Method of making a food product using microbial growth and products thereof

Abstract

Described is a method to prepare a food product, the method comprising steps of, providing a scaffolding substrate, inoculating said scaffolding substrate with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species, preferably a first filamentous fungus species and a second filamentous fungus species, and incubating said scaffolding substrate to allow the at least two microbial species to grow inside, outside, or inside and outside said scaffolding substrate, wherein the at least two microbial species enable a prolific growth of the at least one filamentous fungus species.

Description

Method of making a food product using microbial growth and products thereofTECHNICAL FIELD

[0001] The present application relates to a method of providing a food product by binding pieces of a textured vegetable protein substrate, which adhere to each other through growth of two or more microbial species with at least one filamentous fungi. Thus, a fungus-containing food product is formed.BACKGROUND

[0002] Various approaches are currently applied to produce meat alternative products resulting in tremendous differences in texture and nutritional value. Indeed, few meat substitutes allow to provide the proteins which are recommended for daily intake as well as having an appealing texture. Also, to be able to obtain texturized food similar to meat and / or meat-derived food, it is possible to add several ingredients to link small pieces together, or to give a specific texture to a product or a part of the product.

[0003] Tempeh is a well-known vegan product, which is usually made from soybeans fermented by a filamentous fungus, normally Rhizopus oligosporus. In recent times, the term Tempeh is used more broadly, including the fermentation and binding of grains or food processing by-products in addition to soybeans. One of the disadvantages of currently available tempeh products is that they lack taste and texture if compared to real meat and / or meat-derived foods. Another disadvantage is that the product is not very juicy, nor very positively flavored per se, requiring heavy marinades and usually deep frying as the most common cooking procedure.

[0004] More recent advances have been made by linking textured vegetable proteins with fungi, as featured in various patent applications (US2021045410, US2022132893). In these applications they use a textured substrate that can be made of several pieces of textured vegetable protein (TVP) which are bound together using at least one fungus. One of the limiting parameters in the production of mycelium-based foods is the mycelial density of fungus within the substrate. Growth of the fungus can be limited by available nutrients, lack of oxygen, and / or heat released as the fungus grows which actively ferments the substrate. Competing bacterial and fungal contamination could also jeopardize the myceliation process. Poor fungal development within a substrate leads to low mycelial density which decreases fibrousness, limits liquid and fat absorption and therefore juiciness and texture complexity during mastication, reduces the binding strength between the different parts of the substrate, increases the population of non-wanted microbial species as the fungi also act as a protective culture; the color may deviate from meat products and / or an off-flavor profiles can appear due to competing microbial contamination 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 a meat substitute that can have an appealing texture being capable of replacing pieces of animal meat or meat-derived products such as salamis, pepperonis, chorizos, fuets, or other types of cured and / or fermented meat sausages but also whole cut such as chicken breast or beef tenderloin.

[0006] If traditional strains commonly used in well-defined solid-state fermentation (SSF) processes such as in the production of blue cheese, camembert, tempeh, and koji, are grown on new, untraditional, or unconventional substrates, challenges are normally encountered such as reduced mycelial growth, off flavour formation, decreased product safety, etc, due to uncharacterized growthdynamics and / or metabolic pathways, poorly understood substrate properties, and lack of technological solutions tailored to the new bioprocess requirements.

[0007] The methods presented hereby solve some of the biggest challenges encountered by the previous art by exploiting the prolific growth of a filamentous fungus species enable by another microbial species in co-cultures and / or in sequential cultures not previously disclosed.SUMMARY

[0008] Described herein are various embodiments of methods for preparing a food product using a scaffolding substrate and at least two microbial species one of which is a filamentous fungus species. The uses of the at least two microbial species enable a more prolific growth of at least the filamentous fungus species. The method can generally include providing a scaffolding substrate, such as a textured vegetable protein substrate, that may or may not be subjected to various pre-processing steps used to help promote the at least 2 microbial species to grow inside, outside or inside and outside of the scaffolding substrate, inoculating the textured substrate with said microbial species, the microbial species are inoculated at the same time or separated in time, and growing the microbial species inside, outside, or inside and outside of the scaffolding substrate. In some embodiments, the filamentous fungus species is used to bind together multiple scaffolding substrates and any discrete particle within, to form a larger composite food product. In other embodiments, the filamentous fungus species is used to create a fungi-based skin on the surface of the food product.

[0009] By using at least two microbial species for inoculation, a primary advantage is the ability to enhance the growth of the filamentous fungi. This is achieved by utilizing the first microbial species to facilitate and support better growth conditions for the filamentous fungi species. For example, the inventors discovered that in the preparations and examples provided hereby, specific filamentous fungus species could grow better above another fungal, algal, or bacterial population, possibly due to the activation of aggressive growth genes and associated competitive and expansive behaviours that can stimulate the production of fungal biomass. A better growth is also possible thanks to the fermentation of the substrate by the first microbial species, fermentation will create additional nutrients which are more accessible and specific for the growth of the second fungus species.

[0010] In general, the advantages for the food product can be of various kinds. Some non-limiting examples include an increase in mycelium density, which can enhance the binding of the scaffolding substrate and result in a firmer texture. The final food product described herein may contain two phases: one with the scaffolding substrate and one with the mycelium. A greater density of mycelium can result in a whiter colour in the phase with the mycelium. In addition, an increase in mycelial density can improve juiciness, as more oil and / or water can be absorbed into the food product, which can be released during mastication and thus contributes to juiciness. Another potential advantage of this method is the ability to tailor the taste profile by modifying the microbial species and / or the incubation conditions.[Oil] In one aspect, the outer surface of the food product can have a coating similar to a fungi-based "skin" improving appearance before cooking and after cooking and also enhancing the experience of eating the food product. In another aspect, the interior of the product may have one or more phases including the scaffold substrate and / or the filamentous fungus species enhancing the experience of eating the food product by providing a more complex texture, similar to meat whole cuts or meat products.

[0012] Another advantage that can be implemented is the reduction of the growth of certain microbial species such as unwanted and potentially harmful microbial species for humans or spoilage species. This is achieved for example by the production of antimicrobial metabolites such as organicacids, enzymes, other antagonistic compounds, and / or competitive exclusion of nutrients and space performed by one or more of the inoculates species.

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

[0014] The method describe herein enhances the flavour profile, the colour and / or the texture of the finished products, making them more similar to animal-meat products such as salamis, pepperonis, or other types of fermented meat sausages and / or more similar to whole animal cuts, such as pork belly or other cuts of meats.

[0015] The present invention thus discloses a method to prepare a food product, said method comprising steps of: a) providing a scaffolding substrate; b) inoculating said scaffolding substrate with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species, preferably a first filamentous fungus species and a second filamentous fungus species; and c) incubating said scaffolding substrate to allow the at least two microbial species to grow inside, outside, or inside and outside said scaffolding substrate, wherein the at least two microbial species enable a prolific growth of the at least one filamentous fungus species.

[0016] According to the invention, the method comprising inoculating said scaffolding substrate with a given colony forming units concentration of the at least one filamentous fungus species, wherein the filamentous fungus species present a more prolific growth than it does when said scaffolding substrate is only inoculated with the given colony forming units concentration of the filamentous fungus species and incubated without presence of other one(s) of the at least two microbial species.

[0017] Also, the prolific growth is produced by carrying out step c), the prolific growth comprising an increased mechanical strength, preferably tensile strength.

