High-pressure texturization of frozen fungal biomass and food production methods therefrom

Abstract

The present invention relates to a compressed fungal ingredient dough, comprising fibrous mycelium biomass and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight and wherein said dough characterized by a homogeneous cutting strength of at least 14 N. The present invention further relates to a method for preparing a compressed fungal ingredient dough, the method comprising the steps of (a) providing a fibrous mycelium biomass; (b) forming a fungal ingredient dough comprising the biomass of (a) and a binding agent; (c) freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a compressed fungal ingredient dough. The present invention likewise relates to a compressed fungal ingredient dough obtainable according to the method of the present invention, and to a food product comprising said compressed fungal ingredient dough. Further to that, the present invention relates to a method for mechanically reducing nucleic acid content, in particular RNA content in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen. The present invention likewise relates to a method for mechanically controlling, and in particular reducing, the equivalent umami concentration (EUC) in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

Description

[0001] New PCT-Patent Application

[0002] Mushlabs GmbH

[0003] Vossius Ref.: AJ3269 PCT-0 BS

[0004] High-pressure texturization of frozen fungal biomass and food production methods therefrom

[0005] Field of the invention

[0006] The present invention relates to a compressed fungal ingredient dough, comprising fibrous mycelium biomass and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight and wherein said dough characterized by a homogeneous cutting strength of at least 14 N. The present invention further relates to a method for preparing a compressed fungal ingredient dough, the method comprising the steps of (a) providing a fibrous mycelium biomass; (b) forming a fungal ingredient dough comprising the biomass of (a) and a binding agent; (c) freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a compressed fungal ingredient dough. The present invention likewise relates to a compressed fungal ingredient dough obtainable according to the method of the present invention, and to a food product comprising said compressed fungal ingredient dough. Further to that, the present invention relates to a method for mechanically reducing nucleic acid content, in particular RNA content in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen. The present invention likewise relates to a method for mechanically controlling, and in particular reducing, the equivalent umami concentration (EUC) in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

[0007] Background of the invention

[0008] Consumers are increasingly seeking meat alternatives that replicate the composition and texture of traditional meats without relying on animal sources, thereby reducing the environmental impact associated with livestock farming. Livestock production is a significant contributor to greenhouse gas emissions, deforestation, biodiversity loss, and other ecological issues.

[0009] Plant-based proteins have been the primary focus in developing meat alternatives. However, challenges remain in achieving the desired taste and, more critically, the texture that mimics meat. Plant proteinsoften require extensive processing, as well as a list of long ingredients to attain a meat-like texture, which can compromise the product's appeal and nutritional value. In contrast, mushroom or filamentous fungi, particularly mycelium, offer a promising solution. Mycelium's natural fibrous structure closely resembles the texture of meat, and it is rich in protein and dietary fibre. This inherent property of mycelium reduces the need for additional processing and a long list of additives which can be tremendously reduced, making fungal biomass a viable candidate for meat substitutes.

[0010] Despite its potential, fungal biomass products are facing issues with texture, resulting in products that are either too tough or too soft due to lack of optimal processing methods combined with the right ingredients. By refining cultivation and processing techniques, we aim to produce a non-animal-based, nutritious, and environmentally friendly food product that closely mimics the taste and texture of various meat and fish products, potentially resulting in new food categories. This approach not only aligns with consumer preferences but also contributes to sustainable food production practices. To address this goal, our invention discloses a novel process to optimize the texture of mycelium-based meat alternatives, whether a whole-cut based on pure mycelium or combined with a handful of ingredients to create long fibrous mass with cellular entanglement with short and long fibre length randomised across various locations within the product mimicking the meat fibrillar structures.

[0011] Notably, existing literature lacks evidence of such products obtained via high pressures applied to frozen fungal biomass, mycelium or mycelium dough, distinguishing this method and the resulting products, as it achieves unique meat textures in non-animal protein substitutes.

[0012] For example, WO 2021 / 163216 provides a method for creating a mycelium-based meat substitute (mold species) by compacting mycelium after harvesting under uniaxial pressure to achieve realistic meat textures, wherein a high uniaxial pressure of 25 to 300 psi (1.72 to 20.68 bar) is applied to align fibres orthogonally. WO 2023 / 049392 uses a diluted mold species mycelium mass (diluted with water or any other suitable substance), wherein a pneumatic pressure to the diluted mycelium product is applied at 1 to 100 psi (0.0689 to 6.89 bar) before molding.

[0013] WO2024 / 098132 discloses mycelial biomass production process and use of mycelial biomass in the production of food products. Therein, the biomass is taught to be pressed at a maximum pressure of 100 psi then frozen for storage (or frozen for storage for later pressing, but no pressing while frozen is reported).Summary of the invention

[0014] Accordingly, it was an objective technical problem of the present invention to provide a vegan or vegetarian food product with improved properties, as well as method of producing such product.

[0015] The problem is solved by the embodiments disclosed herein, and as characterized by the claims. In particular, the problem is solved by the provision of the compressed fungal ingredient dough of the present invention, and the method of manufacturing the same.

[0016] Accordingly, the present inventors have demonstrated that a compressed fungal ingredient dough with advantageous properties, including appropriate texture and homogeneity, and reduced nucleic acid content, in particular RNA content, can be obtained in a process comprising the step of pressing a dough comprising the fibrous mycelium biomass and a binding agent at a pressure of at least 75 bar while frozen. Accordingly, the present invention is based, at least in part, on a surprising discovery that the treatment of the dough according to the present invention results in, among others, a reduction in its RNA content.

[0017] The present invention is hereinbelow summarized in the following embodiments.

[0018] In a first embodiment, the present invention relates to a compressed fungal ingredient dough, wherein said compressed fungal ingredient dough comprises fibrous mycelium biomass and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight (i.e., wt. %). It is preferred that said dough has been treated at a pressure of at least 75 bar while frozen.

[0019] In a second embodiment, the present invention relates to a method for preparing a compressed fungal mycelium ingredient dough, the method comprising the steps of

[0020] (a) providing a fibrous mycelium biomass;

[0021] (b) forming a dough comprising the biomass of (a) and a binding agent;

[0022] (c) freezing the dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; and (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a frozen compressed fungal ingredient dough.

[0023] The compressed fungal ingredient dough as obtained in said method may be a frozen compressed fungal ingredient dough.The present invention further relates to the compressed fungal mycelium ingredient dough obtainable according to the method of the second embodiment of the present invention. The present invention likewise relates to a food product (such as whole-cut food product) comprising said compressed fungal mycelium ingredient dough.

[0024] In a third embodiment, the present invention relates to a method for mechanically reducing nucleic acid content, in particular RNA content in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

[0025] In a fourth embodiment, the present invention relates to a compressed fungal ingredient dough, wherein said fungal ingredient dough comprises a chopped fruiting body of an edible mushroom and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight. It is preferred that said dough has been treated at a pressure of at least 75 bar while frozen.

[0026] In a fifth embodiment, the present invention relates to a method for preparing a compressed fungal ingredient dough, the method comprising the steps of

[0027] (a) providing a chopped fruiting body of an edible mushroom;

[0028] (b) forming a dough comprising the biomass of (a) and a binding agent;

[0029] (c) freezing the dough obtained in (b), thereby obtaining a frozen dough; and

[0030] (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen dough of (c) to yield a frozen compressed fungal ingredient dough.

[0031] The compressed fungal ingredient dough as obtained in said method may be a frozen compressed fungal ingredient dough.

[0032] The present invention further relates to a compressed fungal ingredient dough obtainable according to the method of the fifth embodiment of the present invention.

