Method of making food products using microbial growth and products thereof

Abstract

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

Description

Technical Field

[0001] The present application relates to a method for providing a food product by combining textured vegetable protein matrix pieces with at least one filamentous fungus, wherein the matrix pieces are adhered to each other by the growth of two or more microbial species. Thus, a fungus-containing food product is formed. Background Art

[0002] A variety of methods are currently used to produce meat substitute products, resulting in significant differences in their texture and nutritional value. In fact, few meat substitutes can provide the recommended daily intake of protein while also possessing an appealing texture. In addition, to achieve textured foods similar to meat and / or meat-derived foods, various ingredients can be added to connect small pieces together or impart a specific texture to the product or part of the product.

[0003] Tempeh is a well-known vegetarian product typically made from soybeans fermented with a filamentous fungus, usually Rhizopus oligosporus. In recent years, the term tempeh has been used more broadly to encompass fermented and combined cereals or food processing byproducts, 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 products are less juicy and less flavorful on their own, requiring thick marinades and often being fried, which is the most common cooking method.

[0004] Recently, progress has been made in combining textured vegetable proteins with fungi, as described in multiple patent applications (US2021045410, US2022132893). In these applications, a texturized matrix made from multiple pieces of textured vegetable protein (TVP) is used, bound together by at least one fungus. One of the limiting parameters in the production of mycelium-based foods is the density of the fungal mycelium within the matrix. Fungal growth can be limited by available nutrients, lack of oxygen, and / or heat released during fungal growth, which actively ferments the matrix. Contamination by competing bacteria and fungi can also compromise the mycelial formation process. Poor fungal development within the matrix results in low mycelial density, which reduces fiber content, limits liquid and fat absorption, thereby affecting juiciness and textural complexity during chewing, reduces the strength of the bonds between different matrix components, and increases the population of undesirable microbial species (because the fungi also act as a protective culture). Due to competing microbial contamination and the presence of unfermented compounds and / or antinutrients in the vegetable protein portion of the matrix, the product may have a different color than meat products and / or may develop off-flavor characteristics.

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

[0006] When traditional strains commonly used in well-defined solid-state fermentation (SSF) processes (e.g., in the production of blue cheese, Camembert, tempeh, and koji) are cultivated on new, non-traditional, or unconventional substrates, challenges such as reduced mycelial growth, off-flavor formation, and reduced product safety are often encountered. This is 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.

[0007] The methods presented herein address some of the greatest challenges encountered by the prior art by utilizing the prolific growth of a filamentous fungal species with another microbial species in co-culture and / or continuous culture, which has not been previously disclosed. Summary of the Invention

[0008] Various embodiments of the method for preparing food products using a scaffolding substrate and at least two microbial species (one of which is a filamentous fungus) are described herein. Using at least two microbial species can enable at least filamentous fungus species to grow more abundantly. The method generally includes: providing a scaffolding substrate, such as an organized vegetable protein matrix, the matrix may or may not be subjected to a variety of pretreatment steps for helping to promote the growth of at least two microbial species inside, outside, or inside and outside the scaffolding matrix; inoculating the microbial species into the organized matrix, the microbial species can be inoculated or inoculated separately at the same time; and allowing the microbial species to grow inside, outside, or inside and outside the scaffolding matrix. In some embodiments, filamentous fungus species are used to bond together any discrete particles of a plurality of scaffolding matrices and their interiors to form a larger composite food product. In other embodiments, filamentous fungus species are used to produce a fungus-based epidermis on the surface of a food product.

[0009] The major advantage that is used for inoculation by using at least two microbial species is to promote the growth of filamentous fungi.This is to realize by utilizing the first microbial species to promote and support the better growth conditions of filamentous fungi.For example, the inventor finds that, in preparation and embodiment provided herein, specific filamentous fungi species can grow better than other fungi, algae or bacterial populations, and this may be due to the activation of invasive growth gene and relevant competitiveness and expansive behavior, and these behaviors can stimulate the generation of fungal biomass (fungal biomass).Due to the fermentation of the first microbial species to matrix, it is also possible to realize better growth, and fermentation can produce extra nutrients, and it is easier to obtain and more specific for the growth of the second fungal species.

[0010] Overall, the advantages to food products are numerous. Some non-limiting examples include increasing mycelium density, which can enhance the binding of the scaffold matrix and result in a firmer texture. The final food product described herein may comprise two phases: one comprising the scaffold matrix and the other comprising the mycelium. A higher mycelium density can result in a whiter color of the mycelium-containing phase. Additionally, an increase in mycelium density can improve juiciness because more oil and / or water can be absorbed into the food product, which can be released during chewing, thereby increasing juiciness. Another potential advantage of this method is the ability to customize the taste profile by varying the microbial species and / or incubation conditions.

[0011] In one aspect, the outer surface of a food product can be coated with a fungus-based "skin"-like coating, thereby improving the appearance before and after cooking and enhancing the experience of eating the food product. In another aspect, the interior of the product can have one or more phases, including a scaffold matrix and / or filamentous fungal species, to enhance the experience of eating the food product by providing a more complex texture (similar to whole meat or meat products).

[0012] Another advantage that can be implemented is the reduction of the growth of certain microbial species, such as those that are harmful or potentially harmful to humans or spoilage species. This can be achieved, for example, by the production of antimicrobial metabolites (e.g., organic acids, enzymes, and other antagonistic compounds) and / or competitive exclusion of nutrients and space by one or more inoculant species.

[0013] The final food product produced by the methods presented herein is a fermented food product composed of an edible matrix, wherein the fermented food product is more nutritious than the edible matrix, preferably wherein the protein in the edible matrix is ​​more digestible than before fermentation.

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

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

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

[0017] Furthermore, by carrying out step c) a fertile growth is generated, said fertile growth comprising 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, the second microbial species being a filamentous fungal species, the method comprising inoculating the scaffold matrix with a first colony forming unit concentration of the first microbial species, and inoculating the scaffold matrix with a second colony forming unit concentration of the second microbial species, wherein the food product obtained by step c), preferably obtained immediately after step c) without further processing, has a tensile strength greater than the sum of the tensile strengths of the first product and the second product, the first product being obtained by inoculating a first corresponding scaffold matrix with a first colony forming unit concentration of the first microbial species and incubating the first corresponding scaffold matrix to allow the first microbial species to grow inside, outside, or inside and outside the first corresponding scaffold matrix; and the second product being obtained by inoculating a second corresponding scaffold matrix with a second colony forming unit concentration of the second microbial species and incubating the second corresponding scaffold matrix to allow the second microbial species to grow inside, outside, or inside and outside the second corresponding scaffold matrix.