[0018] According to this invention, the at least two microbial species comprising the first microbial species and the second microbial species which is a filamentous fungus species, the method comprising inoculating said scaffolding substrate with a first colony forming units concentration of the first microbial species and inoculating said scaffolding substrate with a second colony forming units concentration of the second microbial species, wherein the food product obtained by step c), preferably immediately after step c) without being subject to further processes, has a tensile strength greater than a sum of tensile strengths of a first product and of a second product, the first product being obtained by inoculating a first corresponding scaffolding substrate with the first colony forming units concentration of the first microbial species, and incubating said first corresponding scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said first corresponding scaffolding substrate; the second product being obtained by inoculating a second corresponding scaffolding substrate with the second colony forming units concentration of the secondmicrobial species, and incubating said second corresponding scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said second corresponding scaffolding substrate.

[0019] According to further non-limitative features of the invention, either taken alone or in any technically feasible combination:• the scaffolding substrate comprises at least one chunky piece or an assembly of chunky pieces, preferably a number of the chunky pieces in the assembly being 2 or more, more preferably an aspect ratio of the chunky pieces, which is defined as a ratio of a longest dimension to a shortest dimension of a respective chunky piece, is 10 or less, 5 or less, 3 or less, 2 or less.• the scaffolding substrate comprises at least one fibre or an assembly of fibers, preferably a number of the fibers in the assembly being 2 or more, more preferably an aspect ratio of the fibers which is defined as a ratio of a longest dimension to a shortest dimension of a respective fiber, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.• the scaffolding substrate comprises at least one sheet or a stack of sheets, preferably a number of the sheets in the stack being 2 or more, more preferably an aspect ratio of the sheets which is defined as a ratio of a longest dimension to a shortest dimension of a respective sheet, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.• the first microbial species is a filamentous fungus species comprising the genus Penicillium and preferably Penicillium nalgiovense and the second microbial species is a filamentous fungus species comprising the Rhizopus genus and preferably Rhizopus oligosporus.• a first colony forming units concentration of the first microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second colony forming units concentration of the second microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108, wherein, preferably the ratio of the first colony forming units concentration to the second colony forming units concentration ranges from 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150.• the first microbial species is a filamentous fungus species comprising the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a filamentous fungus species comprising the genus Aspergillus and preferably Aspergillus oryzae.• a first colony forming units concentration of the first microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second colony forming units concentration of the second microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108, wherein, preferably a ratio of the first colony forming units concentration tothe second colony forming units concentration ranges from 0.01 to 2000, preferably 0.1 to 1000, and more preferably 20 to 500.• step b) comprises steps of o bl) inoculating said scaffolding substrate with a first microbial species; and then, subsequently, o b2) inoculating said scaffolding substrate with a second microbial species, preferably the second microbial species being the filamentous fungus species.• step c) comprises steps of o cl) incubating said scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said scaffolding substrate; and then o c2) incubating said scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said scaffolding substrate,• step cl) is carried out 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 time period 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 time period of less than 72 h, preferably less than 44 h.

[0020] According to this invention the food product produce by this method comprises at least two distinct phases, preferably the two distinct phases can be distinguished by human eye, more preferably a first phase comprising the scaffolding substrate and a second phase comprising the mycelium of the at least one filamentous fungus species, preferably, wherein a ratio of the second phase of the food product, which is define as a ratio of an area occupied by the second phase in a cross section of the food product to a total area of said cross section, range from 0.05 to 0.95 and more preferably from 0.1 to 0.6.BRIEF DESCRIPTION OF THE DRAWINGSFIG.l is a flow diagram illustrating a method for producing a food product according to various embodiments described herein.FIG.2 is a flow diagram 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 nalgiovense.FIG.5 is a photograph of TVP fermented with Rhizopus oligosporus and Penicillium nalgiovense in co-culture.FIG.6 is a photograph of TVP fermented with Rhizopus oligosporus, Magnification.FIG.7 is a photograph of TVP fermented with Penicillium nalgiovense, Magnification.FIG.8 is a photograph of TVP fermented with Rhizopus oligosporus and Penicillium nalgiovense in co-culture.FIG.9 is a photograph of TVP fermented with Penicillium nalgiovense, Magnification 2.FIG.10 is a stress-strain diagram of tensile tests performed on TVP pieces either bound in co-cultural fermentation with the 2 species Rhizopus oligosporus and Penicillium nalgiovense or in mono-cultural fermentation with 1 species Rhizopus oligosporus or 1 species Penicillium nalgiovense.FIG.11 is a photograph of a tensile test device measuring a food product according to one embodiment.FIG.12 is a graphic showing varying tensile strengths of different food product according to various embodiments.DETAILED DESCRIPTION

[0021] FIG. 1 presents a method 100 for preparing a food product according to an embodiment, comprising steps of providing 110 a scaffolding substrate, inoculating 120 the scaffolding substrate with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species, preferably a microbial species and a filamentous fungus species, incubating 130 said scaffolding substrate to allow the at least two microbial species to grow inside, outside, or inside and outside said scaffolding substrate, wherein the at least two microbial species enable a more prolific growth of the at least one filamentous fungus species.

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

[0023] The method 100 uses the effects of a co-culturation of a filamentous fungus species and at least one other microbial species for achieving a prolific growth of the filamentous fungus species. Prolific growth as used in the present application is defined by the increase of at least one characteristic of the mycelium, this can be and not limited to, faster growth, increased mycelium density of at least one filamentous fungus, longer mycelium, larger mycelium, stronger mycelium compared to a growth without the other microbial species. In one embodiment the at least two microbial species can support each other's growth and so both microbial species can achieve to have a prolific growth.

[0024] For the food product the effect of the prolific growth can be characterised by an increased mycelial density and / or an increased tensile strength in the production of meat-like, more specifically, muscle-like fungal-based food products. The tensile strength of a food product may be determined by subjecting said food product to a mechanical force at its extremity and measuring the amount of force required to induce fracture. This value is herein referred to as the tensile strength of the food product. Various types of mechanical forces can be applied to a food product, including but not limited to tension, compression, torsion, bending, and shear. The method 100 also enhances the flavour profile, the texture, the colour and / or the juiciness of the finished products. In some embodiments the finish product is more similar to animal-meat products such as salamis, pepperonis, or other types of fermented meat sausages. In other embodiments, the finished product is more similar to whole animal cuts, such as pork belly or other cuts of meats.

[0025] In step of providing 110 a scaffolding substrate, any substrate suitable for food preparations can be used. In some embodiments, the scaffolding substrate is a vegetable protein substrate containing (poly)saccharides, fibers, mineral salts and / or amino acids necessary for the growth of the microbial species. "Protein" as used in the present application refers to protein isolate, concentrate or flour or combinations thereof, which may also contain other macronutrients, such as carbohydrates, fats, dietary fibres, salts, or residual water. Said isolate, concentrate, flour or combination thereof preferably contains a pure protein content of at least 40 wt%, preferably at least 50 wt%, even morepreferably at least 60 wt%. Preferably, said protein also comprises sufficient carbohydrates to act as a nutrient source for the microbial species.

[0026] In one embodiment the scaffolding substrate comprise only one chunky piece. In another embodiment the scaffolding substrate comprises an assembly of several chunky pieces from 2 to 100 pieces or 100 to 1000 pieces or more. The aspect ratio of the chunky piece which is defined as a ratio of a longest dimension to a shortest dimension of a respective chunky piece is 5 or less, 3 or less, or 2 or less.