[0033] In a sixth embodiment, the present invention relates to method for mechanically reducing RNA content in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

[0034] In a seventh embodiment, the present invention relates to a method for preparing a compressed fungal ingredient dough, the method comprising the steps of(a / b) providing a partially separated fermentation broth;

[0035] (c) freezing the partially separated fermentation broth of (a / b), thereby obtaining a frozen partially separated fermentation broth; and

[0036] (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen partially separated fermentation broth of (c) to yield a frozen compressed fungal ingredient dough.

[0037] The compressed fungal ingredient dough as obtained in said method may be a frozen compressed fungal ingredient dough.

[0038] The present invention further relates to a compressed fungal ingredient dough obtainable according to the method of the seventh embodiment of the present invention. The present invention likewise relates to a food product (such as whole-cut food product) comprising said compressed fungal mycelium ingredient dough.

[0039] Brief description of figures

[0040] The invention is further illustrated by the hereto appended figures. These serve merely illustrative purposes and are in no way to be interpreted as limiting the scope of protection, which is defined by the hereto appended claims.

[0041] Fig. 1 shows A. sample 4 before thawing, in its frozen state (left panel) and after thawing (right panel). It is clearly visible that the product falls apart already upon thawing. B. Sample 4 upon cooking. The product has completely fallen apart into small crumbles.

[0042] Fig. 2 shows sample 6 A. before thawing; B. after thawing; and C. upon cooking.

[0043] Fig. 3 shows cut-through of sample 6 upon cooking, which shows good bite and chewy, fibrous texture.

[0044] It is apparent that the sample shows good texture and structure and is also more dense than other samples.

[0045] Fig. 4 shows the food product wherein the dough was formulated without the use of binder. Accordingly, only the biomass was pressed to 200 bar. The product visibly breaks down in structure and shows gaps, as well as no visible fiber formation.Detailed description of the invention

[0046] As mentioned before, the present invention relates, in one embodiment, to a compressed mycelium ingredient dough.

[0047] It is to be understood that the compressed mycelium ingredient dough, as provided by the present invention, is useful in production of the fungal food product, in particular in the production of a whole-cut food product.

[0048] Accordingly, the present also invention relates to a food product which is made using the compressed mycelium ingredient dough of the present invention. Accordingly, the present invention relates, in one embodiment, to a food product comprising the compressed mycelium ingredient dough of the present invention. Preferably, said food product is a whole-cut food product.

[0049] The term “whole-cut” is well understood to the person skilled in the art. As understood herein, the wholecut food product refers to a vegan or vegetarian food product, which resembles whole muscle meat or seafood. In other words, said whole food product is a meat-replacement product (which is also understood to encompass seafood replacement product), which resembles a whole muscle. Thus, the product of the invention appears to have been made from a single piece of material. Accordingly, the whole-cut food product of the present invention is not meant to resemble a product based on ground ingredients (such as ground meat-based product). For example, when referring to a whole-cut product in the case of meat, a whole-cut could be a steak, roast, or fillet that is sliced or prepared directly from a larger, unprocessed cut of meat like a whole ribeye or tenderloin, rather than being ground, shredded, or further broken down into smaller components. Further examples of whole-cut products include turkey breast and turkey slices. Accordingly, the whole-cut product may also refer to cold-cut products, which originate from whole-cut products, such as mentioned turkey slices or ham slices. Accordingly, it is to be understood that the wholecut food products refer to portions of meat or meat alternatives that consist of a single, intact piece, preserving the natural muscle structure. Examples under this invention include, but are not limited to: steaks, breasts, fillets, chops, cutlets, chunks, ribs, loins, sticks or nuggets designed to imitate animal meats such as beef, chicken, fish, lamb, veal, duck, and others. Additionally, the term whole-cut food product may also refer to dairy alternatives, such as cheese, regardless of if sliced or in block, or other molded forms or confectionery and sweets that could be molded in block or other molded form.As understood herein, the product of the invention (which term relates both to compressed fungal ingredient dough of the invention as well as whole-cut food product of the invention) is a vegetarian product. In other words, the product of the invention preferably does not include any meat or animal tissue. Accordingly, the product of the invention is free (or substantially free) of any meat or animal tissue. More preferably, the product of the invention is a vegan product. This means that the product of the invention, preferably, does not include, any material of animal original. In other words, the product of the invention is free (or substantially free) of any material of animal origin. When referring to being substantially free of, e.g., material of animal origin, a reference may be made to a product that includes not more than 1 % w / w of said material, preferably not more than 0.5% w / w of said material, more preferably not more than 0.1 % w / w of said material. Most preferably, substantially free of particular material means free of said material.

[0050] It is to be understood that the whole-cut food product of the present invention is a fungal-based food product. In other words, the whole-cut food product of the present invention comprises fungal cells (which, as understood herein, may have been processed in any way known to the skilled person, which include, but are not limited to, freezing, hydrating, dehydrating, and / or thermal treatment). In other words, the whole-cut food product of the present invention comprises fungal biomass, comprising fungal cells (which, as understood herein, may have been processed in any way known to the skilled person, which include, but are not limited to, freezing, hydrating, dehydrating, and / or thermal treatment). As understood herein, the source of the fungal cells, as described herein, in the food product of the invention is the compressed fungal ingredient dough of the present invention. Thus, it is preferred that in accordance with the present invention the compressed fungal ingredient dough of the invention comprises fungal cells (which, as understood herein, may have been processed in any way known to the skilled person, which include, but are not limited to, freezing, hydrating, dehydrating, and / or thermal treatment). In other words, the compressed fungal ingredient dough of the present invention comprises fungal biomass, comprising fungal cells (which, as understood herein, may have been processed in any way known to the skilled person, which include, but are not limited to, freezing, hydrating, dehydrating, and / or thermal treatment). Said compressed fungal ingredient dough of the present invention is obtainable using a fungal ingredient dough. Accordingly, as apparent to the skilled person, the fungal components of the product of the present invention (in particular whole-cut food product of the present invention) may be introduced into said product through a fungal ingredient dough, in a form of a compressed fungal ingredient dough, as defined herein.

[0051] The term “dough”, as referred to when making a reference to a fungal ingredient dough, is known to the skilled person. Accordingly, as understood herein, dough is a composition that is viscous and malleable,that can be shaped and manipulated. As apparent to the skilled person, when referring to a dough as a food product ingredient, the dough is typically used as a base for baked or cooked products. The dough is formed by combining its ingredients to create a cohesive mass that can be rolled, kneaded, or stretched into different shapes or forms. Its consistency can vary, being either soft, firm, or elastic, depending on its intended use, its production method and particular ingredient used. The precise formulation of the dough and its characteristics (e.g., viscosity, elasticity, or extensibility) may vary depending on the intended end product.

[0052] Accordingly, any reference to dough may include the composition that is formed by mixing the ingredients and blending them together in a mechanical process, such as kneading, until a uniform and homogeneous consistency is achieved. However, it is preferred that the term “dough” also includes the so obtained composition that has been further processed, in other words which has undergone further manipulations, such as resting, fermenting, freezing, hydrating, dehydrating, or thermal treatment (including cooking, steaming, baking, and other forms of applying elevated temperature to the material).

[0053] The dough as described herein serves as the foundational material from which the final edible product is derived, undergoing transformations during the baking or cooking process. The dough, as described herein, is further compressed in accordance to the invention to yield the compressed fungal ingredient dough according to the invention.

[0054] It follows that the present invention encompasses the embodiments, as described herein, wherein the dough, i.e. , the fungal ingredient dough, in particular said compressed fungal ingredient dough, can be present in its raw form, cooked form, frozen form, boiled form, hydrated form, rehydrated form, and / or fermented form. All these forms are preferably encompassed by the present invention.

