heat released as the fungus grows, actively fermenting the substrate. Competition from bacterial and fungal contaminants can also jeopardize the mycelialization process. Poor growth of fungi within the substrate results in a low mycelial density which reduces fiber quality, limits liquid and fat absorption and therefore limits juiciness and texture complexity during mastication, reduces the bond strength between different parts of the substrate, and increases the population of undesirable microbial species as the fungi also act as a protective culture; the meat product may deviate in color and / or develop off-flavor profiles due to competing microbial contaminants and unfermented compounds and / or antinutrients present in the vegetable protein fraction of the substrate.

[0005] In view of the above, there is a need for meat substitutes that can have an attractive texture that can replace pieces of animal meat or meat-derived products such as salami, pepperoni, chorizo, fuet, or other types of processed and / or fermented meat sausages, as well as whole cuts such as chicken breast or beef tenderloin.

[0006] When commonly used conventional strains in well-defined solid-state fermentation (SSF) processes, such as those used in the production of blue cheese, Camembert, tempeh, and koji, are grown on new, non-traditional, or unconventional substrates, challenges such as reduced mycelial growth, off-flavor development, and decreased product safety are usually encountered due to uncharacterized growth kinetics and / or metabolic pathways, poorly understood substrate properties, and a lack of technological solutions tailored to the requirements of the new bioprocess. The methods presented herein solve some of the greatest challenges encountered in the prior art by taking advantage of the significant growth of a filamentous fungal species enabled by another microbial species in co-culture and / or sequential culture, which has not been previously disclosed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 045410 [Patent Document 2] US Patent Application Publication No. 2022 / 132893 Summary of the Invention [Means for solving the problem]

[0008] Abstract Various embodiments of methods for preparing food products using a scaffold substrate and at least two microbial species, one of which is a filamentous fungal species, are described herein. The use of at least two microbial species allows for more significant growth of at least the filamentous fungal species. The methods generally include providing a scaffold substrate, such as a textured vegetable protein substrate, which may or may not be subjected to various pretreatment steps used to help promote the growth of at least two microbial species inside, outside, or inside and outside of the scaffold substrate; inoculating the textured substrate with the microbial species, either simultaneously or spaced apart; and growing the microbial species inside, outside, or inside and outside of the scaffold substrate. In some embodiments, the filamentous fungal species is used to bond multiple scaffold substrates and any individual particles therein together to form a larger composite food product. In other embodiments, the filamentous fungal species is used to create a fungal-based skin on the surface of the food product.

[0009] A major advantage of using at least two microbial species for inoculation is the enhanced growth of filamentous fungi. This is achieved by utilizing a first microbial species to promote and support better growth conditions for the filamentous fungal species. For example, in the preparations and examples provided herein, the inventors have discovered that certain filamentous fungal species can outgrow another fungal, algae, or bacterial population, likely due to the activation of active growth genes and associated competitive and expansive behaviors that can stimulate the production of fungal biomass. Furthermore, fermentation of the substrate by the first microbial species allows for better growth, and the fermentation creates additional, specific nutrients that are more accessible to the growth of the second fungal species.

[0010] In general, the benefits to food products can be varied. Some non-limiting examples include increased mycelial density, which can enhance the binding of the scaffold matrix and result in a firmer texture. The final food products described herein can contain two phases: a phase with a scaffold matrix and a phase with mycelium. The higher the mycelium density, the whiter the color of the mycelium phase can be. In addition, increased mycelial density can improve juiciness, as more oil and / or moisture can be absorbed into the food and released during chewing, contributing to juiciness. Another potential advantage of this method is the ability to adjust the taste profile by changing the microbial species and / or incubation conditions.

[0011] In one aspect, the exterior surface of the food product can have a fungal-based "skin"-like coating, improving appearance before and after cooking and enhancing the eating experience. In another aspect, the interior of the product can have one or more phases that include a scaffold matrix and / or filamentous fungal species, enhancing the eating experience by providing a more complex texture similar to whole cuts of meat or meat products.

[0012] Another benefit that can be implemented is the reduction of growth of certain microbial species, such as undesirable and potentially harmful microbial species or spoilage species to humans, achieved, for example, by the production of antimicrobial metabolites such as organic acids, enzymes, other antagonistic compounds, and / or competitive exclusion of nutrients and space by one or more of the inoculum species.

[0013] The final food product produced by the methods presented herein is a fermented food product comprised of an edible substrate, wherein the fermented food product is more nutritious than the edible substrate, and preferably the proteins in the edible substrate are more digestible than they were prior to fermentation.

[0014] The methods described herein enhance the flavor profile, color, and / or texture of the final product, making it more similar to animal meat products such as salami, pepperoni, or other types of fermented meat sausages, and / or to whole animal cuts such as pork flank or other cuts of meat.

[0015] Accordingly, the present invention discloses a method for preparing a food product, said method comprising: a) providing a scaffold substrate; b) inoculating the scaffold substrate with at least two microbial species, wherein the at least two microbial species comprise at least one filamentous fungal species, preferably a first filamentous fungal species and a second filamentous fungal species; c) incubating the scaffold substrate to grow at least two microbial species inside, outside, or inside and outside the scaffold substrate; Including, At least two microbial species allow significant growth of at least one filamentous fungal species.

[0016] According to the present invention, the method includes a step of inoculating the scaffold substrate with a predetermined colony forming unit concentration of at least one filamentous fungal species, wherein the filamentous fungal species exhibits more significant growth than when the scaffold substrate is inoculated with only the predetermined colony forming unit concentration of the filamentous fungal species and incubated in the absence of the other one of the at least two microbial species.

[0017] Also, significant growth occurs by carrying out step c), the significant growth including increased mechanical strength, preferably tensile strength.

[0018] According to the present invention, the at least two microbial species comprise a first microbial species and a second microbial species which is a filamentous fungal species, and the method comprises the steps of inoculating the scaffold substrate with a first concentration of colony forming units of the first microbial species and inoculating the scaffold substrate with a second concentration of colony forming units of the second microbial species, wherein the food product obtained by step c) preferably immediately after step c) without being subjected to further processing has a tensile strength greater than the sum of the tensile strengths of the first product and the second product, and wherein the first product has been inoculated with the first microbial species on a first corresponding scaffold substrate. and incubating the first corresponding scaffold substrate to grow a first microbial species inside, outside, or inside and outside of the first corresponding scaffold substrate; and a second product is obtained by inoculating a second corresponding scaffold substrate with a second colony forming unit concentration of a second microbial species and incubating the second corresponding scaffold substrate to grow a second microbial species inside, outside, or inside and outside of the second corresponding scaffold substrate.