[0019] According to further non-limiting features of the present invention, employed individually or in any technically feasible combination: The scaffold matrix comprises at least one thick block or a group of thick blocks, preferably, the number of thick blocks in a group is 2 or more, more preferably, the aspect ratio of the thick blocks (which is defined as the ratio of the longest dimension to the shortest dimension of each thick block) is 10 or less, 5 or less, 3 or less, 2 or less. ●The scaffold matrix comprises at least one fiber or a group of fibers, preferably the number of fibers in a group is 2 or more, more preferably, the aspect ratio of the fibers (which is defined as the ratio of the longest dimension to the shortest dimension of each fiber) is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10000. ●The scaffold matrix comprises at least one sheet or a stack of sheets, preferably, the number of sheets in a stack is 2 or more, more preferably, the aspect ratio of the sheets (which is defined as the ratio of the longest dimension to the shortest dimension of each sheet) is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10000. • The first microbial species is a filamentous fungal species comprising Penicillium, preferably Penicillium nalgiovense, and the second microbial species is a filamentous fungal species comprising Rhizopus, preferably Rhizopus oligosporus. The concentration range of the first colony-forming unit of the first microbial species is 1 × 10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second microbial species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 , wherein preferably, the ratio of the first colony forming unit concentration to the second colony forming unit concentration ranges from 0.01 to 1000, preferably from 0.1 to 500, and more preferably from 50 to 150. • 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. The concentration range of the first colony forming unit of the first microbial species is 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second microbial species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 , wherein preferably, the ratio of the first colony forming unit concentration to the second colony forming unit concentration ranges from 0.01 to 2000, preferably from 0.1 to 1000, and more preferably from 20 to 500. ●Step b) includes the following steps o b1) inoculating the scaffold matrix with a first microbial species; then, o b2) inoculating the scaffold matrix with a second microbial species, preferably, the second microbial species is a filamentous fungal species. Step c) includes the following steps o c1) incubating the scaffold matrix to allow a first microbial species to grow inside, outside, or inside and outside the scaffold matrix; and then c2) incubating the scaffold matrix to allow a second microbial species to grow inside, outside, or inside and outside the scaffold matrix, Step c1) is carried out before or after step b2), preferably, wherein step c1) is carried out at a temperature of 5-50°C, preferably 10-40°C, more preferably 20-30°C, for a duration of less than 48 h, and / or step c2) is carried out at a temperature of 20-45°C, preferably 28-35°C, for a duration of less than 72 h, preferably less than 44 h.

[0020] According to the present invention, the food product produced by the method comprises at least two different phases, preferably, the two different phases are distinguishable by the human eye, more preferably, the first phase comprises the scaffold matrix and the second phase comprises mycelia of at least one filamentous fungal species, preferably, wherein the proportion 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 from 0.05 to 0.95, more preferably from 0.1 to 0.6. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart illustrating a method for producing a food product according to various embodiments described herein. FIG. 2 is a flow chart illustrating a method for producing a food product according to various embodiments described herein. FIG.3 is a photograph of TVP fermented with Rhizopus oligosporus. FIG.4 is a photograph of TVP fermented with Penicillium naldigium. FIG.5 is a photograph of TVP fermented with Rhizopus oligosporus and Penicillium nalgiospermum. FIG.6 is a photograph (enlarged) of TVP fermented with Rhizopus oligosporus. FIG.7 is a photograph (enlarged) of TVP fermented with Penicillium naldig. FIG.8 is a photograph of TVP fermented with Rhizopus oligosporus and Penicillium nalgiospermum in co-culture. FIG.9 is a photograph of TVP fermented with Penicillium naldig (zoom 2). FIG. 10 is a stress-strain graph of tensile tests performed on TVP blocks that were either co-cultured with two species of R. oligosporus and P. nalgiospermum or fermented with either R. oligosporus or P. nalgiospermum alone. FIG. 11 is a photograph of a tensile testing apparatus for measuring the tensile strength of a food product, according to one embodiment. FIG. 12 is a graph showing the different tensile strengths of different food products according to various embodiments. Detailed description

[0021] Figure 1 A method 100 for preparing a food product according to an embodiment is shown, comprising the steps of: providing 110 a scaffold matrix; inoculating 120 the scaffold matrix with at least two microbial species, the at least two microbial species comprising at least one filamentous fungal species, preferably a microbial species and a filamentous fungal species; and incubating 130 the scaffold matrix to allow the at least two microbial species to grow inside, outside, or inside and outside the scaffold matrix, wherein the at least two microbial species are capable of enabling the at least one filamentous fungal species to grow more abundantly.

[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-culturing a filamentous fungal species with at least one other microbial species to achieve robust growth of the filamentous fungal species. As used herein, robust growth is defined as an enhancement of at least one characteristic of the mycelium, which may include, but is not limited to, faster growth rate, higher mycelial density of the at least one filamentous fungus, longer mycelium, larger mycelium, or stronger mycelium, compared to growth without the other microbial species. In one embodiment, the at least two microbial species can mutually support each other's growth, thereby achieving robust growth of both microorganisms.

[0024] For food products, when producing meat-like, more specifically muscle-like, fungus-based food products, the effect of their abundant growth can be characterized by an increase in mycelial density and / or an increase in tensile strength. The tensile strength of a food product can be determined by subjecting the food product to a mechanical force at its end and measuring the amount of force required to cause it to break. This value is referred to as the tensile strength of the food product in this article. Various types of mechanical forces can be applied to the food product, including but not limited to stretching, compression, torsion, bending and shearing. Method 100 can also enhance the flavor characteristics, texture, color and / or juiciness of the finished product. In some embodiments, the finished product is more similar to an animal meat product, such as salami, Italian pepperoni or other types of fermented meat sausages. In other embodiments, the finished product is more similar to a whole piece of animal meat, such as pork belly or other meat pieces.

[0025] In the step of providing 110 a scaffold matrix, any matrix suitable for food preparation can be used. In some embodiments, the scaffold matrix is ​​a plant-based protein matrix containing (poly) sugars, fibers, mineral salts, and / or amino acids required for the growth of microbial species. As used herein, "protein" refers to protein isolates, protein concentrates, protein powders, or combinations thereof, which may also contain other macronutrients, such as carbohydrates, fats, dietary fiber, salts, or residual moisture. The isolates, concentrates, powders, or combinations thereof preferably contain at least 40% by weight of pure protein, preferably at least 50% by weight, and more preferably at least 60% by weight. Preferably, the protein also contains sufficient carbohydrates to serve as a nutrient source for the microbial species.

[0026] In one embodiment, the scaffold matrix comprises only one thick block. In another embodiment, the scaffold matrix comprises a plurality of thick blocks, numbering from 2 to 100, or from 100 to 1000 or more. The aspect ratio of the thick blocks, defined as the ratio of the longest dimension to the shortest dimension of each thick block, is 5 or less, 3 or less, or 2 or less.

[0027] In one embodiment, the scaffold matrix comprises only one elongated mass in one direction. In the context of the present invention, the elongated mass may also be referred to as a "fiber." In another embodiment, the scaffold matrix comprises a plurality of fibers, numbering from 1 to 100, or from 100 to 10,000 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 greater, or 10 or greater, or 100 or greater, or 1,000 or greater, and preferably 10,000 or less.

[0028] In another embodiment, the scaffold matrix comprises only one block extending in two directions, which is referred to herein as a sheet. In another embodiment, the scaffold substrate comprises a plurality of sheets, the number of which is from 1 to 100, or from 100 to 1000 or more. The aspect ratio of the sheet, defined as the ratio of the longest dimension to the shortest dimension of each sheet, is 5 or greater, or 10 or greater, or 100 or greater, or 1000 or greater, and preferably 10,000 or less.

[0029] The texture of a food product can be adjusted by varying the number of pieces it comprises while maintaining the same final size. Thus, the number of pieces can be varied significantly, impacting the final texture of the food product. For example, a small number of pieces can produce a texture similar to a whole block, while a large number of pieces can create a texture similar to salami. The choice of piece number, size, and shape can also affect the chewiness, juiciness, and mouthfeel of the final product. Therefore, by adjusting the number, size, and shape of pieces, a wide variety of textures and mouthfeel can be created for a food product.