[0055] When referring to the dough present in the composition of the present invention as “fungal ingredient dough”, according to the present invention, the fungal ingredient dough comprises fibrous mycelium biomass and a binding agent. It is to be understood that this definition does not exclude the presence of further components in the fungal ingredient dough. The skilled person, when formulating the fungal ingredient dough according to the present invention can include further ingredients that do not interfere with preparation of the dough and its mechanical properties. Examples of such further ingredients are provided herein. It is to be understood that the compositional definitions of the fungal ingredient dough, as provided herein, also apply to the compressed fungal ingredient dough of the present invention.As understood herein, the fungal ingredient dough as provided in the present invention is a compressed fungal ingredient dough. To this end, the term compressed relates to the fact that said fungal ingredient dough has been reduced in its volume due to applied pressure / force. Said fungal ingredient dough may have been compressed when frozen, according to the methods of the present invention. As a result, it is to be understood that at least part of cells that are comprised in the fungal ingredient dough are not intact. Accordingly, it is preferred that at least 10% of cells (by number) are not intact, more preferably at least 20% of cells (by number) are not intact, even more preferably at least 30% of cells (by number) are not intact. Further it is preferred that the compressed fungal ingredient dough of the present invention contains less than 20% by volume of air pockets; more preferably less than 10% by volume, even more preferably less than 5% by volume, preferably as based on a cross-section taken parallel or perpendicular to the longest dimension of the shaped product. Most preferably, as understood herein, the compressed fungal ingredient dough is substantially free of the air pockets, more preferably free of the air pockets. State-of-the-art techniques, such as fluorescence fingerprint (FT), X-ray computed tomography (CT), ultrasonic testing, and optical methods, are known to the skilled person and can be employed to measure air pocket content in compressed products. It is particularly preferred that CT is employed to measure the content of the air pockets in the (compressed) fungal ingredient dough of the present invention. When referring to a compressed fungal ingredient dough, reference may also be made to a pressurized fungal ingredient dough of the invention, or to compacted fungal ingredient dough of the invention.

[0056] The term “fibrous mycelium biomass” is well known to the skilled person. As understood herein, fibrous mycelium biomass refers to the biomass composed of fungal cells that grow in the form of mycelium, a network of thread-like hyphae. Accordingly, this biomass is formed of densely interconnected fungal cells that form a fibrous structure. This is typically achieved by cultivating the mycelium under controlled conditions, so that its growth is encouraged to form a dense, fibrous matrix. This biomass is typically primarily composed of living fungal cells, and its physical properties can be tailored by adjusting growth parameters such as temperature, humidity, and substrate. However, the term also encompasses cells that have been processed by any method known to the skilled person, such as freezing, hydrating, dehydrating, or thermal treatment.

[0057] It is further preferred that the dough of the present invention is substantially free of air pockets. As understood herein, air pockets are enclosed spaces of air (or gas) within the dough. Removing air pockets from dough refers to the process of expelling or redistributing gas bubbles trapped during mixing to achieve a uniform texture and improve dough workability. This can be accomplished through methods such as punching, kneading, folding, or mechanical extrusion, which help to eliminate irregular air pockets,control dough volume, and ensure a consistent crumb structure in the final product. The process contributes to better handling, shaping, and fermentation control, resulting in a more desirable texture and appearance in dough-based products, such as the food product (or the whole-cut food product) of the present invention. As referred to herein, the term “substantially free of air pockets” refers to a situation wherein the skilled person is not capable of recognizing that the air pockets are present in the dough. Preferably, the fungal ingredient dough according to the present invention is free of air pockets. This can be achieved, as apparent to the skilled person, by extensive mixing or kneading in a suitable mixing vessel or apparatus, of the ingredients forming said dough. According to the present invention, the compressed fungal ingredient dough is preferably also substantially free of air pockets, as described herein, more preferably the compressed fungal ingredientdough of the present invention is free of air pockets. It follows that the compressed fungal ingredient dough obtained of the fungal ingredient dough that is free of air pockets would also be free of air pockets.

[0058] As mentioned before, the fungal ingredient dough (and, likewise, the compressed fungal ingredient dough) comprises a binding agent. As it is understood herein, a binding agent is a substance or ingredient used in food formulations to hold or bind together other ingredients, providing structure, consistency, and cohesion to the final product. Binding agents are typically added to compositions to prevent separation of ingredients, enhance texture, and maintain shape during processing or cooking. Common examples of binding agents used in food applications include starches, gums, proteins (such as egg whites or gelatin or egg replacers or whey), and certain natural fibers. The binding agent functions by either forming a gellike network, interacting with moisture, or promoting adhesion among the components of the food mixture, thus contributing to the overall stability and integrity of the product.

[0059] It is to be understood that a reference to a binding agent may be also understood as a reference to at least one binding agent. In the context of the present invention, it is preferred that the binding agent as used in the dough according to the invention comprises a binding agent selected from additive binding agents and nutrient binding agents. Additive binding agents and nutrient binding agents are defined in the following.

[0060] Accordingly, additive binding agent is selected from alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, processed euchema seaweed, locust bean gum, guar gum, tragacanth, gum arabic (acacia gum), xanthan gum, tara gum, gellan gum, sorbitol, mannitol, glycerol, konjac, pectins, cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl methyl cellulose, sodium carboxy methyl cellulose, cellulose gum,enzymatically hydrolysed carboxy methyl cellulose, sodium- potassium and calcium salts of fatty acids, magnesium salts of fatty acids, mono-and diglycerides of fatty acids, acetic acid esters of mono- and diglycerides of fatty acids, lactic acid esters of mono-and diglycerides of fatty acids, citric acid esters of mono-and diglycerides of fatty acids, tartaric acid esters of mono-and diglycerides of fatty acids, microcrystalline cellulose-cellulose Gel, mono and diacetyl tartaric acid esters of mono-and diglycerides of fatty acids, mixed acetic and tartaric acid esters of mono-and diglycerides of fatty acids, sorbitol and mannitol. Further accordingly, additive binding agent may also be selected from psylium husk, polydextrose, oxidized starch, monostarch phosphate, distarch phosphate, phosphate distarch phosphate, acetylated distarch phosphate, acetylated starch, acetylated distarch adipate, hydroxy propyl starch, hydroxy propyl distarch phosphate, starch sodium octenyl succinate, starch-based ingredients, and acetylated oxidized starch.

[0061] Nutrient binding agent is preferably selected from protein rich ingredients (e.g., pea protein isolate, chickpea protein isolate, wheat gluten, egg white powder, potato protein and / or mungbean protein isolate), carbohydrate / dietary fiber rich ingredients (e.g., grain-based flours, grain-based starches, legume-based starches, fruit-based fibers, polysaccharides, starch-based ingredients, psylium husk, inulin, wheat starch, corn starch, potato starch), vitamins / mineral-rich ingredients, and / or lipid rich ingredients (e.g., all types of edible oils and butters). It is to be understood that certain nutrient binding agents may be considered additive binding agents.

[0062] It is particularly preferred that the binding agent is selected from methylcellulose, konjac, carrageenan, pectin, gellan gum, guar gum, gluten, egg white powder, starch, and psyllium husk.

[0063] In one embodiment of the present invention, the binding agent as used in the dough according to the invention comprises a binding agent selected from gluten, methylcellulose, xanthan gum, alginate, locust bean gum, agar-agar, gum arabic, carrageenan, pectin, gellan gum, cellulose gum, egg white powder, konjac, and guar gum. Preferably, the binding agent as used in the dough according to the invention comprises a binding agent selected from gluten, methylcellulose, pectin, and egg white powder. More preferably, the binding agent as used in the dough of the present invention comprises a binding agent selected from gluten and egg white powder. Accordingly, the binding agent as used in the dough according to the invention may be gluten. Alternatively, the binding agent as used in the dough according to the invention may be egg white powder.Further according to the present invention, the binding agent as used in the dough according to the present invention may include more than one binding agent. For example, the binding agent, as used in the dough according to the present invention, may be gluten and egg white powder. In other words, the dough as defined herein, according to the present invention may comprise both gluten and egg white powder as binding agent(s).