[0027] In one embodiment the scaffolding substrate comprise only one elongated piece extending in one direction. Said elongated piece may also be referred to as a "fibre" in the context of the present invention. In another embodiment the scaffolding substrate comprises an assembly of several fibres, from 1 to 100 pieces or 100 to 10000 pieces or more. The aspect ratio of the fibre which is defined as a ratio of a longest dimension to a shortest dimension of a respective fibre is 5 or more, or 10 or more, or 100 or more, or 1000 or more and preferably 10000 or less.

[0028] In another embodiment the scaffolding substrate comprises only one piece extending in two directions, said piece being called sheet in this application. In another embodiment the scaffolding substrate comprise several sheets from 1 to 100 or 100 to 1000 or more. The aspect ratio of the sheets which is defined as a ratio of a longest dimension to a shortest dimension of a respective sheet is 5 or more, or 10 or more, or 100 or more, or 1000 or more and preferably 10000 or less.

[0029] The texture of a food product can be modulated by varying the number of pieces that make it up while maintaining the same final size. Thus, the number of pieces can vary significantly and influence the final texture of the food product. For example, a small number of pieces can give a texture similar to that of a whole cut, while a larger number of pieces can give a texture similar to that of a salami. The choice of the number of pieces, their size and shape can also influence the chewing, juiciness, and mouthfeel of the final product. Thus, by modulating the number of pieces, their size and shape, it is possible to create a wide variety of textures and mouthfeel sensations for food products.

[0030] In some embodiments the scaffolding substrate comprises a plurality of chunky pieces, fibres and / or sheets to mimic salamis, pepperonis, chorizos, fuets, or other types of cured and / or fermented meat sausages. In those embodiments the number of pieces can be between 10 and 10000 or more.

[0031] In some embodiments the scaffolding substrate comprises large chunky pieces, fibre pieces and / or sheet pieces to mimic for example whole cut such as chicken breast or beef tenderloin. In those embodiments the number of pieces can be between 1 and 100 and preferably between 1 and 20 and preferably between 1 and 10.

[0032] In some embodiments the scaffolding substrate was formed by any texturization process, it can be high moisture extrusion cooking (HMEC) or shear cell (SC) processing, where proteins are molten under high temperature, pressure and at moisture contents of 40-80% and subsequently cooled under shear resulting in the formation of a solidified fibrous structure.

[0033] In some embodiments the scaffolding substrate was formed by enzymatic reactions. The proteins can be crosslinked by enzymes such as transglutaminase to form aggregates.

[0034] In some embodiments, the scaffolding substrate includes vegetable protein coated with another layer such as agar so the fungi can grow inside the coating and form a fungi-based skin.

[0035] In a preferred embodiment the scaffolding substrate is a textured vegetable protein (TVP) substrate. TVP substrate is formed using an extrusion process by which a pressurized molten proteinmixture exits the extruder; the sudden drop in pressure causes rapid expansion into a spongy structure. This spongy structure provides pores or channels allowing microbial species to grow both on the surface but also inside the substrate and receive sufficient oxygen and nutrients. The TVP substrate can have any shape, it can be a chunky piece, a fiber, or a sheet. The TVP may be dried after extrusion leading to a lower water content and higher solids (dry matter) and protein content or may be only partly dried or not dried prior to further processing the TVP substrate.

[0036] The substrate, in particular the TVP substrate, typically comprises more than 40% wt of protein in the dry matter, with a preferred embodiment comprising at least 60 wt% of protein in the dry matter, with a more preferred embodiment comprising at least 60%, with an even more preferred embodiment comprising at least 70 wt% in the dry matter.

[0037] This TVP substrate can be rehydrated prior to incubation and thus decreasing the relative weight percentage of protein in the overall substrate 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 soy, pea, fava, beans, oil-seeds, such as rapeseed or sunflower, grains, such as wheat or oat, or any other sources. The TVP substrate may comprise a mixture of different protein sources or a single protein source not blended with other proteins. In a further preferred embodiment, the TVP is composed of a blend of soy protein and pea protein. In a further preferred embodiment, the TVP is composed of a blend of rice protein and pea protein. In a further preferred embodiment, the TVP is composed of a blend of at least 2 plant-based protein sources, preferably wherein at least one is soy or pea. In a further preferred embodiment, the TVP is composed of pea as only protein source, comprising both protein and starch. In another embodiment, the TVP comprises other non-slaughtered ingredients originating from single cell organisms, cellular agriculture, algae, plants or fungi.

[0039] In a preferred embodiment, the scaffolding substrate is made of several pieces of texturized vegetal protein (TVP), wherein these pieces can be chunky pieces, fibers (including thin TVP filaments also known as soy floss as well as spun soy fiber made from these same soy flosses) and / or sheets.

[0040] In another embodiment, the scaffolding substrate can be made by any kind of edible textured vegetable protein product or a combination thereof.

[0041] The arrangement of these pieces can be random; in other words, the pieces of the scaffolding substrate may not face a particular direction or a preferential position, or on the contrary, they can be placed to form a relatively organized structure as well in 1 direction as in 2 directions or in the 3 directions of space. For example, the sheets can be stacked on top of each other to form a multi layers food product to increase the texture.

[0042] Preferably, pressure or vacuum is applied to the pieces of texturized protein to form a scaffolding substrate. The pressure may be applied manually or by means of machines. In this embodiment, the growth of the filamentous fungus species inside and outside the scaffolding substrate during the incubation 130 results for example in the binding of the different pieces of the scaffolding substrate together. Incubation 130 is discussed in more detail below.

[0043] During the inoculation 120, the scaffolding substrate is inoculated with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species. The inoculation can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate.

[0044] The inoculation 120 can be done with an inoculum. Inoculum contain an amount of colony forming units (CFUs) of the microbials species that can be spores, active vegetative hyphae, and active parts of fungal tissue, it may include as well dormant hyphae and parts of fungal tissue, e.g. dehydrated or lyophilized 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 viable and able to multiply for example via binary fission under controlled conditions. The inoculum is introduced into and / or onto a larger volume of the scaffolding substrate in order 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 may contain both the vegetative and reproductive structures of the fungus as well as a spawning substrate. Spawning substrate can be grains, vermiculite, sawdust or other appropriate powdered or granulated substrate that ensures a large amount of colony forming units once the inoculum has been mixed with the final substrate.

[0045] The number of colony forming units in the inoculum and its composition can vary depending on the desired outcome and the characteristics of the microbials species being used. In some embodiments, the inoculum is directly introduced into and / or on the substrate to deliver microbial species both on the surface and / or inside. In other embodiments, the microbial species and / or the spores are in an aqueous suspension and the inoculum is introduced into and on the substrate to deliver microbial species both on the surface but also inside. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. Said fungi are preferably selected from the group consisting of ascomycetes, basidiomycetes, deuteromycetes, oomycetes, and / or zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Sporidiobolus, Staphylococus or Pleurotus. More specifically, the species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camembert!, Penicillium rogueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or a combination thereof.

[0046] In some embodiments the first microbial species is a filamentous fungus species comprising the genus Penicillium and preferably Penicillium nalgiovense and the second microbial species is a filamentous fungus species comprising the genus Rhizopus and preferably Rhizopus oligosporus. The inoculation of the first filamentous fungus species can be performed by spreading colony forming units on the scaffolding substrate. For this embodiment the colony forming units are spores of the respective filamentous fungus species. A first spore concentration of the first filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second spore concentration of the second filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108. Preferably the ratio of the first spore concentration to the second spore concentration ranges from 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150.