[0030] In some embodiments, the scaffold matrix comprises a plurality of thick blocks, fibers and / or sheets to simulate salami, pepperoni, chorizo, catalana, or other types of cured and / or fermented meat sausages. In these embodiments, the number of blocks may range from 10 to 10,000 or more.

[0031] In some embodiments, the scaffold matrix comprises large chunks, fibrous chunks and / or sheet chunks to simulate, for example, whole cuts of meat, such as chicken breast or beef tenderloin. In these embodiments, the number of chunks may be 1 to 100, preferably 1 to 20, and more preferably 1 to 10.

[0032] In some embodiments, the scaffold matrix is ​​formed by any texturizing process, which can be high moisture extrusion cooking (HMEC) or shear cell (SC) processing, in which proteins are melted under high temperature, high pressure and a moisture content of 40-80%, and then cooled under shear to form 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-based protein coated with another layer (eg, agar) so that fungi can grow inside the coating and form a fungus-based skin.

[0035] In a preferred embodiment, the scaffold matrix is ​​a textured vegetable protein (TVP) matrix. The TVP matrix is ​​formed using an extrusion process where a pressurized molten protein mixture flows out of an extruder; a sudden drop in pressure causes it to expand rapidly, forming a sponge-like structure. This sponge-like structure provides pores or channels that allow microbial species to grow on and within the matrix and receive sufficient oxygen and nutrients. The TVP matrix can have any shape and can be a thick block, fiber, or sheet. The TVP can be dried after extrusion to reduce the moisture content and increase the solids (dry matter) and protein content, or it can be only partially dried or not dried at all before further processing the TVP matrix.

[0036] The matrix, in particular the TVP matrix, typically comprises more than 40 wt% protein in dry matter, preferred embodiments comprise at least 60 wt% protein in dry matter, more preferred embodiments comprise at least 60 wt% protein, and even more preferred embodiments comprise at least 70 wt% protein in dry matter.

[0037] The TVP matrix can be rehydrated prior to incubation to reduce the relative weight percentage of protein in the overall matrix by adding water.

[0038] In a preferred embodiment, the TVP matrix comprises one or more protein sources derived from plant-based sources, including legumes or legumes, such as soybeans, peas, beans, beans, oilseeds (such as rapeseed or sunflower seeds), cereals (such as wheat or oats) or any other source. The TVP matrix can contain a mixture of different protein sources, or a single protein source that is not mixed with other proteins. In another preferred embodiment, the TVP consists of a blend of soy protein and pea protein. In another preferred embodiment, the TVP consists of a blend of rice protein and pea protein. In another preferred embodiment, the TVP consists of a blend of at least two plant-based protein sources, preferably at least one of which is soybean or pea. In another preferred embodiment, the TVP consists only of peas as a protein source, comprising protein and starch. In another preferred embodiment, the TVP comprises other non-slaughter ingredients from single-cell organisms, cellular agriculture, algae, plants or fungi.

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

[0040] In another embodiment, the scaffold matrix can be made from any variety or combination of edible texturized vegetable protein products.

[0041] The arrangement of these blocks can be random; in other words, the blocks of the scaffold matrix may not be oriented in a specific direction or preferred position, or instead, they can be arranged along one, two, or three directions in space to form a relatively organized structure. For example, these sheets can be stacked on top of each other to form a multi-layered food product to increase texture.

[0042] Preferably, pressure or vacuum is applied to the organized protein mass to form the scaffold matrix. Pressure can be applied manually or by machine. In this embodiment, during incubation 130, the growth of filamentous fungal species within and outside the scaffold matrix, for example, causes the different pieces of the scaffold matrix to bind together. Incubation 130 will be discussed in more detail below.

[0043] The scaffold matrix is ​​inoculated with at least two microbial species, including at least one filamentous fungal species, during inoculation 120. Inoculation can be performed in any manner, and thus additional subsequent steps may be required to ensure good growth of the microbial species within and / or around the matrix.

[0044] Inoculation 120 can be performed by an inoculum. The inoculum contains a certain amount of colony forming units (CFU) of a microbial species, which can be spores, active vegetative hyphae and active parts of fungal tissue, and can also include dormant hyphae and parts of fungal tissue, such as dehydrated or freeze-dried hyphae and parts of fungal tissue. Colony forming units (CFU) refer to the number of microbial cells (bacteria, spores, active vegetative hyphae and active parts of fungal tissue, etc.) that are alive and can be multiplied under controlled conditions (e.g., by binary fission). The inoculum is introduced into and / or onto a larger volume of the scaffold matrix to initiate the growth of the microbial species on the matrix. The inoculum can take different forms, such as spores, mycelial fragments, fluid or solid culture suspensions, and may contain the vegetative and reproductive structures of the fungus and a spawning substrate. The spawning substrate can be grains, vermiculite, sawdust or other suitable powdered or granular substrates to ensure that a large number of colony forming units are formed after the inoculum is mixed with the final matrix.

[0045] The number of colony forming units in the inoculum and its composition can vary according to the expected results and the characteristics of the microbial species used. In some embodiments, the inoculum is directly introduced into the interior of the substrate and / or onto the substrate to deliver the microbial species to the surface and / or interior of the substrate. In other embodiments, the microbial species and / or spores are in an aqueous suspension and the inoculum is introduced into the interior of the substrate and onto the substrate to deliver the microbial species to the surface and interior of the substrate. The microbial species inoculated can be algae, fungi, bacteria or a combination thereof. The fungi are preferably selected from the class Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes and / or Zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Saccharomyces, Staphylococcus or Pleurotus. More specifically, the species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiosus, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or a combination thereof.

[0046] In some embodiments, the first microbial species is a filamentous fungal species, including Penicillium, preferably Penicillium naldig; the second microbial species is a filamentous fungal species, including Rhizopus, preferably Rhizopus oligosporus. Inoculation of the first filamentous fungal species can be performed by spreading colony-forming units on the scaffold matrix. In this embodiment, the colony-forming units are spores of the corresponding filamentous fungal species. The first spore concentration of the first filamentous fungal species is in the range of 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the concentration of the second spores of the second filamentous fungal species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 Preferably, the ratio of the first spore concentration to the second spore concentration ranges from 0.01 to 1000, preferably from 0.1 to 500, more preferably from 50 to 150.

[0047] In some other embodiments, the first microbial species is a filamentous fungal species, including the genus Lysospora, preferably Lysospora pseudoroseum; 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 performed by spreading colony-forming units on the scaffold matrix. In this embodiment, the colony-forming units are spores of the corresponding filamentous fungal species. The first spore concentration of the first filamentous fungal species is in the range of 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the concentration of the second spores of the second filamentous fungal species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 Preferably, the ratio of the first spore concentration to the second spore concentration ranges from 0.01 to 2000, preferably from 0.1 to 1000, more preferably from 20 to 500.

[0048] During incubation 130, the scaffold matrix is ​​incubated. The microbial species inoculated during inoculation 120 grow and develop within the matrix. To promote growth of the microbial species, this step is performed under controlled parameters (e.g., temperature, humidity, oxygen concentration, and / or time). Preferably, incubation conditions are adjusted to avoid the generation of unpleasant odors, such as those resulting from ammonia formation, accumulation of organic acids, and / or prior spore formation.