[0064] It is further preferred that the fungal ingredient dough (i.e., the compressed fungal ingredient dough) according to the present invention comprises at least 70% w / w of fibrous mycelium biomass and at least 10% w / w of binding agent as defined herein. Thus, the (compressed) fungal ingredient dough according to the present invention may comprise at least 70% w / w, at least 71 % w / w, at least 72% w / w, at least 73% w / w, at least 74% w / w, at least 75% w / w, at least 76% w / w, at least 77% w / w, at least 78% w / w, at least 79% w / w, at least 80% w / w, at least 81 % w / w, at least 82% w / w, at least 83% w / w, at least 84% w / w, at least 85% w / w, at least 86% w / w, at least 87% w / w, at least 88% w / w, at least 89% w / w, or at least 90% w / w of fibrous mycelium biomass. Preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention comprises at least 75 % w / w of fibrous mycelium biomass, more preferably the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention comprises at least 80% w / w of fibrous mycelium biomass, even more preferably the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention comprises at least 85% of fibrous mycelium biomass. Further to that, the fungal ingredient dough (or the compressed fungal ingredient dough) according to the present invention may comprise at least 10% w / w, at least 11% w / w, at least 12% w / w, at least 13% w / w, at least 14% w / w, at least 15% w / w, at least 16% w / w, at least 17% w / w, at least 18% w / w, at least 19% w / w, at least 20% w / w, at least 21 % w / w, at least 22% w / w, at least 23% w / w, at least 24% w / w, at least 25% w / w, at least 26% w / w, at least 27% w / w, at least 28% w / w, at least 29% w / w, at least 30% w / w of binding agent as defined herein. Preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) according to the invention comprises at least 15% w / w of the binding agent. More preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) according to the invention comprises at least 20% w / w of the binding agent. It is further to be understood that the % w / w amount of fibrous mycelium biomass and of the binding agent do not necessarily need to add up to 100% w / w, as other ingredients, as disclosed in the following, may also be present in the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention.

[0065] Optionally, the compressed fungal ingredient dough (or the fungal ingredient dough, or the food product of the present invention), further comprises algae (i.e., algal cells), archaea (i.e., archaeal cells), animalcells, bacteria (i.e., bacterial cells) or a combination thereof. Accordingly, optionally the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention comprises algae, archaea, animal cells, bacteria or a combination thereof. As understood herein said cells (algal cells, archaeal cells, animal cells, bacterial cells) may have been processed in any way known to the skilled person, which include, but are not limited to, freezing, hydrating, dehydrating, and / or thermal treatment.

[0066] As mentioned before, the food product of the invention, in particular the whole-cut food product of the invention, may comprise further components, beyond the compressed fungal ingredient dough. Preferably, the whole-cut food product of the present invention comprises at least one additional component selected from plant-based protein sources, mushrooms and mushroom concentrates, animalbased protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources. However, the present invention also refers to an embodiment wherein the fungal ingredient dough (or the compressed fungal ingredient dough) further comprises at least one additional component selected from plant-based protein sources, mushrooms and mushroom concentrates, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources, as defined herein.

[0067] Accordingly, the product or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may further comprise at least one additional component selected from plant-based protein sources, preferably selected from legumes (such as soybean, pea, chickpea, mung bean, fava bean, lupin bean, lentils, edamame, adzuki beans, preferably fava bean), cereals (such as wheat, rice, oat, quinoa, amaranth, spelt, barley, rye, teff), tubers (such as potato, sweet potato, cassava, preferably potato), nuts and seeds (such as almonds, walnuts, pumpkin seeds, sunflower seeds, chia seeds, hemp seeds, cashews, pistachios, and flaxseeds), vegetables (such as spinach, broccoli, Brussels sprouts, asparagus, kale, artichoke), fruits (e.g., guava, jackfruit, durian), algae (such as algae selected from Spirulina and Chlorella).

[0068] Furthermore, the product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may further comprise at least one additional component selected from mushroom and mushroom concentrates, which is preferably selected from Agaricus spp. (Button mushroom), Honey mushroom, Boletus, Truffle, Morel, Chantarelle, Saffron milk-cap, Oyster mushroom, King oyster mushroom, Shiitake, Straw mushroom, and Enoki.

[0069] Furthermore, the product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may further comprise at least one additional component selectedfrom animal-based protein sources, preferably selected from egg, dairy, meat (such as chicken, turkey, beef, pork, bison, lamb, duck), and seafood (such as fish, prawns, crab, lobster, mussels, oysters, scallops, clams, squid, and octopus).

[0070] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may comprise at least one additional component selected from plant-based fat and oil sources, preferably selected from soybean, rapeseed, canola, coconut, sunflower, avocado, olive, flaxseed, corn, palm, shea butter, macadamia, walnut, and grapeseed, preferably rapeseed and canola).

[0071] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may comprise at least one additional component selected from carbohydrate sources, such as starch, wheat flour, potato flour, rice flour, oat flour, corn flour, tapioca flour, buckwheat flour, millet flour, sorghum flour, or fruit extracts.

[0072] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the invention may comprise at least one additional component selected from fiber sources, such as pea, sugarcane, wheat, cellulose, oats, apple, psyllium husk, flaxseed, chia seeds, inulin, beet fiber, bamboo fiber, acacia fiber, or resistant starch.

[0073] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) according to the invention may further comprise algae. Any algae suitable for use in food applications can be used in the fungal ingredient as referred to herein. For example, the algae as used herein may be a microalgae. Preferably, the microalgae to be used in the product or in the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention is selected from Chlorophyta, Cyanobacteria, Haptophyta, Ochrophyta, Rhodophyta, Trebouxiophyceae, Chlorodentrophyceae, Chlorophyceae, Cyanophyceae, Prymnesiophyceae, Pavlovophyceaea, Bacillariophyceae, Coscinodiscophyceae, Labyrinthulomycetes, Xantophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorellales, Chlamydomonadales, Sphaeropleales, Chlorodendrales, Oscillatoriales, Synechococcales, Isochrysidales, Bacillariales, Thalassiosirales, Biddulphiineae Labyrinthulales, Mischocodcales, Eustigmatales, Porphyridiales, Chlorellaceae, Haematococaceae, Dunaliellaceae, Chlamydomonadaceae, Scenedesmaceae, Chlorodendraceae, Chlorodendraceae, Microcoleaceae, Synechococcaceae, Isochrysidaceae, Pavlovaceae, Bacillariaceae, Phaeodactylaceae, Skeletonemataceae, Thalassiosiaceae, Eupodiscaceae, Thraustochytriaceae, Pleurochloridaceae, Monodopsidaceae, Porphyridiaceae, Chlorella, Haematococcus, Dunaliella, Scenedesmus, Dimorphus,Acuminatus, Tetraselmis, Arthrospira, Synechococcus, Isochrysidus, Nitzschia, Phaedactylum, Odontella, Pseudonana, Schizochytrium, Monodus, Nannochloropsis, and Porphyridium. Preferably, the microalgae to be used in the product or in the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention is selected from Chlorella vulgaris, Chlorella reinhardtii, Haematococcus pluvialis, Dunaliella salina, Scenedesmus dimorphus and acuminatus, Tetraselmis suecica and striates, Arthrospira platensis, Isochrysidus galbana, Nitzschia dissipata, Phaedactylum tricornutum, Odontella aurita, Monodus subterranea, Nannochloropsis gaditana, oceanica and oculate, and Porphyridium cruentum. Alternatively, the algae as used herein may be a macroalgae. Preferably, the macroalgae to be used in the product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention is selected from Rhodophyta, Chlorophyta, Ochrophyta, Bangiophyceae, Florideophyceae, Phaeophyceae, Ulvophyceae, Bangiales, Palmariales, Gigartinales, Ulvales, Laminariales, Bangiaceae, Palmariaceae, Gigartinaceae, Solieriaceae, Ulvaceae, Laminariaceae, Pyropia, Palmaria, Chondrus, Kapaphycus, Eucheuma, Ulva, Sacharina and Laminaria. Preferably, the macroalgae is selected from Bangiaceae yezoensis and haitianensis, Palmaria palmata and mollis, Chondrus crispus, Kapaphycus alvarezii, Euchema cottonii, Ulva lactuca, Sacharina japonica and latissimi, and Laminaria digitata.