[0047] In some other embodiments the first microbial species is a filamentous fungus species comprising the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a filamentous fungus species comprising the genus Aspergillus and preferably Aspergillus oryzae. The inoculation of the first filamentous fungus species can be performed by spreading colony forming units on the scaffolding substrat. For this embodiment the colony forming units are spores of the respective filamentous fungus species. A first spore concentration of the first filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from l x 104to l x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second spore concentration of the second filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108. Preferably a ratio of the first spore concentration to the second spore concentration ranges from 0.01 to 2000, preferably 0.1 to 1000, and more preferably 20 to 500.

[0048] During the incubation 130, the scaffolding substrate is incubated. The microbial species inoculated during the inoculation 120 grows and develops into and on the substrate. In order to facilitate the growth of 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 formation of unpleasant off-flavours, such as resulting from formation of ammonia, organic acid accumulation, and / or before sporulation.

[0049] Incubation 130 can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate. Incubation 130 can be, for example, liquid fermentation or more preferably, solid-state fermentation (SSF). The fermentation conditions and time may be adjusted to the microbial species, the available nutrients, the composition of the substrate and to the desired result. Preferably, the scaffolding is incubated at a temperature between 4 and 70°C, in particular between 10 and 50°C, more preferably between 14°C and 40°C and more preferably between 22°C and 38°C.

[0050] Preferably, the incubation 130 is terminated prior to spore formation.

[0051] Microbial growth 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 growth of the microbial species. Alternatively, microbial growth may be interrupted by decreasing oxygen concentration to below the critical level required by the respective microbial species to grow. Preferably, the growth of the filamentous fungus species is interrupted by heating the food product to above 60°C, preferably above 71°C for at least 1 minute and more preferably more than 30 minutes, whereas the temperature is measured in the centre of the product.

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

[0053] FIG. 2 presents another embodiment for forming a food product, comprising steps of providing a scaffolding substrate 110, inoculating 120a the scaffolding substrate with at least a first microbial species, incubating 130a the scaffolding substrate with optimal conditions to grow the first microbial species, inoculating 120b with at least a second microbial species, preferably the second microbial specie is a filamentous fungus species, incubating 130b the scaffolding substrate with optimal conditions to grow the second microbial species.

[0054] In some embodiments, the inoculation can be carried out in a successive step, a first inoculation 120a of said scaffolding substrate with a first microbial species, and then, subsequently, a second inoculation 120b of said scaffolding substrate with a second microbial species, preferably a filamentous fungus species.

[0055] In some embodiments, the incubation 130 can be carried out in a successive step, for example a first incubation 130a and a second incubation 130b where the two incubations have different parameters to grow the microbial species.

[0056] In inoculation 120a, the scaffolding substrate is inoculated with at least one microbial species. The inoculation can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate.

[0057] The inoculation 120a can be done with an inoculum. Inoculum contain an amount of colony forming units (CFUs) of the microbials species that can be spores, active vegetative hyphae, and active parts of fungal tissue, it may include as well dormant hyphae and parts of fungal tissue, e.g. dehydratedor lyophilized hyphae and parts of fungal tissue. The inoculum is introduced into and / or onto a larger volume of the scaffolding substrate in order 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 may contain both the vegetative and reproductive structures of the fungus as well as a spawning substrate. Spawning substrate can be grains, vermiculite, sawdust or other appropriate powdered or granulated substrate that ensures a large amount of colony forming units once the inoculum has been mixed with the final substrate.The size and composition of the inoculum can vary depending on the desired outcome and the characteristics of the microbial species being used. In some embodiments, the inoculum is directly introduced into and / or on the substrate to deliver microbial species both on the surface and / or inside. In other embodiments, the microbial species and / or the spores are in an aqueous suspension and the inoculum is introduced into and on the substrate to deliver the microbial species both on the surface but also inside. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. Said fungi are preferably selected from the group consisting of ascomycetes, basidiomycetes, deuteromycetes, oomycetes, and / or zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococus, Sporidiobolus or Pleurotus. More specifically, the species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camembert!, Penicillium rogueforti, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus eryngii, or a combination thereof. In some embodiments Penicillium nalgiovense, Lactobacillus, Sporidiobolus pararoseus, Aspergillus Sojae or Saccharomyces are inoculated.

[0058] The first incubation 130a of said scaffolding substrate allows the first microbial species to grow inside, outside, or inside and outside said scaffolding substrate. Incubation 130a can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate. Incubation 130a can be, for example, liquid fermentation or more preferably, solid-state fermentation (SSF). The fermentation conditions and time may be adjusted to the microbial species, the available nutrients, the composition of the substrate and to the desired result. Preferably, the incubation conditions are adjusted to avoid formation of unpleasant off-flavours, such as resulting from formation of ammonia, organic acid accumulation, and / or before sporulation.

[0059] The microbial species inoculated during the inoculation 120a grows and develops into and on the scaffolding substrate. In pursuance to facilitate the growth of microbial species, this step is carried out under controlled parameters, such as temperature, humidity, oxygen concentration and / or time. The incubation 130a can be carried out at a temperature between 5 and 50°C, preferably between 10 and 40°C and preferably between 20 and 30°C, for a time period inferior to 72h, preferably for a time period inferior to 48 h.

[0060] In one embodiment, the incubation 130a is terminated prior to spore formation. Microbial growth 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 growth of the first microbial species. Alternatively, microbial growth may be interrupted by decreasing oxygen concentration to below the critical level required by the microbial species to grow. Alternatively, the growth of the first microbial species is interrupted by heating the food product to above 60°C, preferably above 71°C for at least 1 minute and more preferably more than 30 minutes, whereas the temperature is measured in the centre of the product.

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

[0062] During inoculation 120b, the scaffolding substrate is inoculated a second time with at least one microbial species, containing at least one filamentous fungus species. The inoculation 120b can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate.

[0063] The inoculation 120b can be done with an inoculum. Inoculum contain an amount of colony forming units (CFUs) of the microbials species that can be spores, active vegetative hyphae, and active parts of fungal tissue, it may include as well dormant hyphae and parts of fungal tissue, e.g. dehydrated or lyophilized hyphae and parts of fungal tissue. The inoculum is introduced into and / or onto a larger volume of the scaffolding substrate in order 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 may contain both the vegetative and reproductive structures of the fungus as well as a spawning substrate. Spawning substrate can be grains, vermiculite, sawdust or other appropriate powdered or granulated substrate that ensures a large amount of colony forming units once the inoculum has been mixed with the final substrate.

[0064] The size and composition of the inoculum can vary depending on the desired outcome and the characteristics of the microbial species being used. In some embodiments, the inoculum is directly introduced into and / or on the substrate to deliver microbial species both on the surface and / or inside. In other embodiments, the microbial species and / or the spores are in an aqueous suspension and the inoculum is introduced into and on the substrate to deliver microbial species both on the surface but also inside. The inoculated microbial species can be algae, fungi, bacteria, or a combination thereof. Said fungi are preferably selected from the group consisting of ascomycetes, basidiomycetes, deuteromycetes, oomycetes, and / or zygomycetes, in particular edible species belonging to the genus Rhizopus, Aspergillus, Penicillium, Ganoderma, Staphylococus, Sporidiobolus or Pleurotus. More specifically, the species Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Aspergillus oryzae, Aspergillus luchuensis, Aspergillus sojae, Penicillium nalgiovense, Penicillium camembert!, Penicillium rogueforti, 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 incubation 130b of said scaffolding substrate allows the second microbial species to grow inside, outside, or inside and outside said scaffolding substrate. During the incubation 130b, the scaffolding substrate is incubated. The growing conditions are optimized for the second microbial species. Therefore, the second microbial species grow and develop into and on the substrate. The second microbial species form a matrix in and around the scaffolding substrate to bind the scaffolding substrate together. In one embodiment, the filamentous fungus species form a network that binds to themselves and to the substrate material. For the sake of facilitating 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 can be the same or be different from those used at the first incubation 130a. The growth of the microbial species inoculated during the inoculation 120a can therefore also continue to grow or not.