[0019] According to further non-limiting features of the present invention, the following features are provided, either alone or in any technically feasible combination: The scaffold substrate comprises at least one chunk or collection of chunks, preferably the number of chunks in the collection is two or more, and more preferably the aspect ratio of the chunks, defined as the ratio of the longest dimension to the shortest dimension of each chunk, is 10 or less, 5 or less, 3 or less, or 2 or less. The scaffold matrix comprises at least one fiber or collection of fibers, preferably the number of fibers in the collection is two or more, more preferably the fiber aspect ratio, defined as the ratio of the longest dimension to the shortest dimension of each fiber, is 10 or more, or 100 or more, or 1000 or more, preferably less than 10,000. The scaffold substrate comprises at least one sheet or stack of sheets, preferably the number of sheets in the stack is two or more, more preferably the aspect ratio of the sheets, defined as the ratio of the longest dimension to the shortest dimension of each sheet, is 10 or more, or 100 or more, or 1000 or more, preferably less than 10,000. The first microbial species is a filamentous fungal species including the genus Penicillium, preferably Penicillium nalgiovense, and the second microbial species is a filamentous fungal species including the genus Rhizopus, preferably Rhizopus oligosporus. A first colony-forming unit concentration of the first microbial species, expressed in terms of spores per kg of scaffold substrate, is 1 x 10 4 ~1×10 12 , preferably 1 x 10 6 ~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second colony forming unit concentration of the second microbial species, in units of spores per kg of scaffold substrate, is in the range of 1 x 10 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 Preferably, the ratio of the first colony-forming unit concentration to the second colony-forming unit concentration is in the range of 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150. The first microbial species is a filamentous fungal species including the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a filamentous fungal species including the genus Aspergillus, preferably Aspergillus oryzae. A first colony-forming unit concentration of the first microbial species, expressed in terms of spores per kg of scaffold substrate, of 1 x 10 4 ~1×10 12 , preferably 1 x 106 ~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second colony forming unit concentration of the second microbial species, in units of spores per kg of scaffold substrate, is in the range of 1 x 10 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 Preferably, the ratio of the first colony-forming unit concentration to the second colony-forming unit concentration is in the range of 0.01-2000, preferably 0.1-1000, more preferably 20-500. Step b) b1) inoculating the scaffold substrate with a first microbial species; and then, b2) inoculating the scaffold substrate with a second microbial species, preferably wherein the second microbial species is a filamentous fungal species; Includes: Step c) c1) incubating the scaffold substrate to grow a first microbial species inside, outside, or inside and outside the scaffold substrate; and then c2) incubating the scaffold substrate to grow a second microbial species inside, outside, or inside and outside the scaffold substrate; Includes: Step c1) is carried out before or after step b2), preferably step c1) is carried out for a time period of less than 48 hours at a temperature of 5-50°C, preferably 10-40°C, more preferably 20-30°C, and / or step c2) is carried out for a time period of less than 72 hours, preferably less than 44 hours, at a temperature of 20-45°C, preferably 28-35°C. According to the present invention, the food product produced by this method comprises at least two distinct phases, preferably the two distinct phases are distinguishable by the human eye, more preferably the first phase comprises a scaffold substrate and the second phase comprises mycelium of at least one filamentous fungal species, and preferably the ratio of the second phase of the food product, defined as the ratio of the area occupied by the second phase in a cross-section of the food product to the total area of ​​said cross-section, is in the range of 0.05 to 0.95, more preferably 0.1 to 0.6. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a flow diagram illustrating a method for producing a food product according to various embodiments described herein. [Figure 2] FIG. 2 is a flow diagram illustrating a method for producing a food product according to various embodiments described herein. [Figure 3] FIG. 3 is a photograph of TVP fermented with Rhizopus oligosporus. [Figure 4] FIG. 4 is a photograph of TVP fermented with Penicillium nalgiovense. [Figure 5] FIG. 5 is a photograph of TVP fermented by co-cultivating Rhizopus oligosporus and Penicillium nalgiovense. [Figure 6] FIG. 6 is a close-up photograph of TVP fermented with Rhizopus oligosporus. [Figure 7] FIG. 7 is a close-up photograph of TVP fermented with Penicillium nalgiovense. [Figure 8] FIG. 8 is a photograph of TVP fermented by co-cultivating Rhizopus oligosporus and Penicillium nalgiovense. [Figure 9] FIG. 9 is a 2x magnification photograph of TVP fermented with Penicillium nalgiovense. [Figure 10]FIG. 10 shows stress-strain diagrams of tensile tests performed on TVP strips bound in either co-culture fermentation with two species of Rhizopus oligosporus and Penicillium nalgiovense, or in monoculture fermentation with one species of Rhizopus oligosporus or one species of Penicillium nalgiovense. [Figure 11] FIG. 11 is a photograph of a tensile testing device measuring food products according to one embodiment. [Figure 12] FIG. 12 is a graph illustrating various tensile strengths of different food products according to various embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description FIG. 1 shows a method 100 for preparing a food product according to one embodiment, comprising step 110 of providing a scaffold substrate; step 120 of inoculating the scaffold substrate with at least two microbial species, wherein the at least two microbial species comprise at least one filamentous fungal species, preferably a microbial species and a filamentous fungal species; and step 130 of incubating the scaffold substrate to grow the at least two microbial species inside, outside, or inside and outside the scaffold substrate, wherein the at least two microbial species allow for more significant growth of the at least one filamentous fungal species.

[0022] According to the present invention, the term "microbial species" may encompass various types of microorganisms, including but not limited to bacteria, fungi, algae, molds, yeasts and / or mushrooms.

[0023] Method 100 utilizes the effects of co-cultivating a filamentous fungal species with at least one other microbial species to achieve significant growth of the filamentous fungal species. As used herein, significant growth is defined by an increase in at least one property of the mycelium, which may be, but is not limited to, faster growth of the at least one filamentous fungus, increased mycelial density, longer mycelium, larger mycelium, or stronger mycelium compared to growth without the other microbial species. In one embodiment, significant growth of both microbial species can be achieved because the at least two microbial species can support each other's growth.

[0024] With respect to food products, the significant growth effect can be characterized by increased hyphal density and / or increased tensile strength in the production of meat-like, and more specifically muscle-like, fungal-based food products. The tensile strength of a food product can be determined by subjecting the food product to a mechanical force at its extremity and measuring the amount of force required to induce fracture. This value is referred to herein as the tensile strength of the food product. Various types of mechanical forces can be applied to the food product, including, but not limited to, tension, compression, torsion, bending, and shear. Method 100 also enhances the flavor profile, texture, color, and / or juiciness of the final product. In some embodiments, the final product more closely resembles an animal meat product, such as salami, pepperoni, or other types of fermented meat sausage. In other embodiments, the final product more closely resembles a whole animal cut, such as pork flank or other cuts of meat.

[0025] In step 110 of providing a scaffold substrate, any substrate suitable for food preparation may be used. In some embodiments, the scaffold substrate is a vegetable protein substrate containing (poly)saccharides, fiber, inorganic salts, and / or amino acids necessary for the growth of microbial species. As used in this application, "protein" refers to a protein isolate, concentrate, or flour, or a combination thereof, which may also contain other macronutrients such as carbohydrates, fat, dietary fiber, salt, or residual water. The isolate, concentrate, flour, or combination thereof preferably contains a pure protein content of at least 40 wt%, preferably at least 50 wt%, and even more preferably at least 60 wt%. Preferably, the protein also contains sufficient carbohydrates to act as a nutrient source for microbial species.

[0026] In one embodiment, the scaffold substrate comprises only one chunk. In another embodiment, the scaffold substrate comprises an assembly of several chunks, such as 2-100 pieces, or 100-1000 pieces or more. The aspect ratio of the chunks, defined as the ratio of the longest dimension to the shortest dimension of each chunk, is 5 or less, 3 or less, or 2 or less.

[0027] In one embodiment, the scaffold substrate comprises only one elongated strip extending in one direction. Said strip may also be referred to as a "fiber" in the context of the present invention. In another embodiment, the scaffold substrate comprises an assembly of several fibers, between 1 and 100 strips, or between 100 and 10,000 strips or more. The aspect ratio of the fibers, defined as the ratio of the longest dimension to the shortest dimension of each fiber, is 5 or more, or 10 or more, or 100 or more, or 1,000 or more, preferably 10,000 or less.

[0028] In another embodiment, the scaffold substrate comprises only one piece extending in two directions, said piece being referred to in this application as a sheet. In another embodiment, the scaffold substrate comprises several sheets, between 1 and 100, or between 100 and 1000 or more. The aspect ratio of the sheets, defined as the ratio of the longest dimension to the shortest dimension of each sheet, is 5 or more, or 10 or more, or 100 or more, or 1000 or more, preferably 10,000 or less.

[0029] The texture of a food product can be adjusted by varying the number of pieces that make up the food product while maintaining the same final size. Thus, the number of pieces can be varied significantly to affect the final texture of the food product. For example, a small number of pieces can provide a texture similar to that of whole cut meat, while a larger number of pieces can provide a texture similar to that of salami. The selection of the number of pieces, their size, and shape can also affect the chewiness, juiciness, and mouthfeel of the final product. Thus, by adjusting the number of pieces, their size, and shape, a wide variety of textures and mouthfeel sensations can be created in the food product.

[0030] In some embodiments, the scaffold matrix comprises a plurality of chunks, fibers, and / or sheets to mimic salami, pepperoni, chorizo, hue, or other types of processed and / or fermented meat sausages, in which the number of pieces can be between 10 and 10,000 or more.

[0031] In some embodiments, the scaffold substrate comprises chunks, fiber pieces, and / or sheet pieces, for example to mimic whole cuts such as chicken breast or beef tenderloin, etc. In those embodiments, the number of pieces can be between 1 and 100, preferably between 1 and 20, and preferably between 1 and 10.

[0032] In some embodiments, the scaffold matrix is ​​formed by any texturing process, which may be high moisture extrusion cooking (HMEC) or shear cell (SC) processing, in which proteins are melted under high temperature, pressure, and moisture content of 40-80%, and then cooled under shear, resulting in the formation of a solidified fibrous structure.