[0074] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from preservatives, preferably selected from calcium carbonate, acetic acid, potassium acetate, sodium acetate, calcium acetate, lactic acid, sorbates, and malic acid.

[0075] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from antioxidants and acidity regulators, preferably selected from ascorbic acid, sodium ascorbate, calcium ascorbate, fatty acid esters of ascorbic acid, tocopherol-rich extract, alpha-tocopherol, gammatocopherol, delta-tocopherol, lecithins, sodium lactate, potassium lactate, calcium lactate, citric acid, sodium citrates, potassium citrates, calcium citrates, tartaric acid (L(+)), sodium tartrates, potassium tartrate, sodium potassium tartrate, sodium malate, potassium malate, calcium malates, calcium tartrate, and triammonium citrate.

[0076] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from pH regulators and anticaking agents, preferably selected from sodium carbonate, potassium carbonate,ammonium carbonates, magnesium carbonates, hydrochloric acid, potassium chloride, calcium chloride, magnesium chloride, sulphuric acid, sodium sulphates, potassium sulphates, calcium sulphate, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, calcium oxide, magnesium oxide, fatty acids, gluconic acid, glucono delta-lactone, sodium gluconate, potassium gluconate, and calcium gluconate.

[0077] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from flavor enhancers, preferably selected from glutamic acid, monosodium glutamate, monopotassium glutamate, calcium diglutamate, monoammonium glutamate, magnesium diglutamate, guanylic acid, disodium guanylate, dipotassium guanylate, calcium guanylate, inosinic acid, disodium inosinate, dipotassium inosinate, calcium inosinate, calcium 5’-ribonucleotides, disodium 5’-ribonucleotides, and glycine and its sodium salt.

[0078] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from colours, preferably selected from riboflavins, chlorophylls and chlorophyllins, anthocyanin, betanin, lycopene, copper complexes of chlorophylls and chlorophyllins, terpene compounds such as carotene compounds and xanthophyll compounds, plain caramel, caustic sulphite caramel, ammonia caramel, sulphite ammonia caramel, vegetable carbon, calcium carbonate, iron oxides and hydroxides, curcumin, tartrazine, cellulose gel, cochineal, carminic acid, carmines, azorubine, carmoisine, lutein, a cocoa powder (melanoidin), a beet powder, a tomato extract, a duckweed powder, a spirulina powder, a paprika powder (capsanthin and / or capsorubin), a turmeric powder, a blueberry powder, a strawberry powder, a berrybased colours powder, a heme powder, a lycopene powder, a betanin powder, an alfalfa powder, a saffron powder, a mint powder and an annatto extract.

[0079] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected from glazing agents and sweeteners, preferably selected from isomalt, maltitols, acesulfame potassium, aspartame, cyclamate, saccharin, sucralose, alitame, steviol glycosides, neotame, lactitol, xylitol, and erythritol.

[0080] The product of the present invention or the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention may further comprise at least one additional component selected fromvitamin B12, vitamin B6, vitamin B2, vitamin B3 (also referred to as niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salt (NaCI, further comprising iodine salts in an amount of up to 5% w / w), enzymes (e.g. transglutaminase, amylase), minerals (e.g. salts comprising calcium, iron, and / or potassium, etc.), flavors or flavor components (salt, pepper, garlic, onions, mushroom fruiting body pieces, vegetable pieces, ginger, turmeric, curry, sugars (i.e., sucrose, glucose, mono- or disaccharide), oils, lemon juice, orange juice, herbs and spices, yeast flakes), texturized vegetable proteins, and natural aromatic compounds. As defined herein, herbs and spices include natural aromatic compounds, such as methyl acetate, linalool, limonene, vanillin, etc. or synthetic ones like aprifloren, cinnamyl propionate, cyclohexadecanolide, and ethyl levulinate. Such further additives may improve optical visibility, flavour, nutrients, and provide additional texture.

[0081] As understood herein, the fungal ingredient dough (or the compressed fungal ingredient dough) according to the present invention may comprise up to 20% w / w of additional component(s), such as component selected from plant-based protein sources, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources. Thus, the fungal ingredient dough according to the present invention may comprise 0%w / w, 1%w / w, 2% w / w, 3% w / w, 4% w / w, 5% w / w, 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w, 15% w / w, 16% w / w, 17% w / w, 18% w / w, 19% w / w, or 20% w / w of additional component(s), such as component selected from plant-based protein sources, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources.

[0082] As already mentioned, in certain embodiments of the present invention, these additional components referred to hereinabove may be included in the fungal ingredient dough of the present invention. Accordingly, as mentioned before, the fungal ingredient dough, according to the present invention, may comprise further components, beyond fibrous fungal mycelium and binding agent. Thus, the fungal ingredient dough may comprise at least one additional component, preferably selected from plant-based protein sources, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources, as defined herein.

[0083] Furthermore, according to the present invention, the binding agent utilized in the dough may be derived from microbial sources, specifically through controlled microbial fermentation processes. These processes may involve solid-state fermentation, wherein microorganisms grow on a solid substrate, or submerged fermentation, where they are cultivated in a liquid medium. Both methods yield internal and external biopolymers, including various extracellular polysaccharides (EPS) produced during thecultivation of selected fungal species. Prominent examples of these biopolymers are pullulans, a type of polysaccharide known for its film-forming properties; glucans, which are essential for structural integrity; xanthan-like polysaccharides, recognized for their thickening and stabilizing abilities; and galactomannans, which contribute to improved texture and moisture retention. Accordingly, in one embodiment of the invention the binding agent is an extracellular polysaccharide, preferably selected from pullulans, glucans and galactomannans. In other words, the EPS produced during the fermentation can be used as a binding agent, so that no further binding agent needs to be added to the dough of the present invention.

[0084] In one embodiment of this invention, the binding agent is synthesized directly from the same mycelia that are utilized for the dough formulation, all within a single fermentation step, wherein the extracellular polysaccharide (EPS) concentration ranges between 1 to 20 g / L, more preferably 1 to 10 g / L, even more preferably 1 to 5 g / L. This integration not only streamlines the production process but also ensures that the binding agent is produced as a natural component of the mycelial biomass, enhancing its functional properties within the final product. However, an additional binding agent can still be added. The skilled person is capable of measuring EPS concentration using the techniques that constitute state of the art. Particularly, such techniques include confocal laser scanning microscopy (CLSM), nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, high-performance liquid chromatography (HPLC), optionally preceded by precipitation of said EPS, for example using an alcohol, such as ethanol. As encompassed by the present invention, it is particularly preferred that EPS concentration is measured by using high-performance liquid chromatography, following precipitation using ethanol. The skilled person is perfectly capable of implementing such a measurement, as described herein.