[0066] The incubation 130b can be carried out in any way and consequently additional subsequent steps may be necessary to obtain a good development of the microbial species inside and / or around the substrate. Incubation 130b can be, for example, liquid fermentation or more preferably, solid-state fermentation (SSF). The fermentation conditions and time may be adjusted to the microbial species, the available nutrients, the composition of the substrate and to the desired result. The incubation 130b can be carried out at a temperature of between 20 and 45°C, preferably between 28 and 35°C, for a time period inferior to 72 h, preferably for a time period inferior to 44 h.

[0067] Preferably, the incubation 130b is terminated prior to spore formation. Preferably, the incubation conditions are adjusted to avoid formation of unpleasant off-flavours, such as resulting from formation of ammonia, organic acid accumulation, and / or sporulation.

[0068] Microbial growth 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 growth of the at least one filamentous fungus species. Alternatively, the growth of the filamentous fungus species may be interrupted by decreasing oxygen concentration to below the critical level required by the respective filamentous fungus species to grow. Preferably, the growth of the filamentous fungus species is interrupted by heating the fungus-containing product to above 60°C, preferably above 71°C for at least 1 minute and more preferably more than 30 minutes, whereas the temperature is measured in the centre of the product.

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

[0070] It is understood that it is possible to add other iterations of inoculation steps and / or incubation steps in order to have 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. In particular, it is understood that during the first inoculation 120a Penicillium nalgiovense and Sporidiobolus pararoseus can be inoculate, during the second inoculation 120b Rhizopus oligosporus can be inoculate and during a third inoculation Aspergillus oryzae can be inoculate. In this example, Penicillium nalgiovense allows Rhizopus oligosporus to have a prolific growth and Sporidiobolus pararoseus allows Aspergillus oryzae to have a prolific growth. Penicillium nalgiovense and Sporidiobolus pararoseus grow during the first incubation 130a, whereas a second incubation 130b with optimal condition for Rhizopus oligosporus can bind all the pieces of the scaffolding substrate together, and a third incubation with optimal condition for Aspergillus oryzae can create a fungi-based skin. The fungi-based skin should be present on the surface of the scaffolding substrate with a high density of mycelium which covers the substrate with a thickness between 0.1 and 5 mm.

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

[0072] The shaping can also be used to form a fungi-based skin on the surface of the final product. Indeed, the growth of filamentous microbials species (or filamentous fungus) also occurs on the outer surface of the scaffolding substrate and can proliferate in a volume beyond the scaffolding substrate. By compacting these filaments on the surface of the scaffolding substrate, it is possible to obtain a compact layer made mainly by filaments which create a fungi-based skin on the surface of the product. It is understood that it is possible to use the method 100 several times, using the finished food product from the previous method 100, as a scaffolding substrate for the next use of the method 100, to create different aspects to obtain a finished product that best imitates a piece of meat or a meat product. For example, a first application of method 100 to bind several scaffolding substrates together, then a second application of method 100 to create a fungus-based skin on the surface of the finished product.

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

[0074] In some embodiments the aqueous solution is only composed of water. The ratio between the mass of substrate and the mass of water can be from 1:1 to 1:2 ratio, more preferably from 1:1.5 to 1:1.8.

[0075] In other embodiments the aqueous solution can be composed of water but also color, flavors, any compounds useful to grow the microbial species such as nutrient and / or any compounds that stop the growth and / or eradicate other non-wanted microbial species.

[0076] The hydration is a growth condition for the microbial species to develop and grow into the substrate. It is also the case for non-wanted microbial species. To reduce the number of microbial species present, the scaffolding substrate can be pasteurized, double pasteurized, sterilized, treated by antibacterial compounds and / or treated by an acid. For example, the scaffolding substrate may be sterilized in an autoclave at up to 120°C. Another example is the acidification, the surface of the scaffolding substrate is treated by an acid, preferably a food-grade acid, more preferably lactic acid, acetic acid, malic acid, citric acid, or succinic acid, preferably to reach a pH of below 6, most preferably between 4.5 and 4.6 at the surface. Even more preferably, the pH on the surface is adjusted to ensure growth of the microbial species used and to reduce growth of other microorganisms.

[0077] In another embodiment the scaffolding substrate and the aqueous solution are treated separately to then be brought into contact and thus hydrate the scaffolding substrate.

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

[0079] In another embodiment, the food product has been developed to mimic a meat product. The food product can be shaped to replicate the shape of the meat product it is replacing in order to facilitate easy recognition and identification by consumers.

[0080] In particular, the food product is shaped to closely resemble the size, thickness, and overall form 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 that is similar in size and thickness to a traditional meat whole cut. Similarly, if the meat product being imitated is a salami or a sausage, the food product will have a shape that is similar in size and thickness to a traditional salami or sausage.

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

[0082] In another embodiment, the food product can be shaped using various methods to achieve a desired shape, texture, and appearance. One such method is vacuum shaping, which involves placing the food product in a mold and applying vacuum pressure to shape the product.

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

[0084] Additional methods of shaping the food product can include molding, casting, and cutting, among others. Molding involves pouring the food product into a mold and allowing it to cool and set, 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 in the food product, such as slices or cubes.

[0085] It should be noted that the shaping methods are not limited to the methods described herein, and that 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 scaffolding substrate is placed inside a shell which is larger than the substrate so that a free space is created between the substrate and the inside face of the shell. The mycelium of the fungi will fill, at least partially, the free space and form a fungi-based skin around the scaffolding substrate. The free space can have a thickness between 0.1mm and 5 cm. The substrate can be held by any means, for example by a rod connected to the shell and crossing the substrate right through, the shell can also have one or more supports on which the substrate is placed.

[0087] The food product comprises at least two different phases, preferably the two different phases can be distinguished by human eye, more preferably a first phase comprising the scaffolding substrate and a second phase comprising the mycelium of the at least one filamentous fungus species, preferably, wherein a ratio of the second phase of the food product, which is define as a ratio of an area occupied by the second phase in a cross section of the food product to a total area of said cross section, range from 0.001 to 0.95 and more preferably from 0.1 to 0.6.

[0088] Once the food product is formed, according to the method 100 described above, further processing may be performed to improve taste, preservation and / or transportation. In particular, the food product may be marinated, spiced, smoked, cured, dehydrated, pressed, infused with water and / or oil, steamed, boiled or post-processed in any other way as typically done with animal meat products.

[0089] The food product is designed to provide a satisfying and juicy experience for consumers, similar to that of a traditional meat product, for example by releasing juices when bitten or cut, further enhancing the overall juiciness experience.

[0090] In one embodiment, the fermented food product is more nutritious than the edible substrate, preferably wherein the proteins in the edible substrate are more digestible than prior to fermentation, the fermentation reduces antinutritional factors to enhance bioavailability of micronutrients. Some non-limiting possible examples are higher antioxidant activity, higher total phenolic content, increased soluble proteins, higher ellagic acid (anti-carcinogenic), increased content of L-DOPA and / or decrease in cholesterol.