[0033] In some embodiments, the scaffold matrix is ​​formed by an enzymatic reaction: proteins can be cross-linked to form aggregates by enzymes such as transglutaminase.

[0034] In some embodiments, the scaffold matrix comprises a plant protein coated with another layer, such as agar, so that the fungus can grow inside the coating and form a fungal-based skin.

[0035] In a preferred embodiment, the scaffold substrate is a textured vegetable protein (TVP) substrate. TVP substrates are formed using an extrusion process in which a pressurized molten protein mixture exits an extruder; a sudden drop in pressure causes it to rapidly expand into a sponge-like structure. This sponge-like structure provides pores or channels that allow microbial species to grow not only on the surface but also within the substrate, receiving sufficient oxygen and nutrients. The TVP substrate may have any shape, including chunks, fibers, or sheets. The TVP may be dried after extrusion, resulting in a lower moisture content and higher solids (dry matter) and protein content, or the TVP substrate may be only partially dried or not dried at all before further processing.

[0036] The substrate, particularly the TVP substrate, typically contains more than 40 wt% protein by dry matter, and in preferred embodiments at least 60 wt% protein by dry matter, more preferred embodiments at least 60%, and even more preferred embodiments at least 70 wt% protein by dry matter.

[0037] The TVP substrate is rehydrated prior to incubation, so that the relative weight percentage of protein in the total substrate can be reduced by adding water.

[0038] In a preferred embodiment, the TVP substrate comprises one or more protein sources derived from plant-based sources, including pulses or legumes such as soybeans, peas, broad beans, and beans; oilseeds such as rapeseed or sunflower; grains such as wheat or oats; or any other source. The TVP substrate may comprise a mixture of different protein sources or a single protein source unblended with other proteins. In a further preferred embodiment, the TVP comprises a blend of soy protein and pea protein. In a further preferred embodiment, the TVP comprises a blend of rice protein and pea protein. In a further preferred embodiment, the TVP comprises a blend of at least two plant-based protein sources, preferably at least one of which is soybean or pea. In a further preferred embodiment, the TVP comprises pea as the sole protein source, including both protein and starch. In another embodiment, the TVP comprises other non-slaughtered ingredients derived from unicellular organisms, cellular agriculture, algae, plants, or fungi.

[0039] In a preferred embodiment, the scaffold matrix is ​​made from several pieces of textured vegetable protein (TVP), which can be chunks, fibers (including thin TVP filaments, also known as soy floss, and spun soy fibers made from these same soy floss), and / or sheets.

[0040] In another embodiment, the scaffold matrix may be made of any type of edible textured vegetable protein product or combination thereof.

[0041] The arrangement of these pieces may be random; in other words, the pieces of the scaffolding substrate may not be oriented in a particular direction or preferential position, or conversely, they may be arranged to form a relatively organized structure, whether in one, two, or three directions in space. For example, sheets may be stacked on top of each other to form a multi-layered food product to improve texture.

[0042] Preferably, pressure or vacuum is applied to the textured protein pieces to form the scaffold matrix. Pressure can be applied manually or mechanically. In this embodiment, growth of filamentous fungal species on the interior and exterior of the scaffold matrix during incubation 130 results in, for example, bonding of the different pieces of the scaffold matrix together. Incubation 130 is discussed in more detail below.

[0043] During inoculation 120, the scaffold substrate is inoculated with at least two microbial species, the at least two microbial species including at least one filamentous fungal species. Inoculation may be performed by any means, such that further subsequent steps may be required to obtain successful development of the microbial species within and / or around the substrate.

[0044] Inoculation 120 can be performed using an inoculum. The inoculum contains a certain amount of colony-forming units (CFUs) of a microbial species, which can be spores, active vegetative mycelia, and active fungal tissue portions; it may also include dormant mycelia and fungal tissue portions, e.g., dehydrated or freeze-dried mycelia and fungal tissue portions. Colony-forming units (CFUs) refer to the number of microbial cells (such as bacteria, spores, active vegetative mycelia, and active fungal tissue portions) that are viable under controlled conditions and capable of multiplying, e.g., by binary fission. The inoculum is introduced into and / or onto a larger volume of scaffold substrate to initiate growth of the microbial species on the substrate. The inoculum can take different forms, such as spores, mycelial fragments, or liquid or solid culture suspensions, and can contain both fungal growth and reproductive structures and the inoculum inoculation substrate. The inoculum substrate may be grain, vermiculite, sawdust or other suitable powdered or granular substrate to ensure a large number of colony forming units when the inoculum is mixed with the final substrate.

[0045] The number and composition of colony-forming units in the inoculum can vary depending on the desired results and the characteristics of the microbial species used. In some embodiments, the inoculum is introduced directly into and / or onto the substrate to deliver the microbial species both on the surface and / or inside. In other embodiments, the microbial species and / or spores are in aqueous suspension, and the inoculum is introduced into and onto the substrate to deliver the microbial species not only on the surface but also inside. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. The fungi are preferably selected from the group consisting of Ascomycetes, Basidiomycetes, Fungi Imperfecti, Oomycetes, and / or Zygomycetes, especially edible species belonging to the following genera: Rhizopus, Aspergillus, Penicillium, Ganoderma, Sporidiobolus, Staphylococcus, or Pleurotus. More specifically, the following species: Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or combinations thereof.

[0046] In some embodiments, the first microbial species is a filamentous fungal species including the genus Penicillium, preferably Penicillium nalgiovense, and the second microbial species is a filamentous fungal species including the genus Rhizopus, preferably Rhizopus oligosporus. Inoculation of the first filamentous fungal species can be carried out by spreading colony-forming units on the scaffold substrate. In this embodiment, the colony-forming units are spores of each filamentous fungal species. The first spore concentration of the first filamentous fungal species is 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 12 , preferably 1 x 10 6~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second spore concentration of the second filamentous fungal species is in the range of 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 Preferably, the ratio of the first spore concentration to the second spore concentration is in the range of 0.01-1000, preferably 0.1-500, more preferably 50-150.

[0047] In some other embodiments, the first microbial species is a filamentous fungal species including the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a filamentous fungal species including the genus Aspergillus, preferably Aspergillus oryzae. Inoculation of the first filamentous fungal species can be carried out by spreading colony-forming units on the scaffold substrate. In this embodiment, the colony-forming units are spores of the respective filamentous fungal species. The first spore concentration of the first filamentous fungal species is 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 12 , preferably 1 x 10 6 ~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second spore concentration of the second filamentous fungal species is in the range of 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 Preferably, the ratio of the first spore concentration to the second spore concentration is in the range of 0.01-2000, preferably 0.1-1000, more preferably 20-500.

[0048] During incubation 130, the scaffold substrate is incubated. The microbial species inoculated during inoculation 120 grow and develop in and on the substrate. To promote the growth of the microbial species, this step is carried out under controlled parameters such as temperature, humidity, oxygen concentration, and / or time. Preferably, the incubation conditions are adjusted to avoid the production of ammonia, the accumulation of organic acids, and / or the formation of unpleasant off-flavors that occur prior to sporulation.

[0049] Incubation 130 may be carried out by any means, so that further subsequent steps may be required to obtain good development of the microbial species within and / or around the substrate. Incubation 130 may be, for example, liquid fermentation or, more preferably, solid-state fermentation (SSF). Fermentation conditions and times may be adjusted depending on the microbial species, available nutrients, composition of the substrate, and the desired results. Preferably, the scaffold is incubated at a temperature between 4 and 70°C, particularly between 10 and 50°C, more preferably between 14 and 40°C, and more preferably between 22 and 38°C.

[0050] Preferably, incubation 130 is terminated before sporulation occurs.

[0051] Microbial growth can be interrupted by any means, such as by changing the temperature and / or water activity to below or above the temperature and water activity conditions required for microbial growth. Alternatively, microbial growth can be interrupted by reducing the oxygen concentration below the critical level required for the respective microbial species to grow. Preferably, growth of filamentous fungal species is interrupted by heating the food product to above 60°C, preferably above 71°C, for at least 1 minute, more preferably for more than 30 minutes, with the temperature measured at the center of the product.

[0052] Alternatively, microbial growth is not interrupted. Instead, the food is stored in a refrigerator or freezer until consumption, preferably prior to sporulation or spoilage.