[0085] According to the present invention, the product of the present invention or the compressed fungal ingredient dough of the present invention can be characterized by its reduced nucleic acid content, in particular RNA content. Nucleic acids store, transmit, and express genetic information essential for life. They consist of nucleotide chains, each containing a sugar, phosphate group, and nitrogenous base. The two main types of nucleic acids are DNA and RNA; DNA, with deoxyribose, provides stability, while RNA, with ribose, is more flexible. Both share a phosphate backbone and have purines (adenine, guanine) and pyrimidines (cytosine, thymine in DNA, uracil in RNA) as nitrogenous bases. DNA serves as a blueprint for organisms, while RNA facilitates genetic translation and regulation. As apparent to the skilled person, the RNA content of food products is an important aspect of their nutritional and biochemical composition, though it is not typically highlighted in common food labels. RNA, also referred to as ribonucleic acid,plays a crucial role in the synthesis of proteins within cells. It is present in all living cells, including those of plants and animals, and thus is found in all foods that come from living organisms. The RNA content of food varies based on the type of food, how it is processed, and its source. Excessive RNA content in food may pose several health risks. RNA breakdown produces purines, which are metabolized into uric acid. High uric acid levels can lead to gout and place strain on the kidneys, particularly in individuals with preexisting conditions. Additionally, foods high in RNA often contain high levels of saturated fats and cholesterol, potentially contributing to cardiovascular disease. Overconsumption of RNA may also increase oxidative stress, which could damage DNA and elevate the risk of cancer over time. Therefore, maintaining balanced RNA intake is important to avoid said health issues.

[0086] Accordingly, the present invention provides compressed fungal ingredient dough (as well as the food product comprising the same) which is characterized by low RNA content (or in other words, by reduced nucleic acid content, in particular reduced RNA content). Thus, according to the present invention, said dough that is comprised in the product of the present invention is characterized by RNA content of not more than 2.0% RNA by weight. Accordingly, said dough may be characterized by RNA content of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 % by weight. Preferably, the dough is characterized by RNA content of not more than 1.0 % RNA by weight. More preferably, the dough is characterized by RNA content of not more than 0.8 % RNA by weight. Even more preferably, the dough is characterized by RNA content of not more than 0.5 % RNA by weight. It is to be understood that % RNA by weight values refer to the dry mass of the compressed fungal ingredient dough, or the food product comprising the same (or in other words, reference is made to % RNA by weight with respect to the dry mass).

[0087] As understood herein, the RNA content, measured by % by weight, (i.e., wt %) refers to total content of all forms of RNA. Accordingly, the total RNA content of a sample, for example dough, food product, or biomass, encompasses all forms of RNA-related molecules, including free nucleotides (the basic building blocks of RNA), nucleosides (nucleotides lacking phosphate groups, often resulting from RNA degradation), oligoribonucleotides (short RNA fragments, which can be products of RNA processing or degradation), and polyribonucleotides (long RNA molecules such as mRNA, rRNA, and tRNA, involved in cellular functions like protein synthesis and gene expression). Preferably, the RNA content as referred to herein is measured upon treatment with a nuclease, precipitation of protein from the sample and analysis by using ion-paired HPLC. Accordingly, this total RNA content reflects the full spectrum of RNA in the sample, including both intact and degraded forms, and provides insight into the cellular transcriptional activity, RNA turnover, and metabolic state.Accordingly, the product of the present invention and likewise the compressed fungal ingredient dough of the present invention is characterized by RNA content of not more than 2.0% RNA by weight. Accordingly, said product or said dough may be characterized by RNA content of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 % by weight. Preferably, the product or the dough is characterized by RNA content of not more than 1.0 % RNA by weight. More preferably, the product or the dough is characterized by RNA content of not more than 0.8 % RNA by weight. Even more preferably, the product or the dough is characterized by RNA content of not more than 0.5 % RNA by weight.

[0088] Thus, the present invention relates to a food product (in particular whole-cut food product), comprising a compressed fungal ingredient dough, wherein said compressed fungal ingredient dough comprises fibrous mycelium biomass and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight. The present invention likewise refers to a compressed fungal ingredient dough, comprising fibrous mycelium biomass and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight.

[0089] The present invention is based, at least in part, on a surprising discovery that the low content of RNA in the dough, as described herein, can be achieved by treating the dough of the invention at high pressure for a short time, when frozen. The treatment is as described herein.

[0090] Accordingly, as provided by the present invention, the compressed fungal ingredient dough (which is understood to be comprised in the food product according to the invention), as referred to herein, has been treated at a pressure of at least 75 bar while frozen.

[0091] As understood herein, if the object has been treated at a particular pressure, it refers to a situation wherein the pressure is applied to the object, i.e., wherein certain force has been applied to this object over a specified surface area. In other words, treatment at a certain pressure may also refer to pressing with a certain pressure. As understood herein, the pressure is applied to the object by using a pressure applying device. Pressure-applying devices are known to the skilled person and several types of presses are utilized in biomass processing. These include hydraulic presses which employ hydraulic cylinders to generate force through fluid pressure, offering precise control over pressure and speed, making them suitable for high-force industrial applications. Further commonly used are pneumatic presses that use compressed air to exert force, ideal for operations requiring rapid, repetitive motions with lower forcerequirements. Mechanical presses operate through mechanical means such as cams, toggles, or gears to apply force, known for high-speed operation and consistency over multiple cycles. Electric presses utilize electric motors to drive the pressing mechanism, providing precise control over force and speed, and are energy efficient. Electro-mechanical presses combine electrical and mechanical components to apply force, offering versatility and control, suitable for applications requiring both speed and precision. Screw presses use a screw mechanism to generate force, allowing for gradual application of pressure, making them suitable for controlled, steady force applications, often used in dewatering processes. High-pressure systems are used in specialized application requiring extremely high pressure, such as certain forming processes.

[0092] As it is understandable to the skilled person, herein the pressure is applied to a potentially irregularly shaped object, due to the irregularity of the shape, conventional methods of calculating pressure through a uniform contact area are not directly applicable, and specialized techniques, such as the use of pressure mapping sensors, 3D surface scanning, soft conformable pressure sensors, and finite element analysis (FEA), may be employed to measure and analyse the distribution of pressure across the object. Such an approach would allow for the accurate measurement of localized and average pressure values by capturing the force applied to the varying contours of the objects surface. The pressure is then calculated by dividing the applied force by the contact area derived through these advanced measurement techniques. As such approach would be impractical in industrial settings, unless specifically indicated to the contrary, the pressure recited herein as applied to the dough corresponds to an operating pressure of pressure applying system.

[0093] As understood herein, the fungal ingredient dough comprised in the product, as referred to herein, has preferably been treated at a pressure of at least 75 bar while frozen. Thereby, the compressed fungal ingredient dough is formed. Preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 80 bar while frozen, thereby becoming compressed fungal ingredient dough. More preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 90 bar while frozen, thereby becoming compressed fungal ingredient dough. Even more preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 100 bar while frozen, thereby becoming compressed fungal ingredient dough. Again more preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 110 bar while frozen (such as a pressure of at least 120 bar, at least 130 bar, at least 140 bar, or at least 150 bar), thereby becoming compressed fungal ingredient dough. Still more preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 150 bar while frozen (such as a pressureof at least 160 bar, at least 170 bar, at least 180 bar, at least 190 bar or at least 200 bar), thereby becoming compressed fungal ingredient dough. Even more preferably, the fungal ingredient dough, as referred to herein, has been treated at a pressure of at least 200 bar while frozen, thereby becoming compressed fungal ingredient dough.