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

[0092] In one embodiment, the food product contains the DNA and RNA of each microbial species inoculated during the method 100. The presence and genetic identity of at least two microbial species in the food product can be determined upon analysis of the final product through chain polymerase reaction (PCR) amplification of specific genetic fragments, more preferably via qPCR-analysis, and incombination with genetic sequencing (e.g. Sanger sequencing), the quantity of microbial DNA and / or RNA from each species inoculated during the 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 microbial species inoculated during the method 100. The amount of DNA and / or RNA from the microbial species inoculated is expected to exceed 10 times the quantity of DNA and / or RNA of other microbial species.Example 1

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

[0095] Pea protein-based TVP pieces with a size between 4-15 mm were hydrated with 170 g water and 2 g lactic acid per 100 g of TVP product, and pasteurized in a vacuum package at 90 °C for 30 min. From the prepared TVP pieces three series A, B, and C were produced.• Series A was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 107spores per kg substrate to start a mono-culture incubation with one species.• Series B was inoculated with Penicillium nalgiovense at a spore concentration of 9 x 108per kg substrate to start a mono-culture incubation with one species.• Series C was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 107spores per kg substrate and Penicillium nalgiovense at a spore concentration of 9 x 108per kg substrate to start a co-culture incubation with two species.

[0096] The TVP pieces were inoculated by homogenously covering their surfaces with the powdered starter cultures through mechanical mixing. 30 g of the inoculated TVP pieces were placed into vented petri dishes and incubated in an incubator at 30 °C and a relative humidity of 98 % for 18.5 h. The fermented TVP pieces were then photographed and observed under a microscope. As can be seen in FIG.5, after incubation the TVP pieces are covered in an aerial hyphae which form a mycelium network that appears like white fuzz on the TVP surface. FIG.3 and FIG.6 depict the incubation results of Series A. FIG.4, FIG.7, and FIG.9 depict the incubation results of Series B. FIG.5 and FIG.8 depict the incubation results of Series C. The fungal mycelium formed in the mono-cultural incubation in Series A and B, shown in FIG.3 and FIG.6, and respectively in FIG.4, FIG.7, and FIG.9, is less dense compared to the fungal mycelium formed in the co-cultural incubation in Series C shown in FIG.5 and FIG.8. Thus, a higher mycelium density is achieved with co-culture which translates into increased control of texture in a food product produced therefrom. This example demonstrates how wanted growth characteristics such as mycelium density and faster growth are promoted by co-cultural incubation.Example 2

[0097] Bodies consisting of individual TVP pieces bound by mycelium in co-culture or mono-culture were produced and compared in terms of mycelium binding strength in tensile tests. 100 g of pea protein-based TVP pieces with a size between 4-15 mm were hydrated with 170 g water and 5 g of an 80% lactic acid solution, and pasteurized in a vacuum package at 92°C for 30 min. From the prepared TVP pieces three series A, B and C were produced.• Series A was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 107spores per kg substrate and Penicillium nalgiovense at a spore concentration of 9 x 108per kg substrate to start a co-culture incubation with two species.• Series B was inoculated with Rhizopus oligosporus at a spore concentration of 1.2 x 107spores per kg substrate to start a mono-culture incubation with one species.• Series C was inoculated with Penicillium nalgiovense at a spore concentration of 9 x 108per kg substrate to start a mono-culture incubation with one species.

[0098] The TVP pieces were inoculated by homogenously covering their surfaces with the powdered starter cultures through mechanical mixing. The inoculated TVP pieces from series A, B and C were then shaped into a cylindrical body of 20 mm diameter and 180 mm length with a perforated polyethylene film on the outside. The cylindrical bodies were incubated in an incubator at a temperature of 30 °C and a relative humidity of 95 % for 32 h. While the TVP pieces were held together by the polyethylene film prior to incubation, the pieces in Series A and B adhered to each other after incubation through mycelial growth fed by nutrients available in the pieces. Series C did not show any binding due to the hyphae of the inoculated Penicillium nalgiovense being too short to bridge between the individual TVP pieces (cf. FIGS. 7 and 9). The polyethylene film was then removed, the cylindrical bodies were vacuum-packaged, and heat treated at 90 °C for 20 min. From the cylindrical bodies rectangular pieces were cut of 11x17x50 mm. The rectangular pieces were then tensile tested in a Texture Analyzer ZwickRoell 2.5 kN zwicki RetroLine, gap set to 10 m, preload at 0.1 N, and extension speed of 200 mm / min. The samples were elongated until they fractured along the middle. The samples fractured along the mycelium phase which binds together the individual TVP pieces. It could be assumed that the cross-sectional area remained close to constant during the tensile test. The stress was calculated by dividing the force N applied to pull the sample apart by the cross-sectional area. The results are displayed in a stress-strain diagram, as can be seen in FIG.10. In the case of series C with Penicillium nalgiovense no binding of the TVP pieces into a coherent body after incubation was achieved. Because of this series C could not be fixed in the experimental set up and was not tensile tested. Thus, no stress-response could be measured. The mechanical reaction of Series C is depicted representatively in FIG.10. The region of fracture of series A is marked with a dashed rectangle named "a", the region of fracture of series B is marked with a dashed rectangle named "b", and the representative region of fracture of series C is marked with a dashed rectangle named "c". The results show that pieces bound in co-cultural incubation with the 2 species Rhizopus oligosporus and Penicillium nalgiovense in series A fractured at higher stresses than pieces bound in mono-cultural incubation with 1 species Rhizopus oligosporus or 1 species Penicillium nalgiovense in series B respectively series C. If the fracture stresses of the monocultural incubation series B and C were to be added up, they remain smaller than the fracture stresses of series A. Thus, this example demonstrates that the combination of the two strains in a co-incubation leads to a more prolific growth of the Rhizopus oligosporus resulting in an increased binding capacity. Increased binding capacity can be used to tailor the texture in a food product.Example 3

[0099] To create the prototypes, TVP pieces were hydrated with distilled water and lactic acid to achieve 60% moisture content. The hydrated TVP pieces were then vacuumed and pasteurized in the VWB2 water bath for 35 minutes at 95°C. After cooling to room temperature, the TVP pieces were inoculated with the desired strains under the BioVanguard Green Line laminar flow. The inoculum and the TVP pieces were shaken in the vacuum bag itself to ensure equal distribution of the spores and the raw material. Several combinations of fungi are inoculated: a first combination with Rhizopus delemar and Penicillium nalgiovense, a second combination with Rhizopus oligosporus and Penicillium nalgiovense, a third combination with Rhizopus delemar and Rhizopus oligosporus, a fourth combination with Rhizopus delemar with Rhizopus oligosporus and Penicillium nalgiovense and also two control prototypes being inoculated with only Rhizopus delemar or only Rhizopus oligosporus. Sextuplicate of substrate bars were prepared in 3D-printed molds with a size of 14 cm long, 28 mm thick and 25 mm wide. Each bar shape was filled with 80 g of hydrated and inoculated TVP. After fillingthe molds, inoculated TVP were put into the incubator for fermentation. At the end of the incubation cycle, the prototypes were harvested, refrigerated, and used for mechanical testing.