[0053] FIG. 2 presents another embodiment for forming a food product, comprising step 110 of providing a scaffold substrate, step 120a of inoculating the scaffold substrate with at least a first microbial species, step 130a of incubating the scaffold substrate under optimal conditions to grow the first microbial species, step 120b of inoculating at least a second microbial species, preferably the second microbial species being a filamentous fungal species, and step 130b of incubating the scaffold substrate under optimal conditions to grow the second microbial species.

[0054] In some embodiments, inoculation may be carried out in successive steps of a first inoculation 120a of the scaffold substrate with a first microbial species, followed by a second inoculation 120b of the scaffold substrate with a second microbial species, preferably a filamentous fungal species.

[0055] In some embodiments, incubation 130 may be performed in successive steps, for example, a first incubation 130a and a second incubation 130b, where the two incubations have different parameters for growing the microbial species.

[0056] In inoculation 120a, the scaffold substrate is inoculated with at least one microorganism. Inoculation may be performed by any means, which may result in the need for further subsequent steps to obtain good growth of the microbial species within and / or around the substrate.

[0057] Inoculation 120a can be performed using an inoculum. The inoculum contains a quantity of colony-forming units (CFUs) of the microbial species, which can be spores, active vegetative mycelia, and active fungal tissue portions. It may also include dormant mycelia and fungal tissue portions, e.g., dehydrated or freeze-dried mycelia and fungal tissue portions. The inoculum is introduced into and / or onto a larger volume of scaffold substrate to initiate growth of the microbial species on the substrate. The inoculum can take different forms, such as spores, mycelial fragments, or liquid or solid culture suspensions, and can contain both fungal growth and reproductive structures as well as the inoculum substrate. The inoculum substrate can be grain, vermiculite, sawdust, or other suitable powdered or granular substrate, ensuring a large quantity of colony-forming units when the inoculum is mixed with the final substrate. The size and composition of the inoculant can vary depending on the desired results and the characteristics of the microbial species used. In some embodiments, the inoculant is introduced directly into and / or onto the substrate to deliver the microbial species both on the surface and / or inside. In other embodiments, the microbial species and / or spores are in aqueous suspension, and the inoculant is introduced into and onto the substrate to deliver the microbial species not only on the surface but also inside. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. The fungi are preferably selected from the group consisting of Ascomycetes, Basidiomycetes, Fungi Imperfecti, Oomycetes, and / or Zygomycetes, especially edible species belonging to the following genera: Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococcus, Sporidiobolus, or Pleurotus. More specifically, the following species are inoculated: Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or combinations thereof. In some embodiments, Penicillium nalgiovense, Lactobacillus, Sporidiobolus pararoseus, Aspergillus sojae, or Saccharomyces are inoculated.

[0058] The first incubation 130a of the scaffold substrate allows a first microbial species to grow inside, outside, or inside and outside the scaffold substrate. Incubation 130a may be performed by any means, and as a result, additional subsequent steps may be required to obtain successful development of the microbial species within and / or around the substrate. Incubation 130a may be, for example, liquid fermentation or, more preferably, solid-state fermentation (SSF). Fermentation conditions and time may be adjusted depending on the microbial species, available nutrients, substrate composition, and desired results. Preferably, incubation conditions are adjusted to avoid ammonia production, accumulation of organic acids, and / or the formation of unpleasant off-flavors that occur prior to sporulation.

[0059] During inoculation 120a, the inoculated microbial species grow and develop within and on the scaffold substrate. To facilitate the growth of the microbial species, this step is carried out under controlled parameters such as temperature, humidity, oxygen concentration, and / or time. Incubation 130a can be carried out for a time period of less than 72 hours, preferably less than 48 hours, at a temperature between 5 and 50°C, preferably between 10 and 40°C, and preferably between 20 and 30°C.

[0060] In one embodiment, incubation 130a is terminated before sporulation. Microbial growth may be interrupted by any means, such as by changing the temperature and / or water activity to below or above the temperature and water activity conditions required for growth of the first microbial species. Alternatively, microbial growth may be interrupted by reducing the oxygen concentration below a critical level required for microbial species growth. Alternatively, growth of the first microbial species may be interrupted by heating the food product to above 60°C, preferably above 71°C, for at least 1 minute, more preferably for more than 30 minutes, with the temperature measured at the center of the product.

[0061] In a preferred embodiment, the growth of the microbial species is not inhibited and the microbial species is allowed to continue growing until the end of incubation 130b.

[0062] During inoculation 120b, the scaffold substrate is inoculated a second time with at least one microbial species, including at least one filamentous fungal species. Inoculation 120b may be performed in any manner, which may result in further subsequent steps being required to obtain good development of the microbial species within and / or around the substrate.

[0063] Inoculation 120b can be performed using an inoculum. The inoculum contains a certain amount of colony-forming units (CFUs) of the microbial species, which can be spores, active vegetative mycelia, and active fungal tissue portions. It may also include dormant mycelia and fungal tissue portions, such as dehydrated or freeze-dried mycelia and fungal tissue portions. The inoculum is introduced into and / or onto a larger volume of scaffold substrate to initiate growth of the microbial species on the substrate. The inoculum can take different forms, such as spores, mycelial fragments, or liquid or solid culture suspensions, and can contain both fungal growth and reproductive structures as well as the inoculum substrate. The inoculum substrate can be grain, vermiculite, sawdust, or other suitable powdered or granular substrate, ensuring a large amount of colony-forming units when the inoculum is mixed with the final substrate.

[0064] The size and composition of the inoculant can vary depending on the desired results and the characteristics of the microbial species used. In some embodiments, the inoculant is introduced directly into and / or onto the substrate to deliver the microbial species both to the surface and / or interior. In other embodiments, the microbial species and / or spores are in aqueous suspension, and the inoculant is introduced into and onto the substrate to deliver the microbial species not only to the surface but also to the interior. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. The fungi are preferably selected from the group consisting of Ascomycetes, Basidiomycetes, Fungi Imperfecti, Oomycetes, and / or Zygomycetes, especially edible species belonging to the following genera: Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococcus, Sporidiobolus, or Pleurotus. More specifically, the following species are inoculated: Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or combinations thereof. In some embodiments, Rhizopus oligosporus is inoculated. In some embodiments, Aspergillus oryzae is inoculated.

[0065] The second incubation 130b of the scaffold substrate allows a second microbial species to grow inside, outside, or inside and outside the scaffold substrate. During incubation 130b, the scaffold substrate is incubated. Growth conditions are optimized for the second microbial species. Thus, the second microbial species grows and develops within and on the substrate. The second microbial species forms a matrix within and around the scaffold substrate, binding the scaffold substrate together. In one embodiment, a filamentous fungal species forms a network that binds itself to the substrate material. To promote the growth of the selected microbial species, this step is carried out under controlled parameters, such as temperature, humidity, oxygen concentration, and / or time. The parameters should be adapted to maximize the growth of the second microbial species; these conditions may be the same or different from those used in the first incubation 130a. Thus, the growth of the microbial species inoculated during inoculation 120a may or may not continue.

[0066] Incubation 130b may be carried out by any means, and as a result, further subsequent steps may be required to obtain good growth of the microbial species within and / or around the substrate. Incubation 130b may be, for example, liquid fermentation or, more preferably, solid-state fermentation (SSF). Fermentation conditions and time may be adjusted depending on the microbial species, available nutrients, substrate composition, and desired results. Incubation 130b may be carried out for a time of less than 72 hours, preferably less than 44 hours, at a temperature between 20 and 45°C, preferably between 28 and 35°C.

[0067] Preferably, incubation 130b is terminated before sporulation occurs. Preferably, incubation conditions are adjusted to avoid the formation of ammonia, accumulation of organic acids, and / or unpleasant off-flavors that may result from sporulation.

[0068] Microbial growth may be interrupted by any means, such as by changing the temperature and / or water activity to below or above the temperature and water activity conditions required for growth of at least one filamentous fungal species. Alternatively, growth of the filamentous fungal species may be interrupted by reducing the oxygen concentration below the critical level required for growth of each filamentous fungal species. Preferably, growth of the filamentous fungal species is interrupted by heating the fungus-containing product to above 60°C, preferably above 71°C, for at least 1 minute, more preferably for more than 30 minutes, the temperature being measured at the center of the product.