[0049] Incubation 130 can be performed in any manner, and thus additional subsequent steps may be required to ensure good growth of the microbial species within and / or around the matrix. Incubation 130 can be, for example, a liquid fermentation or, more preferably, a solid-state fermentation (SSF). Fermentation conditions and time can be adjusted depending on the microbial species, available nutrients, matrix components, and desired results. Preferably, the scaffold is incubated at a temperature of 4 to 70° C., particularly between 10 and 50° C., more preferably between 14 and 40° C., and even more preferably between 22 and 38° C.

[0050] Preferably, incubation 130 is terminated prior to sporulation.

[0051] The growth of the microorganisms can be interrupted by any means, for example, by changing the temperature and / or water activity to below or above the temperature and water activity conditions required for the growth of the microbial species. Alternatively, the growth of the microorganisms can be interrupted by reducing the oxygen concentration to below the critical level required for the growth of the corresponding microbial species. Preferably, the growth of the 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, wherein the temperature is measured at the center of the product.

[0052] Alternatively, the growth of the microorganisms is not interrupted. Instead, the food product is stored in a refrigerator or freezer until consumed, preferably before sporulation or spoilage occurs.

[0053] Figure 2 Another embodiment of forming a food product is presented, comprising the steps of providing a scaffold matrix 110; inoculating 120a the scaffold matrix with at least a first microbial species; incubating 130a the scaffold matrix under optimal conditions to grow the first microbial species; inoculating 120b with at least a second microbial species, preferably a filamentous fungal species; and incubating 130b the scaffold matrix under optimal conditions to grow the second microbial species.

[0054] In some embodiments, inoculation can be performed in sequential steps, first inoculating 120a the scaffold matrix with a first microbial species, and then subsequently inoculating 120b the scaffold matrix with a second microbial species, preferably a filamentous fungal species.

[0055] In some embodiments, incubation 130 can be performed in sequential steps, such as 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 matrix is ​​inoculated with at least one microbial species. Inoculation can be performed in any manner, and thus additional subsequent steps may be required to ensure good growth of the microbial species within and / or around the matrix.

[0057] Inoculation 120a can be carried out by inoculum. The inoculum contains a certain amount of colony forming units (CFU) of microbial species, and the microbial species colony can be spores, active vegetative hyphae and the active part of fungal tissue, and can also include the part of dormant hyphae and fungal tissue, such as dehydrated or freeze-dried hyphae and fungal tissue. The inoculum is introduced into and / or on the larger volume of the scaffold matrix to start the growth of the microbial species on the matrix. The inoculum can take different forms, such as spores, mycelium fragments, fluid or solid culture suspensions, and may contain the vegetative structure and reproductive structure of the fungus and the bacterial species matrix. The bacterial species matrix can be grains, vermiculite, sawdust or other suitable powdered or granular matrix, to ensure that the inoculum forms a large amount of colony forming units after mixing with the final matrix. The size and composition of the inoculum can vary depending on the desired outcome and the characteristics of the microbial species used. In some embodiments, the inoculum is directly introduced into the interior of the substrate and / or onto the substrate to deliver the microbial species to the surface and / or interior of the substrate. In other embodiments, the microbial species and / or spores are in an aqueous suspension and the inoculum is introduced into the interior of the substrate and onto the substrate to deliver the microbial species to the surface and interior of the substrate. The inoculated microbial species can be algae, fungi, bacteria or a combination thereof. The fungi are preferably selected from the class Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes and / or Zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococcus, Saccharomyces or Pleurotus. More specifically, species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgi, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or a combination thereof. In some embodiments, Penicillium nalgi, Lactobacillus, Sporangiophora pseudoroseum, Aspergillus sojae, or a yeast is inoculated.

[0058] Incubation 130a for the first time of described scaffold matrix allows the first microorganism species to grow inside, outside or inside and outside of described scaffold matrix.Hatching 130a can be carried out in any way, therefore may need extra subsequent step, to guarantee that matrix inside and / or surrounding microorganism species good growth.Hatching 130a can be for example liquid fermentation or more preferably solid state fermentation (SSF).Fermentation conditions and time can be adjusted according to microorganism species, available nutrients, matrix components and expected result.Preferably, regulate incubation conditions to avoid producing unpleasant odor, for example, the odor produced before sporulation by the formation of ammonia, the accumulation of organic acid and / or.

[0059] During inoculation 120a, the inoculated microbial species grows and develops within the scaffold matrix. To promote growth of the microbial species, this step is performed under controlled parameters (e.g., temperature, humidity, oxygen concentration, and / or time). Incubation 130a can be performed at a temperature between 5 and 50°C, preferably between 10 and 40°C, and preferably between 20 and 30°C, for less than 72 hours, preferably less than 48 hours.

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

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

[0062] During inoculation 120b, the scaffold matrix is ​​inoculated a second time with at least one microbial species, including at least one filamentous fungal species. Inoculation 120b can be performed in any manner, and thus additional subsequent steps may be required to ensure good growth of the microbial species within and / or around the matrix.

[0063] Inoculation 120b can be carried out by inoculum.Inoculum contains a certain amount of colony forming units (CFU) of microbial species, and the microbial species colony can be the active part of spores, active vegetative hyphae and fungal tissue, and can also include the part of dormant hyphae and fungal tissue, such as the part of dehydrated or freeze-dried hyphae and fungal tissue.Inoculum is introduced into and / or on the larger volume of scaffold matrix, to start the growth of microbial species on matrix.Inoculum can take different forms, such as spores, mycelium fragments, fluid or solid culture suspension, and may contain the vegetative structure and reproductive structure of fungi and bacterial species matrix.The bacterial species matrix can be grain, vermiculite, sawdust or other suitable powdery or granular matrix, to ensure that inoculum forms a large amount of colony forming units after mixing with the final matrix.

[0064] The size and composition of the inoculum can vary depending on the desired outcome and the characteristics of the microbial species used. In some embodiments, the inoculum is directly introduced into the interior of the substrate and / or onto the substrate to deliver the microbial species to the surface and / or interior of the substrate. In other embodiments, the microbial species and / or spores are in an aqueous suspension and the inoculum is introduced into the interior of the substrate and onto the substrate to deliver the microbial species to the surface and interior of the substrate. The inoculated microbial species can be algae, fungi, bacteria or a combination thereof. The fungi are preferably selected from the class Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes and / or Zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococcus, Saccharomyces or Pleurotus. More specifically, the species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium naldii, Penicillium camemberti, Penicillium roqueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or a combination thereof. In some embodiments, Rhizopus oligosporus is inoculated. In some embodiments, Aspergillus oryzae is inoculated.

[0065] The second time hatching 130b of described scaffold matrix allows the second kind of microbial species to grow inside, outside or inside and outside of described scaffold matrix.In the hatching 130b process, scaffold matrix will be hatched.Growth conditions are optimized for the second kind of microbial species.Therefore, the second kind of microbial species grows and develops inside matrix and on matrix.The second microbial species forms matrix (matrix) inside and around scaffold matrix, scaffold matrix is ​​combined together.In one embodiment, filamentous fungus species forms a network that is combined with self and matrix material.In order to promote the growth of selected microbial species, this step is carried out under controlled parameters (such as temperature, humidity, oxygen concentration and / or time).Should adjust parameters to maximize the growth of the second microbial species, these conditions can be identical or different with the condition used in hatching 130a for the first time.Therefore, the growth of the microbial species inoculated during inoculation 120a also can continue or stop.