[0094] As understood herein, the term “frozen” refers to a situation wherein the temperature of the dough at its each point is below the freezing point of water, in particular below 0 °C. The skilled person would understand that such a condition is achieved when the dough (or, in general any object) is kept at a particular temperature for sufficiently long time. Preferably, the dough is kept (i.e., incubated at, or its temperature is as provided) during application of pressure at a temperature of between -80°C and -4°C , preferably between -50°C and -4°C , more preferably between -30°C and -4°C. Accordingly, the dough may be kept during the application of pressure at a temperature of -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11 °C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, or -4°C. Preferably, the dough is kept at a temperature of between -20°C and -15°C. Accordingly, the dough may be kept at a temperature of -20°C, -19°C, -18°C, -17°C, -16°C, or -15°C. Freezing of dough in order to achieve such temperature can be performed in different ways known to the skilled person. It is particularly preferred that the freezing of the dough is performed using a plate freezing assembly, which is a specialized system designed for the rapid and efficient freezing of perishable materials, typically food products, by direct contact with cold metal plates. The assembly consists of a series of closely spaced, temperature-controlled plates that apply uniform pressure to the material to facilitate freezing. The material is placed between the plates, and a refrigerant is circulated through the plates to rapidly lower the temperature of the material, causing it to freeze. The design of the plate freezing assembly ensures consistent heat transfer, even freezing, and minimal dehydration or damage to the material. This type of assembly is commonly used in food processing, pharmaceutical manufacturing, and other industries where rapid freezing is required to preserve product quality, texture, and nutritional value. The assembly may include mechanisms for adjusting plate pressure, controlling refrigerant flow, and accommodating different product sizes and shapes for efficient freezing.

[0095] The compressed fungal ingredient dough, as referred to herein, may be provided in its frozen form. Alternatively, the compressed fungal ingredient dough may be not frozen (or, in other words, one may refer to such fungal ingredient dough as thawed fungal ingredient dough). It is preferred that, according to the present invention, the compressed fungal ingredient dough is frozen. It is further to be understood that said compressed fungal ingredient dough may be defined by its cutting strength. It is to be understood that said cutting strength is not to be determined on the frozen compressed fungal ingredient dough, buton the cooked (or thawed and cooked) compressed fungal ingredient dough, as referred to herein. For thawing, it is preferred that the compressed fungal ingredient dough is kept (or in other words, rested) at room temperature for certain amount of time, preferably up to 48 hours (more preferably 24 - 48 hours).

[0096] The skilled person recognizes that specific pressure-applying equipment needs to be used in order to achieve such pressure. A system capable of delivering a pressure of 75 bars or more typically involves high-pressure hydraulic, pneumatic, or specialized pressing machinery. High-pressure hydraulic systems, such as hydraulic presses and pumps, can generate pressures well above 75 bars and are commonly used in industrial applications like metal forming, molding, and material testing. Pneumatic systems with air boosters or high-pressure compressors can also achieve pressures exceeding 75 bars, often used in applications like spray painting, cleaning, and automation. Additionally, cold isostatic pressing systems can apply uniform high-pressure fluid to compress materials, achieving pressures well over 75 bars, commonly used in powder metallurgy and ceramics. Similarly, high-pressure molding and forming equipment can generate the required pressure for processes such as injection molding or die casting. Furthermore, gas compression systems used in industries like energy and petrochemicals rely on high-pressure compressors to compress gases to 75 bars or higher. These systems are essential for various industrial processes requiring significant pressure to achieve specific material properties or manufacturing outcomes.

[0097] It is further preferred that the fungal ingredient dough has been pressed while block-frozen at a pressure of at least 75 bar. Accordingly, as disclosed herein, the biomass or the fungal ingredient dough can be processed in two primary ways. It can be crumbled and frozen before being placed into the mold, where high pressure is applied to achieve the desired shape. Alternatively, the fresh biomass or the fungal ingredient dough can be frozen as a block and then sawed (cut) into smaller pieces for further processing under high pressure. The second method, which involves freezing the biomass or the dough in a block before pressurization, is preferred over the first method due to its operational ease. Crumbled pieces do not mechanically link together as effectively as those that are frozen as a single block. Freezing in block form enhances the interaction between the dough and ice crystal formation, which contributes to the creation of fibrillar structures.

[0098] As encompassed by the present invention, the pressure of at least 75 bar, as referred to herein and as defined herein, is preferably to be applied for a short time. Preferably, the pressure is to be applied for a time of up to 10 s. Accordingly, the pressure of at least 75 bar, as defined herein, may be applied for a time of 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s. More preferably the pressure of at least 75 bar isapplied for up to 5 s (such as for 1 s, for 2 s, for 3 s, for 4 s, or for 5 s). Even more preferably, the pressure of at least 75 bar is applied for up to 2 s. Again more preferably, the pressure of at least 75 bar is applied for up to 1 s. Thus, it is to be understood that the pressure of at least 75 bar, as defined herein, may also be applied for a time of less than 1s, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 s).

[0099] It is further preferred that the pressure is applied unidirectionally. As shown by the present inventors, unidirectional application of pressure results in the plurality of mycelium fibers being aligned in a single direction. This can be achieved, for example, by application of an electro-mechanical press. Thus, preferably, according to the present invention, the pressure is applied by an electro-mechanical press.

[0100] According to the present invention, it is preferred that molding process involves applying a force in a range between 50kN and 200 kN, more preferably between 90 kN and 150 kN to shape the biomass material. The applied force is distributed across molds of varying surface areas, resulting in specific pressures tailored to the desired product shape and density. The skilled person is aware of the fact that particular pressure can be achieved by manipulating the force applied and the surface area to which said force is applied. For example, for a block shape with an area of approximately 136 cm2, forces of 90 kN, 120 kN, and 150 kN yield pressures of 66 bar, 88 bar, and 110 bar respectively. For smaller units or pieces with an estimated surface area of 23 cm2(approximately six per block), the corresponding pressures significantly increase to 396 bar, 528 bar, and 660 bar. This demonstrates that the pressure of 75 bar used according to the present invention is easily achievable to the skilled person. Such values can be directly linked to the mold geometry and targeted compaction characteristics, providing technical flexibility in adapting the process to different product formats, including 2D and 3D shapes.

[0101] Accordingly, encompassed by the present invention is a method for producing the compressed fungal ingredient dough, comprising applying a compressive force in the range of 90 to 150 kN to a mold having a surface area between approximately 20 and 140 cm2, thereby achieving a pressure in the range of 66 to 660 bar to form a shaped article. Preferably, the pressure is at least 75 bar. For example, to achieve a minimum pressure of 75 bar using a force of 90 kN, the mold surface area must not exceed 120 cm2.

[0102] As understood herein, it is preferred that the compressed fungal ingredient dough of the present invention is homogeneously textured. As understood herein, the term "homogeneously textured" preferably refers to a material or product that exhibits a uniform and consistent texture throughout its entire structure, with no significant variation in physical properties such as density or composition. In a homogeneously textured product, the distribution of components is evenly spread. This uniformity in texture ensures that thematerial or product behaves predictably under various conditions and maintains a stable quality throughout its use or processing. In the context of food products, a homogeneously textured product would exhibit an even consistency, without areas that are noticeably different in texture, hardness, or moisture content.

[0103] Texture of a food product can be defined, as apparent to the skilled person, using its cutting strength. As referred to herein, cutting strength refers to the amount of force, typically measured in newtons, required to penetrate and cut through the food product using a standard cutting tool under defined conditions (e.g., blade type, speed, and angle). This property is indicative of the firmness, cohesiveness, and overall texture of the food, which can influence its mouthfeel, consumer preference, and mechanical processing characteristics.