[0069] Alternatively, microbial growth is not interrupted. Instead, the food is stored in a refrigerator or freezer until consumption, preferably prior to sporulation or spoilage.

[0070] It is understood that other iterations of the inoculation and / or incubation steps can be added to allow for the presence of more bacterial species with additive effects between them. It is also understood that more than one microbial species can be inoculated in any inoculation step. In particular, it is understood that Penicillium nalgiovense and Sporidiobolus pararoseus can be inoculated during the first inoculation 120a, Rhizopus oligosporus can be inoculated during the second inoculation 120b, and Aspergillus oryzae can be inoculated during the third inoculation. In this example, Penicillium nalgiovense allows Rhizopus oligosporus to grow significantly, and Sporidiobolus pararoseus allows Aspergillus oryzae to grow significantly. Penicillium nalgiovense and Sporidiobolus pararoseus grow during the first incubation 130a, while a second incubation 130b under conditions optimal for Rhizopus oligosporus can bind all the pieces of the scaffold substrate together, and a third incubation under conditions optimal for Aspergillus oryzae can create a fungal-based epidermis. The fungal-based epidermis should be present on the surface of the scaffold substrate with a dense mycelium covering the substrate with a thickness between 0.1 and 5 mm.

[0071] In another embodiment, the scaffold substrate is shaped before, during and / or after any incubation step. The purpose of shaping can be multiple, for example, shaping can be used to give the final product its shape.

[0072] Molding can also be used to form a fungal-based skin on the surface of the final product. Indeed, growth of filamentous microbial species (or filamentous fungi) can occur on the outer surface of the scaffold substrate and can grow beyond the scaffold substrate in volume. By compressing these filaments onto the surface of the scaffold substrate, a compact layer created primarily by the filaments can be obtained, creating a fungal-based skin on the surface of the product. It is understood that method 100 can be used several times, using the final food product from a previous method 100 as a scaffold substrate for subsequent uses of method 100 to create different versions of a final product that optimally mimics a piece of meat or meat product. For example, a first application of method 100 to bond several scaffold substrates together, followed by a second application of method 100 to create a fungal-based skin on the surface of the final product.

[0073] In some embodiments, the scaffold substrate may be prepared prior to inoculation 120 or inoculation 120a. During the step of preparing the scaffold substrate, the scaffold substrate may be hydrated with an aqueous solution and treated to reduce and / or eradicate the number of other microbial species present inside and / or outside the scaffold substrate that may constitute a source of contaminants. The water activity of the scaffold substrate is adjusted to conditions desirable for microbial growth, preferably a water activity between 0.8 and 1.0, more preferably between 0.9 and 1.0, and even more preferably between 0.96 and 1.

[0074] In some embodiments, the aqueous solution consists solely of water. The ratio of the mass of the substrate to the mass of water may be in the range of 1:1 to 1:2, more preferably 1:1.5 to 1:1.8.

[0075] In other embodiments, the aqueous solution may be composed not only of water but also of any compounds useful for growing microbial species, such as colorants, flavors, nutrients, and / or any compounds that stop the growth and / or eradicate other undesirable microbial species.

[0076] Hydration is a growth condition for microbial species to develop and grow within the substrate. This also applies to undesirable microbial species. To reduce the number of microbial species present, the scaffold substrate can be pasteurized, double pasteurized, sterilized, treated with antimicrobial compounds, and / or treated with acid. For example, the scaffold substrate can be sterilized in an autoclave at up to 120°C. Another example is acidification, in which the surface of the scaffold substrate is treated with an acid, preferably a food-grade acid, more preferably lactic acid, acetic acid, malic acid, citric acid, or succinic acid, to reach a pH of less than 6 at the surface, most preferably between 4.5 and 4.6. Even more preferably, the pH on the surface is adjusted to ensure the growth of the microbial species used and reduce the growth of other microorganisms.

[0077] In another embodiment, the scaffold substrate and aqueous solution are processed separately and then contacted to hydrate the scaffold substrate.

[0078] In another embodiment, the scaffold substrate is shaped before, during, and / or after hydration and / or sterilization. The purpose of shaping can be multiple, for example, shaping can be used to increase the contact surface between the scaffold substrate and air. In another embodiment, shaping can be performed after any inoculation to incorporate spores and / or vegetative forms of microbial species and / or to give the final product its shape.

[0079] In another embodiment, food products are developed to mimic meat products: they may be shaped to replicate the shape of the meat product they are replacing for ease of recognition and identification by consumers.

[0080] In particular, the food product is shaped to closely resemble the size, thickness, and overall shape of the meat product it is imitating. For example, if the meat product being imitated is a whole cut, the food product will have a shape similar in size and thickness to a conventional whole cut of meat. Similarly, if the meat product being imitated is salami or sausage, the food product will have a shape similar in size and thickness to a conventional salami or sausage.

[0081] The food product also takes into account any unique characteristics of the meat product being imitated. For example, if the meat product has a distinctive texture or pattern on its surface and / or interior, the food product is designed to replicate that texture and pattern as accurately as possible.

[0082] In another embodiment, the food product may be formed using a variety of methods to achieve the desired shape, texture, and appearance. One such method is vacuum forming, which involves placing the food product in a mold and applying vacuum pressure to shape the product.

[0083] Another method of shaping food products is mechanical shaping, which involves using mechanical forces to shape the product. This can be accomplished through the use of presses, extruders, or other similar devices. Mechanical shaping can be particularly useful for shaping food products that require a high degree of consistency and uniformity in the shape and size of the food product.

[0084] Further methods of shaping food products can include molding, casting, and cutting, among others. Molding involves pouring the food product into a mold and allowing it to cool and solidify, while casting involves a similar process but with a liquid or semi-liquid food product. Cutting can be used to create precise shapes and sizes of food products, such as slices or cubes.

[0085] It should be noted that the shaping methods are not limited to those described herein, and other methods and variations of the above methods may be used as well. The particular shaping method used will depend on various factors, such as the type of food product, the desired shape and texture, and the manufacturing process used to produce the food product.

[0086] In another embodiment, the scaffold substrate is placed inside a shell larger than the substrate, creating a free space between the substrate and the inner surface of the shell. The fungal mycelium at least partially fills the free space, forming a fungal-based skin around the scaffold substrate. The free space may be between 0.1 mm and 5 cm thick. The substrate may be held in place by any means, such as a rod connected to the shell and penetrating the substrate, and the shell may also have one or more supports on which the substrate is placed.

[0087] The food product comprises at least two distinct phases, preferably the two distinct phases are distinguishable by the human eye, more preferably a first phase comprises a scaffold substrate and a second phase comprises mycelium of at least one filamentous fungal species, preferably wherein the ratio of the second phase of the food product, defined as the ratio of the area occupied by the second phase in a cross-section of the food product to the total area of ​​said cross-section, is in the range of 0.001 to 0.95, more preferably 0.1 to 0.6.

[0088] Once the food product has been formed according to the method 100 described above, further processing may be performed to improve taste, shelf life, and / or transportability. In particular, the food product may be marinated, spiced, smoked, aged, dehydrated, pressed, injected with water and / or oil, steamed, boiled, or post-processed in any other manner as is typically done with animal-based meat products.

[0089] The food products are designed to provide the consumer with a satisfying and juicy sensation similar to that of conventional meat products, for example, by releasing meat juices when chewed or cut, further enhancing the overall sensation of juiciness.

[0090] In one embodiment, the fermented food is more nutritious than the edible substrate, preferably where the proteins in the edible substrate are more digestible than before fermentation, and fermentation reduces antinutritional factors and improves the bioavailability of micronutrients. Some non-limiting possible examples are higher antioxidant activity, higher total phenolic content, increased soluble protein, higher ellagic acid (anticarcinogenic), increased L-DOPA content, and / or reduced cholesterol.

[0091] The food product can also be used to make other products, including sausages, which can be produced by adding a thin layer of fungus around the finished food product to form a fungus-based food product with a fungus-based skin. This thin layer can be produced, for example, by liquid fermentation, then collected, processed, and finally deposited all around the finished food product. This thin layer can have a thickness of between 0.1 mm and 5 mm and can be composed of at least one fungus species.