[0100] The tensile strength of the bars was measured using the ZwickiLine Texture Analyser. The setup can be seen in Fig. 11. The bar prototypes were analyzed with the tensile test, measuring the maximum tensile strength (Fmax) of the bar prototype in Megapascal (MPa). The Texture Analyzer measured the value of Fmax by dividing the maximum tensile force by the cross-sectional area of the bar prototype. For the analysis, the bar prototype was fixed into the clamps of the texture analyser with a grip separation of 2.2 mm and at initial separation of the clamps of 50 mm. The pre-load was 0.2 Newton (N) and the speed of the extension was 0.5 mm / s.

[0101] Results are presented on Fig. 12 and show that all combinations comprising at least one Rhizopus spp. and Penicillium nalgiovense present an increase of tensile strength on average 30%. Thus, these examples demonstrate that several combinations of two strains in a co-incubation lead to a more prolific growth of the Rhizopus strains resulting in an increased binding capacity of the food product.The invention is described in the following aspects:1. A method to prepare a food product, the method comprising steps of: a) providing a scaffolding substrate; b) inoculating said scaffolding substrate with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species; and c) incubating said scaffolding substrate to allow the at least two microbial species to grow inside, outside, or inside and outside said scaffolding substrate.2. The method of the preceding aspect, wherein the at least two microbial species enable a prolific growth, which is preferably produced by carrying out step c), the prolific growth comprising an increased mechanical strength, preferably tensile strength or an increased mycelium density or an increased tensile strength and an increased mycelium density.3. The method of any preceding aspect, wherein the at least two microbial species enable a prolific growth of the at least one filamentous fungus species.4. The method of any preceding aspect, wherein the method comprising inoculating said scaffolding substrate with a given number of colony forming units of the at least one filamentous fungus species, wherein the filamentous fungus species grows more densely than it does when said scaffolding substrate is only inoculated with the given number of colony forming units of the filamentous fungus species and incubated without presence of other one(s) of the at least two microbial species.5. The method of any preceding aspect, wherein the at least two microbial species comprising a first microbial species and a second microbial species which is a filamentous fungus species, the method comprising inoculating said scaffolding substrate with a first number of colony forming units of the first microbial species and inoculating said scaffolding substrate with a second number of colony forming units of the second microbial species,wherein the food product obtained by step c), preferably immediately after step c) without being subject to further processes, has a tensile strength greater than a sum of tensile strengths of a first product and of a second product, the first product being obtained by inoculating a first corresponding scaffolding substrate with the first number of colony forming units of the first microbial species, and incubating said first corresponding scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said first corresponding scaffolding substrate; the second product being obtained by inoculating a second corresponding scaffolding substrate with the second number of colony forming units of the second microbial species, and incubating said second corresponding scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said second corresponding scaffolding substrate.6. The method of any preceding aspect, wherein the scaffolding substrate is made from proteins from at least one plant source, preferably one protein source is pea protein, preferably the scaffolding substrate contains at least 50% protein in the dry matter, preferably 60% or more in the dry matter.7. The method of any preceding aspect, wherein the scaffolding substrate is made from TVP, preferably the TVP scaffolding substrate is made with one protein source which is pea protein, preferably the TVP scaffolding substrate contains at least 50% of pea protein in the dry matter, preferably 60% or more of pea protein in the dry matter.8. The method of any preceding aspect, wherein the scaffolding substrate comprises at least one chunky piece or an assembly of chunky pieces, preferably a number of the chunky pieces in the assembly being 2 or more.9. The method of the preceding aspect, wherein an aspect ratio of the chunky pieces, which is defined as a ratio of a longest dimension to a shortest dimension of a respective chunky piece, is 10 or less, 5 or less, 3 or less, or 2 or less.10. The method of aspect 8 or 9, wherein an average aspect ratio of the chunky pieces, which is defined as an aspect ratio of 15 randomly selected chunky pieces, the aspect ratio being defined as a ratio of a longest dimension to a shortest dimension of a respective chunky piece, is 10 or less, 5 or less, 3 or less, or 2 or less.11. The method of any preceding aspect, wherein the scaffolding substrate comprises at least one fiber or an assembly of fibers, preferably a number of the fibers in the assembly being 2 or more.12. The method of aspect 11, wherein an aspect ratio of the fibers which is defined as a ratio of a longest dimension to a shortest dimension of a respective fiber, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.13. The method of any preceding aspect, wherein the scaffolding substrate comprises at least one sheet or a stack of sheets, preferably a number of the sheets in the stack being 2 or more.14. The method of the preceding aspect, wherein an aspect ratio of the sheets which is defined as a ratio of a longest dimension to a shortest dimension of a respective sheet, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.15. The method of any of aspects 8-14, wherein the chunky pieces, fibers and / or the sheets are bound together by growth of the at least one filamentous fungus species.16. The method of any of aspects 8-15, wherein the chunky pieces, fibers and / or the sheets can be placed in an organized manner, for example all the longest dimension of the chunky pieces and / or fibers and / or the sheets can be substantially aligned along the same direction.17. The method of any of aspects 8-15, wherein the chunky pieces and / or the sheets can be placed in a non-organized manner, for example all the longest dimension and / or the shortest dimension of the chunky pieces and / or fiber and / or the sheets are not substantially aligned along the same direction.18. The method of any preceding aspect, wherein other one(s) of the at least two microbial species comprise a fungus species, an algae species and / or a bacterium species, preferably a fungus species, more preferably a filamentous fungus species.19. The method of any preceding aspect, wherein the first microbial species is a first filamentous fungus species comprising the genus Penicillium and preferably Penicillium nalgiovense and the second microbial species is a second filamentous fungus species comprising the Rhizopus genus and preferably Rhizopus oligosporus and / or Rhizopus delemar.20. The method of the preceding aspect, wherein a first colony forming units concentration of the first filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second colony forming units concentration of the second filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108.21. The method of the preceding aspect, wherein a ratio of the first colony forming units concentration to the second colony forming units concentration ranges from 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150.22. The method of any preceding aspect, wherein the first microbial species is a first filamentous fungus species comprising the genus Sporidiobolus, preferably Sporidiobolus pararoseus, and the second microbial species is a second filamentous fungus species comprising the genus Aspergillus, preferably Aspergillus oryzae.23. The method of the preceding aspect, wherein a first colony forming units concentration of the first filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and asecond colony forming units concentration of the second filamentous fungus species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x IO10, and more preferably 1 x 107to 1 x 108.24. The method of the preceding aspect, wherein a ratio of the first colony forming units concentration to the second colony forming units concentration ranges from 0.01 to 2000, preferably 0.1 to 1000, and more preferably 20 to 500.25. The method of any preceding aspect, wherein step b) comprises steps of bl) inoculating said scaffolding substrate with a first microbial species; and then, subsequently, b2) inoculating said scaffolding substrate with a second microbial species, preferably the second microbial species being the filamentous fungus species.26. The method of the preceding aspect, wherein step c) comprises steps of cl) incubating said scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said scaffolding substrate; and then c2) incubating said scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said scaffolding substrate.27. The method of aspect 25 in accordance with aspect 26, wherein step cl) is carried out before or after step b2).28. The method of any of aspects 25-27, wherein the first microbial species comprises the genus Penicillium, the genus Lactobacillus, the genus Sporidiobolus and / or the genus Aspergillus, preferably Penicillium nalgiovense, Lactobacillus plantarum, Sporidiobolus pararoseus, Aspergillus sojae, Aspergillus luchuensis and / or Saccharomyces cerevisiae.29. The method of any of aspects 25-28, wherein the second microbial species comprises the genus Rhizopus, the genus Penicillium and / or the genus Aspergillus, preferably Rhizopus oligosporus, Rhizopus delemar, Rhizopus oryzae, Penicillium nalgiovense and / or Aspergillus oryzae.30. The method of any 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 time period of less than 72 h and preferably less than 48 h, and / or step c2) is carried out at a temperature of 20-45°C, preferably 28-35°C, for a time period of less than 72 h, preferably less than 44 h.31. The method of any preceding aspect, wherein step c) comprises forming a fungi-based skin surrounding said scaffolding substrate, preferably by steps of: placing said scaffolding substrate in a shell to form a confined space between said scaffolding substrate and the shell, and carrying out step c) to allow the at least two microbial species to grow within the confined space, preferably a thickness of the confined space ranges from 0.1 mm to 5 cm, more preferably 0.5 mm to 2.5 cm, even more preferably 1 mm to 1 cm.32. The method of any preceding aspect, wherein the scaffolding substrate comprises a textured vegetable protein (TVP).33. The method of any preceding aspect, further comprising sterilizing and / or pasteurizing the scaffolding substrate before step b).34. The method of any preceding aspect, further comprising hydrating the scaffolding substrate by an aqueous solution such that the scaffolding substrate has a water activity above 0.8 during steps b) and / or c).35. The method of any preceding aspect, wherein step c) is performed under solid state fermentation conditions.36. The method of any preceding aspect, further comprising shaping the scaffolding substrate before and / or after step b).37. A food product prepared by the method of any preceding aspect.38. A food product comprising a scaffolding substrate and mycelium of at least one filamentous fungus species grown inside, outside, or inside and outside the scaffolding substrate, the scaffolding substrate being formed by one or more pieces of textured vegetal protein (TVP).39. The food product of any of the two preceding aspects, wherein said food product comprises at least two different phases, preferably the two different phases can be distinguished by human eye, more preferably a first phase comprising the scaffolding substrate and a second phase comprising the mycelium of the at least one filamentous fungus species.40. The food product of the preceding aspect, wherein a ratio of the second phase of the food product, which is define as a ratio of an area occupied by the second phase in a cross section of the food product to a total area of said cross section, range from 0.001 to 0.95 and more preferably from 0.1 to 0.6.Various aspects, embodiments or examples of the invention have been described for purposes of illustration. The invention, however, should not be unduly limited by any of the details of the above disclosure, as a skilled person in the art will appreciate that changes and modifications that do not contradict the principle of the invention may be made and are still covered by the invention. In particular, the invention shall not be construed to be limited to the embodiments described above with reference to the drawings. Rather, the scope of protection of the invention is determined solely by the appended claims and their equivalents.