[0066] Hatch 130b can be carried out in any way, therefore may need extra subsequent step, to ensure that the microorganism species inside and / or around the substrate are well grown.Hatch 130b can be for example liquid fermentation or more preferably solid state fermentation (SSF).Fermentation conditions and time can be adjusted according to microorganism species, available nutrients, substrate components and expected results.Hatch 130b can be between 20 to 45 ℃, preferably at a temperature between 28 to 35 ℃, for a duration less than 72h, preferably a duration less than 44h.

[0067] Preferably, the incubation is terminated 130b prior to spore formation. Preferably, the incubation conditions are adjusted to avoid the generation of unpleasant odors, such as those resulting from the formation of ammonia, accumulation of organic acids, and / or spore formation.

[0068] The growth of the microorganisms can be interrupted by any means, for example, by changing the temperature and / or water activity to below or above the temperature and water activity conditions required for the growth of at least one filamentous fungal species. Alternatively, the growth of the filamentous fungal species can be interrupted by reducing the oxygen concentration to below the critical level required for the growth of the corresponding filamentous fungal species. Preferably, the growth of the filamentous fungal species is interrupted by heating the product containing the fungi to above 60°C, preferably above 71°C, for at least 1 minute, more preferably for more than 30 minutes, wherein the temperature is measured at the center of the product.

[0069] Alternatively, the growth of the microorganisms is not interrupted. Instead, the food product is stored in a refrigerator or freezer until consumed, preferably before sporulation or spoilage occurs.

[0070] It is understood that other repeated inoculation steps and / or incubation steps can be added to obtain more bacterial species with additional effects between them. It is also understood that in any inoculation step, more than one microbial species can be inoculated. Specifically, it should be understood that during the first inoculation 120a, Penicillium nalgiospermum and S. pseudorosodium can be inoculated, during the second inoculation 120b, Rhizopus oligosporus can be inoculated, and during the third inoculation, Aspergillus oryzae can be inoculated. In the present example, Penicillium nalgiospermum allows Rhizopus oligosporus to grow abundantly, and S. pseudorosodium allows Rhizopus oligosporus to grow abundantly. Penicillium nalgiospermum and S. pseudorosodium grow during the first incubation 130a, and the second incubation 130b with the optimal conditions of Rhizopus oligosporus can combine all the blocks of the scaffold matrix together, and the third incubation with the optimal conditions of Aspergillus oryzae can produce a fungus-based epidermis. The fungus-based epidermis should appear on the scaffold matrix surface and have a high density of mycelium, covering a thickness of the matrix between 0.1 and 5 mm.

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

[0072] Molding can also be used to form a fungus-based epidermis on the surface of the final product. In fact, the growth of filamentous microbial species (or filamentous fungi) also occurs on the outer surface of the scaffold matrix and can proliferate in the space outside the scaffold matrix. By compacting these filaments on the surface of the scaffold matrix, a dense layer consisting mainly of filaments can be obtained, which forms a fungus-based epidermis on the surface of the product. It is understood that method 100 can be used multiple times, using the finished food product from the previous method 100 as the scaffold matrix for the next method 100 to create different appearances, thereby obtaining a finished product that best simulates a piece of meat or meat product. For example, the first application of method 100 combines multiple scaffold matrices together, and then the second application of method 100 produces a fungus-based epidermis on the surface of the finished product.

[0073] In some embodiments, the scaffold matrix can be prepared prior to inoculation 120 or inoculation 120a. During the step of preparing the scaffold matrix, the scaffold matrix can be hydrated with an aqueous solution to reduce the number of and / or eliminate other microbial species present within and / or outside the scaffold matrix that may constitute a source of contamination. The water activity of the scaffold matrix is ​​adjusted based on the conditions required for microbial growth, preferably 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 only of water. The mass ratio of the matrix to the mass of water can be 1:1 to 1:2, more preferably 1:1.5 to 1:1.8.

[0075] In other embodiments, the aqueous solution may consist of water and may also consist of colorants, flavorants, any compounds that aid the growth of microbial species (e.g., nutrients), and / or any compounds that prevent the growth of and / or eliminate other undesirable microbial species.

[0076] Hydration is the growth condition for the development and growth of microbial species in the matrix. This is also true for unwanted microbial species. In order to reduce the number of microbial species present, the scaffold matrix can be pasteurized, double pasteurized, sterilized, treated with antimicrobial compounds and / or treated with acid. For example, the scaffold matrix can be sterilized in an autoclave up to 120°C. Another example is acidification, which uses an acid (preferably a food grade acid, more preferably lactic acid, acetic acid, malic acid, citric acid or succinic acid) to treat the surface of the scaffold matrix, preferably making the surface pH value lower than 6, most preferably between 4.5 and 4.6. More preferably, the surface pH value is adjusted to ensure the growth of the microbial species used and reduce the growth of other microorganisms.

[0077] In another embodiment, the scaffold matrix and the aqueous solution are treated separately and then brought into contact to hydrate the scaffold matrix.

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

[0079] In another embodiment, the food product is developed to simulate a meat product. The food product can be shaped into the same shape as the meat product it replaces to facilitate easy recognition and identification by consumers.

[0080] Specifically, the shape of the food product should 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 piece, the shape of the food product should be similar in size and thickness to a traditional whole piece of meat. Similarly, if the meat product being imitated is a salami or sausage, the shape of the food product should be similar in size and thickness to a traditional salami or sausage.

[0081] The food product will also take into account any unique characteristics of the meat product being imitated. For example, if the meat product has a unique texture or pattern on the surface and / or interior, the food product design will replicate that texture and pattern as closely as possible.

[0082] In another embodiment, the food product can be formed using a variety of methods to achieve the desired shape, texture, and appearance. One 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 force to shape the product. This can be achieved using a press, extruder, or other similar equipment. Mechanical shaping is particularly suitable for shaping foods that require a high degree of consistency and uniformity in shape and size.

[0084] Other methods for shaping food products include molding, casting, and cutting. Molding involves pouring the food product into a mold and allowing it to cool and solidify. Casting is a similar process, but for fluid or semi-fluid food products. Cutting can be used to create precise shapes and sizes in food products, such as slicing or dicing.

[0085] It should be noted that the forming method is not limited to the method described herein, and other methods and variations of the above methods may also be used. The specific forming method depends on many factors, such as the type of food product, the desired shape and texture, and the manufacturing process for producing the food product.

[0086] In another embodiment, the scaffold matrix is ​​placed within a shell that is larger than the substrate so that a free space is formed between the substrate and the inner surface of the shell. The fungal mycelium will at least partially fill this free space and form a fungal-based epidermis around the scaffold matrix. The thickness of the free space can be between 0.1 mm and 5 cm. The substrate can be fixed by any means, such as by rods connected to the shell and passing through the substrate. The shell can also have one or more supports for placing the substrate.

[0087] The food product 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 matrix and the second phase comprises mycelia of at least one filamentous fungal species, preferably, wherein the proportion 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 from 0.001 to 0.95, more preferably from 0.1 to 0.6.

[0088] After the food product is formed according to the above method 100, it can be further processed to improve taste, storage and / or transportation. Specifically, the food product can be pickled, seasoned, smoked, pickled, dehydrated, pressed, soaked in water and / or oil, steamed, boiled, or processed in any other way commonly used for animal meat products.