[0092] In one embodiment, the food product contains DNA and RNA from each microbial species inoculated during method 100. The presence and genetic identity of at least two microbial species in the food product can be determined by analysis of the final product via polymerase chain reaction (PCR) amplification of specific gene fragments, more preferably by qPCR-analysis, and in combination with gene sequencing (e.g., Sanger sequencing). The amount of microbial DNA and / or RNA from each species inoculated during method 100 is expected to exceed 0.0001% of the total product mass.

[0093] In another embodiment, the food product can contain a majority amount of microbial DNA and / or RNA from the microbial species inoculated during method 100. The amount of DNA and / or RNA from the inoculated microbial species is expected to be more than 10 times the amount of DNA and / or RNA of other microbial species. [Example]

[0094] Example 1 Mycelial growth and attachment was observed on TVP pieces fermented with either Rhizopus oligosporus, Penicillius nalgiovense or a combination thereof.

[0095] Pea protein-based TVP pieces with sizes between 4 and 15 mm were hydrated with 170 g of water and 2 g of lactic acid per 100 g of TVP product and pasteurized at 90 °C for 30 min in a vacuum package. Three series, A, B, and C, were produced from the prepared TVP pieces. Series A: 1.2 x 10 per kg of substrate 7 Single-species monoculture incubations were initiated by inoculation with Rhizopus oligosporus at a spore concentration of 1 spore. Series B: 9 x 10 per kg of substrate 8 Single-species monoculture incubations were initiated by inoculation with Penicillium nalgiovense at a spore concentration of 1000 spores. Series C, 1.2 x 10 per kg of substrate 7 Rhizopus oligosporus at a spore concentration of 9 x 10 spores per kg of substrate 8 Co-culture incubation of the two species was initiated by inoculation with Penicillium nalgiovense at a spore concentration of 10 ...

[0096] TVP pieces were inoculated by evenly covering the surface of the TVP pieces with the powdered starter culture through mechanical mixing. 30 g of the inoculated TVP pieces were placed in a breathable Petri dish and incubated in an incubator at 30°C and 98% relative humidity for 18.5 hours. The fermented TVP pieces were then photographed and observed under a microscope. As can be seen in Figure 5, after incubation, the TVP pieces were covered with aerial mycelia, which formed a fluffy-looking mycelial network on the TVP surface. Figures 3 and 6 show the incubation results for Series A. Figures 4, 7, and 9 show the incubation results for Series B. Figures 5 and 8 show the incubation results for Series C. The fungal mycelia formed in the monoculture incubations of Series A and B, shown in Figures 3 and 6, and Figures 4, 7, and 9, respectively, were less dense than the fungal mycelia formed in the coculture incubations of Series C, shown in Figures 5 and 8. Thus, higher mycelium densities are achieved by co-culture, which leads to improved texture control in the food products produced from that mycelium density. This example demonstrates how desirable growth characteristics such as mycelium density and more rapid growth can be promoted by co-culture incubation.

[0097] Example 2 Bodies consisting of individual TVP pieces bound to mycelium in co-culture or monoculture were prepared and compared for mycelium binding strength in tensile tests. 100 g of pea protein-based TVP pieces, measuring between 4 and 15 mm, were hydrated with 170 g of water and 5 g of 80% lactic acid solution and pasteurized at 92°C for 30 minutes in a vacuum package. Three series, A, B, and C, were prepared from the prepared TVP pieces. Series A: 1.2 x 10 per kg of substrate 7 Rhizopus oligosporus at a spore concentration of 9 x 10 spores per kg of substrate 8 Co-culture incubation of the two species was initiated by inoculation with Penicillium nalgiovense at a spore concentration of 10 ... Series B: 1.2 x 10 per kg of substrate 7 Single-species monoculture incubations were initiated by inoculation with Rhizopus oligosporus at a spore concentration of 1 spore. Series C, 9 x 10 per kg of substrate 8 Single-species monoculture incubations were initiated by inoculation with Penicillium nalgiovense at a spore concentration of 1000 spores.

[0098] The TVP pieces were inoculated by uniformly covering the surface of the TVP pieces with a powdered starter culture through mechanical mixing. The inoculated TVP pieces from Series A, B, and C were then molded into cylindrical bodies measuring 20 mm in diameter and 180 mm in length, with perforated polyethylene film attached to the outside. The cylindrical bodies were incubated in an incubator at 30°C and 95% relative humidity for 32 hours. Although the TVP pieces were held together by the polyethylene film before incubation, the pieces from Series A and B adhered to each other after incubation due to hyphal growth fed by available nutrients within the pieces. Series C did not show any bonding because the inoculated Penicillium nalgiovense hyphae were too short to bridge individual TVP pieces (see Figures 7 and 9). The polyethylene film was then removed, and the cylindrical bodies were vacuum-packaged and heat-treated at 90°C for 20 minutes. Rectangular pieces measuring 11 × 17 × 50 mm were cut from the cylindrical bodies. The rectangular pieces were then tensile tested in a ZwickRoell 2.5kN zwicki RetroLine Texture Analyzer with a 10m gap setting, a 0.1N preload, and an extension rate of 200mm / min. The samples were elongated along the center until fracture. The samples fractured along the mycelium layer that bound the individual TVP pieces together. It can be assumed that the cross-sectional area remained approximately constant during the tensile test. The stress was calculated by dividing the force (N) applied to separate the samples by the cross-sectional area. The results are displayed in a stress-strain diagram, as can be seen in Figure 10. In the case of Series C with Penicillium nalgiovense, bonding of the TVP pieces into a cohesive body was not achieved after incubation. For this reason, Series C could not be fixed in the experimental setup and was not tensile tested. Therefore, the stress response could not be measured. The mechanical response of Series C is representatively shown in Figure 10. The fractured areas of Series A are marked with dashed rectangles designated by "a", the fractured areas of Series B are marked with dashed rectangles designated by "b", and the representative fractured areas of Series C are marked with dashed rectangles designated by "c".The results show that pieces bound in co-culture incubations of two species of Rhizopus oligosporus and Penicillium nalgiovense in Series A broke at a higher stress than pieces bound in mono-culture incubations of one species of Rhizopus oligosporus in Series B or one species of Penicillium nalgiovense in Series C. When the breaking stresses of mono-culture incubations Series B and C are added together, they remain less than the breaking stress of Series A. Thus, this example demonstrates that combining two strains in a co-incubation results in more pronounced growth of Rhizopus oligosporus, resulting in increased binding capacity. Increased binding capacity can be used to adjust food texture.

[0099] Example 3 To create the prototype, TVP pieces were hydrated with distilled water and lactic acid to achieve a moisture content of 60%. The hydrated TVP pieces were then vacuumed and pasteurized in a VWB2 water bath at 95°C for 35 minutes. After cooling to room temperature, the TVP pieces were inoculated with the desired strain under a BioVanguard Green Line laminar airflow. The inoculum and TVP pieces were shaken within the vacuum bag itself to ensure even distribution of spores and raw materials. Several combinations of fungi were inoculated: the first with Rhizopus delemar and Penicillium nalgiovense; the second with Rhizopus oligosporus and Penicillium nalgiovense; the third with Rhizopus delemar and Rhizopus oligosporus; the fourth with Rhizopus delemar, Rhizopus oligosporus, and Penicillium nalgiovense; and two control prototypes inoculated with only Rhizopus delemar or only Rhizopus oligosporus. Six substrate rods were fabricated in 3D-printed molds measuring 14 cm in length, 28 mm in thickness, and 25 mm in width. Each rod shape was filled with 80 g of hydrated, inoculated TVP. After filling the mold, the inoculated TVP was placed in an incubator for fermentation. At the end of the incubation cycle, the prototypes were collected, refrigerated, and used for mechanical testing.

[0100] The tensile strength of the bars was measured using a ZwickiLine Texture Analyzer. The setup can be seen in Figure 11. The bar prototypes were analyzed in a tensile test to measure the ultimate tensile strength (Fmax) of the bar prototypes in megapascals (MPa). The texture analyzer measured the Fmax value by dividing the maximum tensile force by the cross-sectional area of ​​the bar prototype. For analysis, the bar prototypes were secured in the clamps of the texture analyzer with a grip separation of 2.2 mm and an initial clamp separation of 50 mm. The preload was 0.2 Newtons (N) and the extension rate was 0.5 mm / s.