Claims

CLAIMS1. A method to prepare a food product, the method comprising steps of: a) providing a scaffolding substrate; b) inoculating said scaffolding substrate with at least two microbial species, the at least two microbial species comprising at least one filamentous fungus species, preferably a first filamentous fungus species and a second filamentous fungus species; and c) incubating said scaffolding substrate to allow the at least two microbial species to grow inside, outside, or inside and outside said scaffolding substrate, wherein the at least two microbial species enable a prolific growth of the at least one filamentous fungus species.

2. The method of claim 1, the method comprising inoculating said scaffolding substrate with a given colony number of forming units of the at least one filamentous fungus species, wherein the filamentous fungus species present a more prolific growth than it does when said scaffolding substrate is only inoculated with the given number of colony forming units of the filamentous fungus species and incubated without presence of other one(s) of the at least two microbial species.

3. The method of any preceding claim, wherein the prolific growth is produced by carrying out step c), the prolific growth comprising an increased mechanical strength, preferably tensile strength or an increased mycelium density or an increased tensile strength and an increased mycelium density.

4. The method of any preceding claim, wherein the at least two microbial species comprising the first microbial species and the second microbial species which is a filamentous fungus species, the method comprising inoculating said scaffolding substrate with a first number of colony forming units of the first microbial species and inoculating said scaffolding substrate with a second number colony forming units of the second microbial species, wherein the food product obtained by step c), preferably immediately after step c) without being subject to further processes, has a tensile strength greater than a sum of tensile strengths of a first product and of a second product, the first product being obtained by inoculating a first corresponding scaffolding substrate with the first number of colony forming units of the first microbial species, and incubating said first corresponding scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said first corresponding scaffolding substrate; the second product being obtained by inoculating a second corresponding scaffolding substrate with the second number of colony forming units of the second microbial species, and incubating said second corresponding scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said second corresponding scaffolding substrate.

5. The method of any preceding claim, wherein the scaffolding substrate comprises at least one chunky piece or an assembly of chunky pieces, preferably a number of the chunky pieces in the assembly being 2 or more, more preferably an aspect ratio of the chunky pieces, which is defined as a ratio of a longest dimension to a shortest dimension of a respective chunky piece, is 10 or less, 5 or less, 3 or less, 2 or less.

6. The method of any preceding claim, wherein the scaffolding substrate comprises at least one fibre or an assembly of fibers, preferably a number of the fibers in the assembly being 2 or more, more preferably an aspect ratio of the fibers which is defined as a ratio of a longest dimension to a shortest dimension of a respective fiber, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.

7. The method of any preceding claim, wherein the scaffolding substrate comprises at least one sheet or a stack of sheets, preferably a number of the sheets in the stack being 2 or more, more preferably an aspect ratio of the sheets which is defined as a ratio of a longest dimension to a shortest dimension of a respective sheet, is 10 or more, or 100 or more, or 1000 or more, and preferably less than 10000.

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

9. The method of the preceding claim, wherein a first colony forming units concentration of the first microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second colony forming units concentration of the second microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108, wherein, preferably the ratio of the first colony forming units concentration to the second colony forming units concentration ranges from 0.01 to 1000, preferably 0.1 to 500, and more preferably 50 to 150.

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

11. The method of the preceding claim, wherein a first colony forming units concentration of the first microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1012, preferably from 1 x 10sto 1 x 1010, and more preferably from 1 x 108to 1 x 109, and a second colony forming units concentration of the second microbial species, in terms of spores per kg of the scaffolding substrate, ranges from 1 x 104to 1 x 1013, preferably 1 x 105to 1 x 1010, and more preferably 1 x 107to 1 x 108, wherein, preferablya ratio of the first colony forming units concentration to thesecond colony forming units concentration ranges from 0.01 to 2000, preferably 0.1 to 1000, and more preferably 20 to 500.

12. The method of any preceding claim, wherein step b) comprises steps of bl) inoculating said scaffolding substrate with a first microbial species; and then, subsequently, b2) inoculating said scaffolding substrate with a second microbial species, preferably the second microbial species being the filamentous fungus species.

13. The method of the preceding claim, wherein step c) comprises steps of cl) incubating said scaffolding substrate to allow the first microbial species to grow inside, outside, or inside and outside said scaffolding substrate; and then c2) incubating said scaffolding substrate to allow the second microbial species to grow inside, outside, or inside and outside said scaffolding substrate, wherein step cl) is carried out 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 time period 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 time period of less than 72 h, preferably less than 44 h.

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

15. The food product of claim 14, wherein said food product comprises at least two distinct phases, preferably the two distinct phases can be distinguished by human eye, more preferably a first phase comprising the scaffolding substrate and a second phase comprising the mycelium of the at least one filamentous fungus species, preferably, wherein a ratio of the second phase of the food product, which is define as a ratio of an area occupied by the second phase in a cross section of the food product to a total area of said cross section, range from 0.001 to 0.95 and more preferably from 0.01 to 0.6.

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