[0089] The food product is designed to provide consumers with a satisfying juiciness experience, similar to traditional meat products, such as releasing juices when biting or cutting, further enhancing the overall juiciness experience.

[0090] In one embodiment, the fermented food product is more nutritious than the edible matrix, preferably wherein the protein in the edible matrix is ​​more digestible than before fermentation, and fermentation reduces anti-nutritional factors, thereby increasing the bioavailability of micronutrients. Some non-limiting possible examples are higher antioxidant activity, higher total phenolic content, increased soluble protein, higher ellagic acid (anti-cancer), 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 made by adding a thin layer of fungi around a finished food product to form a fungus-based food product with a fungus-based skin. For example, the thin layer can be produced by liquid fermentation, then harvested, processed, and finally deposited around the finished food product. The thin layer can be between 0.1 mm and 5 mm thick and can be composed of at least one fungus.

[0092] In one embodiment, the food product contains DNA and RNA from each of the 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 polymerase chain reaction (PCR) amplification of specific gene fragments (more preferably, by qPCR analysis) of the final product in combination with gene sequencing (e.g., Sanger sequencing). It is expected that the amount of microbial DNA and / or RNA from each species inoculated during method 100 will exceed 0.0001% of the total product mass.

[0093] In another embodiment, the food product may contain a majority of microbial DNA and / or RNA from the microbial species inoculated during method 100. It is expected that the amount of DNA and / or RNA from the inoculated microbial species will exceed the amount of DNA and / or RNA from the other microbial species by a factor of 10. Example 1

[0094] Mycelial growth and binding were observed on TVP blocks fermented with Rhizopus oligosporus, Penicillius nalgiovense or a combination thereof.

[0095] Pea protein-based TVP chunks with a size of 4-15 mm were hydrated with 170 g of water and 2 g of lactic acid per 100 g of TVP product and then pasteurized at 90° C. for 30 min in vacuum packaging. Series A was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 10 per kg of substrate. 7 to start a monoculture incubation of one species. Series B was inoculated with Penicillium nalgiformis at a spore concentration of 9 x 10 per kg of substrate.8 to start a single culture incubation of a species Series C was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 10 per kg of substrate. 7 and inoculated with Penicillium nalgiovense at a spore concentration of 9 x 10 8 to start the co-culture incubation of the two species.

[0096] The powdered starter culture was evenly mixed and spread on the surface of the TVP block for inoculation. 30 g of the inoculated TVP block was placed in a ventilated culture dish and incubated in an incubator at 30°C and 98% relative humidity for 18.5 hours. The fermented TVP block was then photographed and observed under a microscope. Figure 5 As shown, after incubation, the TVP blocks were covered with aerial hyphae, which formed a mycelial network on the TVP surface and looked like white fuzz. Figure 3 and Figure 6 The results of the culture of series A are shown. Figure 4 、 Figure 7 and Figure 9 The results of the culture of series B are shown. Figure 5 and Figure 8 Shown are the culture results of series C. Figure 3 and Figure 6 as well as Figure 4 、 Figure 7 and Figure 9 Shown are the fungal mycelia formed by single culture incubation in series A and B, respectively. Figure 5 and Figure 8 The fungal mycelium formed by the co-culture incubations in Series C shown is less dense. Therefore, co-cultivation can achieve higher mycelial density, allowing for better control over the texture of the resulting food product. This example demonstrates how co-cultivation can promote desirable growth characteristics, such as mycelial density and faster growth rates. Example 2

[0097] Composites consisting of individual TVP blocks were prepared, formed by binding mycelia under co-culture or monoculture conditions, and the bond strength of the mycelia was compared using tensile testing. 100 g of pea protein-based TVP blocks ranging in size from 4 to 15 mm were hydrated with 170 g of water and 5 g of 80% lactic acid solution, then pasteurized at 92°C for 30 min in vacuum packaging. Three series, A, B, and C, were prepared from these TVP blocks. Series A was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 10 per kg of substrate. 7 and inoculated with Penicillium nalgiformis at a spore concentration of 9 x 108 to start the co-culture incubation of the two species. Series B was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 10 per kg of substrate. 7 to start a monoculture incubation of one species. Series C was inoculated with Penicillium nalgiformis at a spore concentration of 9 x 10 per kg of substrate. 8 to start a monoculture incubation of one species.

[0098] The powdered starter culture was inoculated by uniformly covering the surface of the TVP blocks with mechanical mixing. The inoculated TVP blocks of series A, B and C were then formed into cylinders with a diameter of 20 mm and a length of 180 mm and wrapped with a layer of perforated polyethylene film. The cylinders were incubated in an incubator at a temperature of 30°C and a relative humidity of 95% for 32 hours. Although the TVP blocks were held together by the polyethylene film before incubation, the blocks of series A and B adhered to each other after incubation by the growth of mycelia supplied by the nutrients available in the blocks. Series C did not show any binding because the hyphae of the inoculated Penicillium nalgiosus were too short to bridge between the individual TVP blocks (see Figure 7 and 9 ). The polyethylene film was then removed and the cylinder was vacuum packed and heat treated at 90°C for 20 minutes. Rectangular blocks of 11x17x50 mm were cut from the cylinder. The rectangular blocks were then placed in a ZwickRoell 2.5kN zwicki RetroLine texture analyzer for tensile testing with a gap setting of 10m, a preload of 0.1N and a tensile speed of 200mm / min. The sample was stretched until it broke along the middle. The sample broke along the mycelium phase, which held the individual TVP blocks together. It can be assumed that the cross-sectional area remained nearly constant during the tensile test. The stress is calculated by dividing the force (N) used to pull the sample apart by the cross-sectional area. The results are displayed in a stress-strain diagram, as shown Figure 10 As shown. In Series C containing Penicillium naldigium, the TVP fragments failed to form a cohesive complex after incubation. Consequently, Series C could not be fixed in the experimental setup, and tensile testing could not be performed. Consequently, the stress response could not be measured. Figure 10The mechanical responses of series C are representatively depicted. The fracture regions of series A are marked with dashed rectangles and designated "a," the fracture regions of series B are marked with dashed rectangles and designated "b," and the representative fracture regions of series C are marked with dashed rectangles and designated "c." The results show that the stress at which the blocks combined with two species of Rhizopus oligosporus and Penicillium natans in series A fractured during co-culture incubation was greater than the stress at which the blocks combined with one species of Rhizopus oligosporus or one species of Penicillium natans in series B and C fractured during monoculture incubation. If the fracture stresses of the monoculture series B and C were added, they were still less than the fracture stress of series A. Therefore, this example shows that the combination of the two strains in the co-incubation resulted in more abundant growth of Rhizopus oligosporus, thereby improving the binding capacity. The improved binding capacity can be used to adjust the texture of food products. Example 3

[0099] To create the prototypes, TVP blocks were hydrated with distilled water and lactic acid to a moisture content of 60%. The hydrated TVP blocks were vacuum-packed and pasteurized in a VWB2 waterbath at 95°C for 35 minutes. After cooling to room temperature, the desired strains were inoculated onto the TVP blocks in a BioVanguard Green Line laminar flow purifier. The inoculum and TVP blocks were shaken in the vacuum bag to ensure even distribution of spores and feedstock. Several fungal combinations were inoculated: the first combination consisted of Rhizopus delavis and Penicillium nalgium; the second consisted of Rhizopus oligosporus and Penicillium nalgium; the third consisted of Rhizopus delavis and Rhizopus oligosporus; and the fourth consisted of Rhizopus delavis, Rhizopus oligosporus, and Penicillium nalgium. Two control prototypes were also inoculated with either Rhizopus delavis alone or Rhizopus oligosporus alone. Six matrix strips, 14 cm long, 28 mm thick, and 25 mm wide, were prepared in a 3D-printed mold. Each strip was filled with 80 grams of hydrated and inoculated TVP. After filling the mold, the inoculated TVP was placed in an incubator for fermentation. At the end of the incubation period, the prototypes were harvested, refrigerated, and used for mechanical testing.