[0101] The results are presented in Figure 12 and show that all combinations containing at least one Rhizopus spp. and Penicillium nalgiovense exhibited an average 30% increase in tensile strength. Thus, these examples demonstrate that some combinations of two strains in co-incubation result in more pronounced growth of the Rhizopus strain, resulting in increased food binding capacity. The present invention is described in the following aspects: 1. A method for preparing a food product, comprising: a) providing a scaffold substrate; b) inoculating the scaffold substrate with at least two microbial species, wherein the at least two microbial species include at least one filamentous fungal species; c) incubating the scaffold substrate to grow at least two microbial species inside, outside, or inside and outside the scaffold substrate; A method comprising: 2. The method of the preceding aspect, wherein at least two microbial species are allowed to grow significantly, preferably resulting from carrying out step c), wherein the significant growth comprises increased mechanical strength, preferably tensile strength or increased mycelial density, or increased tensile strength and increased mycelial density. 3. The method of any of the preceding aspects, wherein the at least two microbial species allow significant growth of at least one filamentous fungal species. 4. The method comprises the step of inoculating the scaffold substrate with a predetermined number of colony forming units of at least one filamentous fungal species;

[0023] Aspect 11. The method of any preceding aspect, wherein the filamentous fungal species grows more densely than when the scaffold substrate is inoculated with only a predetermined number of colony forming units of the filamentous fungal species and incubated in the absence of the other one of the at least two microbial species. 5. The at least two microbial species include a first microbial species and a second microbial species that is a filamentous fungal species; the method comprising the steps of inoculating the scaffold substrate with a first number of colony forming units of a first microbial species and inoculating the scaffold substrate with a second number of colony forming units of a second microbial species; the food product obtained by step c) preferably has a tensile strength immediately after step c) without being subjected to further processing that is greater than the sum of the tensile strengths of the first product and the second product, The first product is inoculating a first corresponding scaffold substrate with a first number of colony forming units of a first microbial species; incubating the first compliant scaffold substrate to grow a first microbial species inside, outside, or inside and outside of the first compliant scaffold substrate; is obtained by The second product is inoculating a second corresponding scaffold substrate with a second number of colony forming units of a second microbial species; incubating the second compliant scaffold substrate to grow a second microbial species inside, outside, or inside and outside of the second compliant scaffold substrate; 10. The method of any preceding aspect, wherein the method is obtained by 6. The method of any of the preceding aspects, wherein the scaffold matrix is ​​made from protein from at least one plant source, preferably one protein source is pea protein, and preferably the scaffold matrix contains at least 50% protein in dry matter, preferably 60% or more protein in dry matter. 7. The method of any of the preceding aspects, wherein the scaffold matrix is ​​made from TVP, preferably wherein the TVP scaffold matrix is ​​made with one protein source being pea protein, preferably wherein the TVP scaffold matrix contains at least 50% pea protein in dry matter, preferably 60% or more pea protein in dry matter. 8. The method of any of the preceding aspects, wherein the scaffold substrate comprises at least one chunk or collection of chunks, preferably the number of chunks in the collection is two or more. 9. The method of the preceding embodiment, wherein the aspect ratio of the chunks, defined as the ratio of the longest dimension to the shortest dimension of each chunk, is 10 or less, 5 or less, 3 or less, or 2 or less. 10. The method of embodiment 8 or 9, wherein the average aspect ratio of the chunks, defined as the aspect ratio of 15 randomly selected chunks, where the aspect ratio is defined as the ratio of the longest dimension to the shortest dimension of each chunk, is 10 or less, 5 or less, 3 or less, or 2 or less. 11. The method of any of the preceding aspects, wherein the scaffold matrix comprises at least one fiber or assembly of fibers, preferably the number of fibers in the assembly is two or more. 12. The method of embodiment 11, wherein the aspect ratio of the fibers, defined as the ratio of the longest dimension to the shortest dimension of each fiber, is 10 or more, or 100 or more, or 1,000 or more, preferably less than 10,000. 13. The method of any of the preceding embodiments, wherein the scaffold substrate comprises at least one sheet or stack of sheets, preferably the number of sheets in the stack is two or more. 14. The method of the preceding embodiment, wherein the aspect ratio of the sheets, defined as the ratio of the longest dimension to the shortest dimension of each sheet, is 10 or more, or 100 or more, or 1000 or more, preferably less than 10,000. 15. The method of any of aspects 8-14, wherein the pieces, fibers and / or sheets are bound together by the growth of at least one filamentous fungal species. 16. The method of any of aspects 8-15, wherein the chunks, fibers, and / or sheets can be arranged in an organized manner, e.g., the longest dimensions of all of the chunks and / or fibers and / or sheets can be aligned along substantially the same direction. 17. The method of any of aspects 8-15, wherein the chunks and / or sheets may be arranged in an unorganized manner, e.g., wherein the longest and / or shortest dimensions of all of the chunks and / or fibers and / or sheets are not aligned along substantially the same direction. 18. The method of any of the preceding aspects, wherein the other one of the at least two microbial species comprises a fungal species, an algal species and / or a bacterial species, preferably a fungal species, more preferably a filamentous fungal species. 19. The method of any of the preceding aspects, wherein the first microbial species is a first filamentous fungal species comprising the genus Penicillium, preferably Penicillium nalgiovense, and the second microbial species is a second filamentous fungal species comprising the genus Rhizopus, preferably Rhizopus oligosporus and / or Rhizopus delemar. 20. A first colony-forming unit concentration of a first filamentous fungal species is 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 12 , preferably 1 x 10 6 ~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second colony forming unit concentration of the second filamentous fungal species is in the range of 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 The method of the preceding aspect, wherein the 21. The method according to the preceding aspect, wherein the ratio of the first colony forming unit concentration to the second colony forming unit concentration is in the range of 0.01-1000, preferably 0.1-500, more preferably 50-150. 22. The method of any of the preceding aspects, wherein the first microbial species is a first filamentous fungal species comprising the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a second filamentous fungal species comprising the genus Aspergillus, preferably Aspergillus oryzae. 23. A first colony-forming unit concentration of a first filamentous fungal species is 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 12 , preferably 1 x 10 6 ~1×10 10 , more preferably 1 × 10 8 ~1×10 9 and a second colony forming unit concentration of the second filamentous fungal species is in the range of 1 x 10 spores per kg of scaffold substrate. 4 ~1×10 13 , preferably 1 x 10 5 ~1×10 10 , more preferably 1 × 10 7 ~1×10 8 The method of the preceding aspect, wherein the 24. The method according to the preceding aspect, wherein the ratio of the first colony forming unit concentration to the second colony forming unit concentration is in the range of 0.01-2000, preferably 0.1-1000, more preferably 20-500. 25. Step b) is b1) inoculating the scaffold substrate with a first microbial species; and then, b2) inoculating the scaffold substrate with a second microbial species, preferably wherein the second microbial species is a filamentous fungal species; 10. The method of any preceding aspect, comprising: 26. Step c) is c1) incubating the scaffold substrate to grow a first microbial species inside, outside, or inside and outside the scaffold substrate; and then c2) incubating the scaffold substrate to grow a second microbial species inside, outside, or inside and outside the scaffold substrate; 2. The method of claim 1, comprising: 27. The method according to aspect 25 according to aspect 26, wherein step c1) is performed before or after step b2). 28. The method of any one of aspects 25 to 27, wherein the first microbial species comprises a species of the genus Penicillium, Lactobacillus, Sporidiobolus and / or Aspergillus, preferably Penicillium nalgiovense, Lactobacillus plantarum, Sporidiobolus pararoseus, Aspergillus sojae, Aspergillus luchuensis and / or Saccharomyces cerevisiae. 29. The method of any one of aspects 25 to 28, wherein the second microbial species comprises a species of the genus Rhizopus, Penicillium and / or Aspergillus, preferably Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Penicillium nalgiovense and / or Aspergillus oryzae. 30. step c1) is carried out for a time of less than 72 hours, preferably less than 48 hours, at a temperature of 5 to 50°C, preferably 10 to 40°C, preferably 20 to 30°C, and / or A method according to any of aspects 25 to 29, wherein step c2) is carried out for a time period of less than 72 hours, preferably less than 44 hours, at a temperature of 20 to 45°C, preferably 28 to 35°C. 31. Step c) preferably comprises: disposing the scaffold substrate within a shell to form a restricted space between the scaffold substrate and the shell; performing step c) to grow at least two microbial species within the confined space; Thus, forming a fungal-based skin surrounding the scaffold substrate, The method of any of the preceding aspects, wherein the thickness of the restricted space is preferably in the range of 0.1 mm to 5 cm, more preferably 0.5 mm to 2.5 cm, and even more preferably 1 mm to 1 cm. 32. The method of any preceding aspect, wherein the scaffold matrix comprises textured vegetable protein (TVP). 33. The method of any preceding aspect, further comprising sterilizing and / or pasteurizing the scaffold substrate prior to step b). 34. The method of any preceding aspect, further comprising hydrating the scaffold substrate with an aqueous solution during steps b) and / or c), such that the scaffold substrate has a water activity of greater than 0.8. 35. The method of any of the preceding aspects, wherein step c) is carried out under solid-state fermentation conditions. 36. The method of any preceding aspect, further comprising shaping the scaffold matrix before and / or after step b). 37. A food product prepared by the method of any of the preceding aspects. 38. A food product comprising a scaffold matrix and mycelium of at least one filamentous fungal species grown inside, outside, or inside and outside the scaffold matrix, wherein the scaffold matrix is ​​formed by one or more textured vegetable protein (TVP) pieces. 39. The food product of any of the two preceding aspects, wherein the food product comprises at least two distinct phases, preferably the two distinct phases are distinguishable by the human eye, and more preferably a first phase comprises a scaffold substrate and a second phase comprises mycelium of at least one filamentous fungal species. 40. The food product of any of the preceding embodiments, wherein the ratio of the second phase of the food product, defined as the ratio of the area occupied by the second phase in a cross-section of the food product to the total area of ​​said cross-section, is in the range of 0.001 to 0.95, more preferably 0.1 to 0.6. Various aspects, embodiments, or examples of the present invention have been described for illustrative purposes. However, the present invention should not be unduly limited by any of the details of the above disclosure, as those skilled in the art will understand that changes and modifications that do not violate the principles of the present invention may be made and still be encompassed by the present invention. In particular, the present invention should not be construed as being limited to the embodiments described above with reference to the drawings. Rather, the scope of protection of the present invention is determined solely by the appended claims and their equivalents.

Claims

1. 1. A method for preparing a food product, said method comprising: a) providing a scaffold substrate; b) inoculating the scaffold substrate with at least two microbial species, wherein the at least two microbial species comprise at least one filamentous fungal species, preferably a first filamentous fungal species and a second filamentous fungal species; c) incubating the scaffold substrate to grow the at least two microbial species inside, outside, or inside and outside the scaffold substrate; Including, The method, wherein said at least two microbial species allow significant growth of said at least one filamentous fungal species.

2. the method comprising the step of inoculating the scaffold substrate with predetermined colony forming units of the at least one filamentous fungal species; 2. The method of claim 1, wherein the scaffold substrate is inoculated with only the predetermined number of colony forming units of the filamentous fungal species and exhibits more significant growth than when incubated in the absence of the other one of the at least two microbial species.

3. 10. The method of any of the preceding claims, wherein said significant growth results from carrying out step c), said significant growth comprising increased mechanical strength, preferably tensile strength or increased mycelium density, or increased tensile strength and increased mycelium density.

4. the at least two microbial species comprising a first microbial species and a second microbial species that is a filamentous fungal species; the method comprising the steps of inoculating the scaffold substrate with a first number of colony forming units of the first microbial species and inoculating the scaffold substrate with a second number of colony forming units of the second microbial species; wherein the food product obtained by step c) has, preferably immediately after step c) without being subjected to further processing, a tensile strength greater than the sum of the tensile strengths of the first product and the second product, The first product is inoculating a first corresponding scaffold substrate with the first number of colony forming units of the first microbial species; incubating the first compliant scaffold substrate to grow the first microbial species inside, outside, or inside and outside of the first compliant scaffold substrate; is obtained by The second product is inoculating a second corresponding scaffold substrate with the second number of colony forming units of the second microbial species; incubating the second compliant scaffold substrate to grow the second microbial species inside, outside, or inside and outside of the second compliant scaffold substrate; 10. The method of any of the preceding claims, obtained by

5. the scaffold matrix comprises at least one chunk or cluster of chunks, preferably the number of chunks in the cluster is two or more; More preferably, the aspect ratio of each chunk, defined as the ratio of the longest dimension to the shortest dimension of the chunk, is 10 or less, 5 or less, 3 or less, 2 or less.

6. the scaffold matrix comprises at least one fiber or assembly of fibers, preferably the number of fibers in the assembly is two or more; 10. A method according to any preceding claim, wherein the aspect ratio of said fibres, defined as the ratio of the longest dimension to the shortest dimension of each fibre, is 10 or more, or 100 or more, or 1000 or more, preferably less than 10,000.

7. the scaffold substrate comprises at least one sheet or stack of sheets, preferably the number of sheets in the stack is two or more; 10. A method according to any preceding claim, wherein the aspect ratio of said sheets, defined as the ratio of the longest dimension to the shortest dimension of each sheet, is more preferably 10 or more, or 100 or more, or 1000 or more, preferably less than 10,000.

8. 10. The method of any of the preceding claims, wherein the first microbial species is a filamentous fungal species comprising the genus Penicillium, preferably Penicillium nalgiovense, and the second microbial species is a filamentous fungal species comprising the genus Rhizopus, preferably Rhizopus oligosporus and / or Rhizopus delemar.

9. a first colony forming unit concentration of the first microbial species, in terms of spores per kg of the scaffold substrate, of 1 x 10 ４ ~1 x 10 １２ , preferably 1 × 10 ６ ~1 x 10 １０ , more preferably 1 × 10 ８ ~1 x 10 ９ and a second colony forming unit concentration of the second microbial species is in the range of 1 x 10 spores per kg of the scaffold substrate. ４ ~1 x 10 １３ , preferably 1 × 10 ５ ~1 x 10 １０ , more preferably 1 × 10 ７ ~1 x 10 ８ and preferably wherein the ratio of said first colony forming unit concentration to said second colony forming unit concentration is in the range of 0.01 to 1000, preferably 0.1 to 500, more preferably 50 to 150.

10. 10. The method of any preceding claim, wherein the first microbial species is a filamentous fungal species comprising the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a filamentous fungal species comprising the genus Aspergillus, preferably Aspergillus oryzae.

11. a first colony forming unit concentration of the first microbial species, in terms of spores per kg of the scaffold substrate, of 1 x 10 ４ ~1 x 10 １２ , preferably 1 × 10 ６ ~1 x 10 １０ , more preferably 1 × 10 ８ ~1 x 10 ９ and a second colony forming unit concentration of the second microbial species is in the range of 1 x 10 spores per kg of the scaffold substrate. ４ ~1 x 10 １３ , preferably 1 × 10 ５ ~1 x 10 １０ , more preferably 1 × 10 ７ ~1 x 10 ８ and preferably wherein the ratio of said first colony forming unit concentration to said second colony forming unit concentration is in the range of 0.01 to 2000, preferably 0.1 to 1000, more preferably 20 to 500.

12. Step b) b1) inoculating the scaffold substrate with a first microbial species; and then, b2) inoculating the scaffold substrate with a second microbial species, preferably wherein the second microbial species is the filamentous fungal species; 10. A method according to any preceding claim, comprising:

13. Step c) c1) incubating the scaffold substrate to grow the first microbial species inside, outside, or inside and outside the scaffold substrate; and c2) incubating the scaffold substrate to grow the second microbial species inside, outside, or inside and outside the scaffold substrate; Including, Step c1) is performed before or after step b2), Preferably, step c1) is carried out for a time of less than 48 hours at a temperature of 5 to 50°C, preferably 10 to 40°C, more preferably 20 to 30°C; and / or 10. The method according to the preceding claims, wherein step c2) is carried out for a time of less than 72 hours, preferably less than 44 hours, at a temperature of 20-45°C, preferably 28-35°C.

14. 10. A food product prepared by the method of any preceding claim.

15. the food product comprises at least two distinct phases, preferably the two distinct phases are distinguishable by the human eye, more preferably a first phase comprising the scaffold substrate and a second phase comprising the mycelium of the at least one filamentous fungal species, Preferably, 15. The food product of claim 14, wherein the ratio of the area of ​​the second phase of the food product, defined as the ratio of the area occupied by the second phase in a cross-section of the food product to the total area of ​​the cross-section, is in the range of 0.001 to 0.95, more preferably 0.01 to 0.6.

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