[0100] The tensile strength of the bars was measured using a ZwickiLine texture analyzer. Figure 11 As shown. The bar prototype was subjected to a tensile test analysis to measure its maximum tensile strength (Fmax) in megapascals (MPa). The texture analyzer calculates Fmax by dividing the maximum tensile force by the cross-sectional area of ​​the bar prototype. During analysis, the bar prototype was secured in the texture analyzer's grips with a 2.2 mm gap between the grips and an initial grip spacing of 50 mm. The preload force was 0.2 Newtons (N) and the tensile speed was 0.5 mm / s.

[0101] The results are as follows Figure 12As shown, all combinations containing at least one Rhizopus species and Penicillium nalgiospermum showed an average increase of 30% in tensile strength. Thus, these examples show that various combinations of the two strains in co-incubations can lead to more abundant growth of the Rhizopus strains, thereby improving the binding capacity of the food product. The present invention is described from the following aspects: 1. A method for preparing a food product, the method comprising the steps of: a) providing a scaffold matrix; b) inoculating the scaffold matrix with at least two microbial species, the at least two microbial species comprising at least one filamentous fungal species; and c) incubating the scaffold matrix to allow the at least two microbial species to grow inside, outside, or inside and outside the scaffold matrix. 2. The method according to the preceding aspect, wherein the at least two microbial species are capable of achieving vigorous growth, which is preferably produced by performing step c), said vigorous growth comprising increased mechanical strength, preferably tensile strength or increased mycelium density or increased tensile strength and increased mycelium density. 3. The method according to any one of the preceding aspects, wherein the at least two microbial species enable the abundant growth of the at least one filamentous fungal species. 4. The method according to any of the preceding aspects, wherein the method comprises inoculating the scaffold matrix with a given number of colony forming units of at least one filamentous fungal species, wherein the filamentous fungal species grows more densely than when the scaffold matrix is ​​inoculated with only a given number of colony forming units of the filamentous fungal species and incubated in the absence of a plurality of the other one of the at least two microbial species. 5. The method according to any of the preceding aspects, wherein the at least two microbial species comprise a first microbial species and a second microbial species, the second microbial species being a filamentous fungal species, The method comprises inoculating the scaffold matrix with a first number of colony forming units of a first microbial species and inoculating the scaffold matrix with a second number of colony forming units of a second microbial species, wherein the food product obtained by step c), preferably immediately after step c), without further processing, has a tensile strength greater than the sum of the tensile strengths of the first product and the second product, The first product is obtained by the following steps: inoculating a first corresponding scaffold matrix with a first number of colony forming units of a first microbial species, and incubating the first corresponding scaffold matrix to allow a first microbial species to grow inside, outside, or inside and outside of the first corresponding scaffold matrix; The second product is obtained by the following steps: inoculating a second corresponding scaffold matrix with a second number of colony forming units of a second microbial species, and The second corresponding scaffold matrix is ​​incubated to allow a second microbial species to grow inside, outside, or inside and outside of the second corresponding scaffold matrix. 6. The method according to any of the preceding aspects, wherein the scaffold matrix is ​​made of protein from at least one plant source, preferably one protein source is pea protein, preferably the scaffold matrix contains at least 50% protein in dry matter, preferably 60% or more protein in dry matter. 7. The method according to any of the preceding aspects, wherein the scaffold matrix is ​​made of TVP, preferably the TVP scaffold matrix is ​​made of a protein source, the protein source being pea protein, preferably 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 according to any of the preceding aspects, wherein the scaffold matrix comprises at least one thick block or a group of thick blocks, preferably, the number of thick blocks in a group is 2 or more. 9. The method of the preceding aspect, 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 aspect 8 or 9, wherein the average aspect ratio of the chunks (defined as the aspect ratio of 15 randomly selected chunks, wherein the aspect ratio is defined as the ratio of the longest dimension to the shortest dimension of the corresponding chunks) is 10 or less, 5 or less, 3 or less, or 2 or less. 11. The method according to any of the preceding aspects, wherein the scaffold matrix comprises at least one fiber or a group of fibers, preferably a number of fibers in a group is 2 or more. 12. A method according to aspect 11, wherein the aspect ratio of the fibers (defined as the ratio of the longest dimension to the shortest dimension of the corresponding fibers) is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10000. 13. The method according to any of the preceding aspects, wherein the scaffold matrix comprises at least one sheet or a stack of sheets, preferably the number of sheets in a stack is 2 or more. 14. A method according to the preceding aspect, wherein the aspect ratio of the sheets (defined as the ratio of the longest dimension to the shortest dimension of the respective sheets) is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10000. 15. The method according to any one of aspects 8 to 14, wherein the thick pieces, fibers and / or sheets are bound together by the growth of at least one filamentous fungal species. 16. The method according to any one of aspects 8 to 15, wherein the chunks, fibers and / or sheets may be placed in an ordered manner, for example all longest dimensions of the chunks and / or fibers and / or sheets may be aligned in substantially the same direction. 17. The method according to any one of aspects 8 to 15, wherein the chunks and / or sheets may be placed in a disordered manner, for example all longest dimensions and / or shortest dimensions of the chunks and / or fibers and / or sheets are not aligned in substantially the same direction. 18. The method according to any one of the preceding aspects, wherein the other one or more species of the at least two microbial species comprise fungal species, algal species and / or bacterial species, preferably fungal species, more preferably filamentous fungal species. 19. The method according to any one of the preceding aspects, wherein the first microbial species is a first filamentous fungal species, including Penicillium, preferably Penicillium nalgiospermum, and the second microbial species is a second filamentous fungal species, including Rhizopus, preferably Rhizopus oligosporus and / or Rhizopus delae. 20. The method according to the preceding aspect, wherein the concentration of the first colony forming units of the first filamentous fungal species is in the range of 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second filamentous fungal species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 . 21. The method according to the preceding aspects, wherein the ratio of the first colony forming unit concentration to the second colony forming unit concentration ranges from 0.01 to 1000, preferably from 0.1 to 500, more preferably from 50 to 150. 22. The method according to any of the preceding aspects, wherein the first microbial species is a filamentous fungal species comprising the genus Lysospora, preferably Lysospora pseudoroseum, and the second microbial species is a filamentous fungal species comprising the genus Aspergillus, preferably Aspergillus oryzae. 23. The method according to the preceding aspect, wherein the concentration of the first colony forming units of the first filamentous fungal species is in the range of 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second filamentous fungal species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 . 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 ranges from 0.01 to 2000, preferably from 0.1 to 1000, more preferably from 20 to 500. 25. The method according to any one of the preceding aspects, wherein step b) comprises the following steps b1) inoculating the scaffold matrix with a first microbial species; then, b2) inoculating the scaffold matrix with a second microbial species, preferably, the second microbial species is a filamentous fungal species. 26. The method according to the preceding aspect, wherein step c) comprises the following steps c1) incubating the scaffold matrix to allow a first microbial species to grow inside, outside, or inside and outside the scaffold matrix; and then c2) incubating the scaffold matrix to allow a second microbial species to grow inside, outside, or inside and outside of the scaffold matrix. 27. The method of aspect 25 according to aspect 26, wherein step c1) is performed before or after step b2). 28. The method of any one of aspects 25-27, wherein the first microbial species comprises Penicillium, Lactobacillus, Sporophore and / or Aspergillus, preferably Penicillium nalgesicola, Lactobacillus plantarum, Sporobacterium pseudoroseum, Aspergillus sojae, Aspergillus ryukyuus and / or Saccharomyces cerevisiae. 29. The method according to any one of aspects 25 to 28, wherein the second microbial species comprises Rhizopus, Penicillium and / or Aspergillus, preferably Rhizopus oligosporus, Rhizopus delavirdii, Rhizopus oryzae, Penicillium nalgiosus and / or Aspergillus oryzae. 30. The method according to any one of aspects 25-29, wherein Step cl) is carried out at a temperature of 5-50°C, preferably 10-40°C, preferably 20-30°C, for a duration of less than 72 hours and preferably less than 48 hours, and / or Step c2) is carried out at a temperature of 20-45°C, preferably 28-35°C, and for a duration of less than 72 hours, preferably less than 44 hours. 31. The method according to any one of the preceding aspects, wherein step c) comprises forming a fungus-based skin around the scaffold matrix, preferably by the following steps: placing the scaffold matrix in a shell to form a closed space between the scaffold matrix and the shell, and performing step c) to allow at least two microbial species to grow within the confined space, Preferably, the thickness of the enclosed space ranges from 0.1 mm to 5 cm, more preferably from 0.5 mm to 2.5 cm, even more preferably from 1 mm to 1 cm. 32. The method of any preceding aspect, wherein the scaffold matrix comprises textured vegetable protein (TVP). 33. The method according to any one of the preceding aspects, further comprising sterilizing and / or pasteurizing the scaffold matrix prior to step b). 34. The method according to any one of the preceding aspects, further comprising hydrating the scaffold matrix by an aqueous solution such that the scaffold matrix has a water activity greater than 0.8 during steps b) and / or c). 35. The method according to any one of the preceding aspects, wherein step c) is performed under solid state fermentation conditions. 36. The method according to any one of the preceding aspects, further comprising shaping the scaffold matrix before and / or after step b). 37. A food product prepared by the method of any preceding aspect. 38. A food product comprising a scaffold matrix formed from one or more pieces of textured vegetable protein (TVP) and mycelia of at least one filamentous fungal species growing within, outside, or both within and outside the scaffold matrix. 39. The food product according to any one 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, more preferably the first phase comprises a scaffold matrix and the second phase comprises mycelia of at least one filamentous fungal species. 40. A food product according to the preceding aspects, wherein the proportion of the second phase of the food (defined as the ratio of the area occupied by the second phase in the cross-section of the food to the total area of ​​the cross-section) ranges from 0.001 to 0.95, more preferably from 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 disclosed above, as those skilled in the art will understand that changes and modifications can be made without violating the principles of the present invention and still fall within the scope of the present invention. In particular, the present invention should not be construed as being limited to the embodiments described above with reference to the accompanying drawings. Instead, the scope of the present invention is determined solely by the appended claims and their equivalents.

Claims

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

2. The method according to claim 1, The method comprises inoculating the scaffold matrix with a given number of colony forming units of at least one filamentous fungal species, in, The filamentous fungal species exhibits more abundant growth than when the scaffold matrix is ​​inoculated with only a given number of colony forming units of the filamentous fungal species and incubated in the absence of a plurality of the other one of the at least two microbial species.

3. A method according to any one of the preceding claims, wherein The luxuriant growth is produced by carrying out step c), said luxuriant growth comprising increased mechanical strength, preferably tensile strength or increased mycelium density or increased tensile strength and increased mycelium density.

4. A method according to any one of the preceding claims, wherein The at least two microbial species comprise a first microbial species and a second microbial species, the second microbial species being a filamentous fungal species, The method comprises inoculating the scaffold matrix with a first number of colony forming units of a first microbial species and inoculating the scaffold matrix with a second number of colony forming units of a second microbial species, in, the food product obtained by step c), preferably immediately after step c), without further processing, has a tensile strength greater than the sum of the tensile strengths of the first product and the second product, The first product is obtained by the following steps: inoculating a first corresponding scaffold matrix with a first number of colony forming units of a first microbial species, and incubating the first corresponding scaffold matrix to allow a first microbial species to grow inside, outside, or inside and outside of the first corresponding scaffold matrix; The second product is obtained by the following steps: inoculating a second corresponding scaffold matrix with a second number of colony forming units of a second microbial species, and The second corresponding scaffold matrix is ​​incubated to allow a second microbial species to grow inside, outside, or inside and outside of the second corresponding scaffold matrix.

5. The method according to any one of the preceding claims, wherein the scaffold matrix comprises at least one thick block or a group of thick blocks, preferably a number of thick blocks in a group is 2 or more, 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.

6. The method according to any one of the preceding claims, wherein the scaffold matrix comprises at least one fiber or a group of fibers, preferably a number of fibers in a group of 2 or more, More preferably, the aspect ratio of the fibers, defined as the ratio of the longest dimension to the shortest dimension of the respective fiber, is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10,000.

7. The method according to any one of the preceding claims, wherein the scaffold matrix comprises at least one sheet or a stack of sheets, preferably the number of sheets in a stack is 2 or more, More preferably, the aspect ratio of the sheets, defined as the ratio of the longest dimension to the shortest dimension of the respective sheets, is 10 or greater, or 100 or greater, or 1000 or greater, and preferably less than 10,000.

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

9. The method according to the preceding claim, wherein: The concentration range of the first colony forming unit of the first microbial species is 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second microbial species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 , wherein the ratio of the first colony forming unit concentration to the second colony forming unit concentration is preferably in the range of 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150.

10. The method of any one of the preceding claims, 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. The method according to the preceding claim, wherein The concentration range of the first colony forming unit of the first microbial species is 1×10 4 to 1×10 12 , preferably 1×10 6 to 1×10 10 , more preferably 1×10 8 to 1×10 9 , and the second colony-forming unit concentration of the second microbial species ranges from 1 × 10 4 to 1×10 13 , preferably 1×10 5 to 1×10 10 , more preferably 1×10 7 to 1×10 8 , wherein the ratio of the first colony forming unit concentration to the second colony forming unit concentration is preferably in the range of 0.01 to 2000, preferably 0.1 to 1000, and more preferably 20 to 500.

12. The method according to any one of the preceding claims, wherein step b) comprises the following steps b1) inoculating the scaffold matrix with a first microbial species; then, b2) inoculating the scaffold matrix with a second microbial species, preferably, the second microbial species is a filamentous fungal species.

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

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

15. The food product according to claim 14, wherein the food product comprises at least two distinct phases, preferably the two distinct phases are distinguishable by the human eye, more preferably the first phase comprises the scaffold matrix and the second phase comprises mycelia of the at least one filamentous fungal species. Preferably, The proportion of the second phase of the food product is defined as the ratio of the area occupied by the second phase in the cross section of the food product to the total area of ​​the cross section, which ranges from 0.001 to 0.95, more preferably from 0.01 to 0.6.

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