[0104] The cutting strength of the compressed fungal ingredient dough of the invention may be defined in absolute terms. Accordingly, it is preferred that the fungal ingredient dough of the present invention is characterized by a homogeneous cutting strength of at least 14 N. It is to be understood that the cutting strength requirement relates to the cooked compressed fungal ingredient dough of the present invention (or alternatively to food product, in particular whole-cut food product, of the present invention). In particular, should the compressed fungal ingredient dough of the invention (or the product of the invention) be provided in its frozen form, its cutting strength is preferably to be determined when the dough or product is thawed, or in other words, not frozen (i.e., its temperature is brought over the freezing point of water, preferably its temperature is brought over 4 °C) and then cooked. It is further preferred that the cutting strength of at least 14 N, as preferably determined herein, is a cutting strength of at least 15 N, more preferably of at least 20 N, even more preferably of at least 25 N, again more preferably of at least 30 N. Accordingly, the cutting strength of the compressed fungal ingredient dough, as defined herein, may for example be 14 N, 15 N, 16 N, 17 N, 18 N, 19 N, 20 N, 21 N, 22 N, 23 N, 24 N, 25 N, 26 N, 27 N, 28 N, 29 N, 30 N, 31 N, 32 N, 33 N, 34 N, or 35 N. Said cutting strength may also be higher than 35 N.

[0105] It is to be understood that, preferably, the cutting strength is to be determined on the (thawed) product, wherein the product has been cooked. According to the present invention, the determination of the cutting strength may be performed if the compressed fungal ingredient dough was cooked sous vide in water at a temperature of between 70 and 90°C, preferably at 80°C, for a time of between 5 to 15 minutes, preferably for a time of 10 minutes. Alternatively, also in accordance with the present invention, the determination of the cutting strength may be performed if the compressed fungal ingredient dough wascooked by pan-frying for a time of 5 to 10 minutes at a temperature of between 160 and 180°C. It is to be understood that pan-drying is to be performed in a non-stick pan.

[0106] Accordingly, it is preferred that the fungal ingredient dough of the present invention is characterized by a homogeneous cutting strength of at least 14 N, as herein defined, if measured when the compressed fungal ingredient dough is cooked sous vide in water at a temperature of between 70 and 90°C, preferably at 80°C, for a time of between 5 to 15 minutes, preferably for a time of 10 minutes, wherein, if the compressed fungal ingredient dough is frozen, it is thawed prior to cooking.

[0107] Alternatively, it is preferred that the fungal ingredient dough of the present invention is characterized by a homogeneous cutting strength of at least 14 N, as herein defined, if measured when the compressed fungal ingredient dough is cooked by pan-frying for a time of 5 to 10 minutes at a temperature of between 160 and 180°C, wherein, if the compressed fungal ingredient dough is frozen, it is thawed prior to cooking.

[0108] In one embodiment, the fungal ingredient dough of the present invention is characterized by a homogeneous cutting strength of at least 14 N, as herein defined, if measured when the compressed fungal ingredient dough is cooked sous vide in water at a temperature of between 70 and 90°C, preferably at 80°C, for a time of between 5 to 15 minutes, preferably for a time of 10 minutes, or when the compressed fungal ingredient dough is cooked by pan-frying for a time of 5 to 10 minutes at a temperature of between 160 and 180°C, optionally wherein, if the compressed fungal ingredient dough is frozen, it is thawed prior to cooking.

[0109] As further encompassed by the present invention, the cutting strength of the product of the present invention (including the compressed fungal ingredient dough of the invention) may also be determined in relative terms. For example, a comparison between the food product (such as whole-cut food product) of the invention, which has been treated at a pressure of at least 75 bar while frozen, as disclosed herein, and a product that has been made using the same components and has been texturized at atmospheric pressure, can be performed. It is to be understood that, preferably, for comparison purposes, both products are cooked in the same way, in particular are cooked sous vide in water at a temperature of 80°C for 10 minutes. Accordingly, it is preferred that the product is characterized by cutting strength increased by at least 15% compared to a product texturized at atmospheric pressure instead of being treated at a pressure of at least 75 bar while frozen. Preferably, the product is characterized by cutting strength increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% compared to a product texturized at atmospheric pressure instead of being treated at a1

[0110] pressure of at least 75 bar while frozen. Particularly preferred is the embodiment wherein the product is characterized by cutting strength increased by at least 50% compared to a product texturized at atmospheric pressure instead of being treated at a pressure of at least 75 bar while frozen. As it is to be understood herein, the product texturized at atmospheric pressure is a product which has been pressed at a pressure of 1 to 20 bar, preferably at a pressure of 17 bar, with a hydraulic press for 300 seconds.

[0111] The product of the present invention, and likewise the compressed fungal ingredient dough of the present invention, may also be referred to as high-pressure-texturized while frozen, referring to the way the product of the invention is obtained.

[0112] It is further preferred that the compressed fungal ingredient dough the present invention comprises a plurality of aligned mycelium fibers. Accordingly, it has been demonstrated by the present inventors that aligning and entangling mycelial fibers allows creating fungal-based texturized products that mimic the fibrous structure of meat. The alignment and entanglement of mycelial fibers can be achieved through specific processing techniques, such as the application of pressure and controlled freezing, as disclosed herein. These processes influence the mechanical properties of the mycelium, resulting in textures with desirable bite and chew characteristics similar to those of animal meat. The mycelium's filamentous network structure, composed of branching hyphae, allows for the formation of a dense, interconnected fiber network that provides structural integrity and mechanical strength.

[0113] As understood herein, the food product of the present invention may also be defined with respect to the content of the dough (or, more specifically, the compressed fungal ingredient dough). It is preferred that the compressed fungal ingredient dough constitutes at least 80% w / w of the food product of the invention. Accordingly, the compressed fungal ingredient dough may constitute 80% w / w, 81 % w / w, 82% w / w, 83% w / w, 84% w / w, 85% w / w, 86% w / w, 87% w / w, 88% w / w, 89% w / w, 90% w / w, 91% w / w, 92% w / w, 93% w / w, 94% w / w, 95% w / w, 96% w / w, 97% w / w, 98% w / w, 99% w / w or 100% w / w of the food product (i.e., preferably the whole-cut food product) of the present invention.

[0114] The food product of the present invention may also be defined with respect to the content of fungal mycelium in said product. As understood herein, the fungal mycelium is a major component of the compressed fungal ingredient dough, and as such is also a significant component of the food product of the present invention. Accordingly and preferably, the fungal mycelium constitutes at least 70% by weight of the food product of the present invention. Thus, the fungal mycelium may constitute at least 70% by weight, at least 71% by weight, at least 72% by weight, at least 73% by weight, at least 74% by weight,at least 75% by weight, at least 76% by weight, at least 77% by weight, at least 78% by weight, at least 79% by weight, at least 80% by weight, at least 81% by weight, at least 82% by weight, at least 83% by weight, at least 84% by weight, at least 85% by weight, at least 86% by weight, at least 87% by weight, at least 88% by weight, at least 89% by weight, or at least 90% by weigh of the food product of the invention. Preferably, the fungal mycelium constitutes at least 80% by weight of the food product of the present invention. Thus, the fungal mycelium may constitute at least 80% by weight, at least 81% by weight, at least 82% by weight, at least 83% by weight, at least 84% by weight, at least 85% by weight, at least 86% by weight, at least 87% by weight, at least 88% by weight, at least 89% by weight, or at least 90% by weight of the food product of the invention.

[0115] The fungal ingredient dough (or the compressed fungal ingredient dough) may further be characterized by its protein content. The term protein, as referred to herein in the context of a food ingredient, is known to the skilled person, and preferably refers to a macronutrient composed of amino acids linked by peptide bonds. It is preferred that the fungal ingredient dough (or the compressed fungal ingredient dough) is characterized by the protein content of at least 20% w / w, more preferably at least 30 % w / w. As understood herein, this value refers to a value with respect to the dry mass of the fungal ingredient dough (or the compressed fungal ingredient dough). More preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) is characterized by the protein content of at least 35% w / w of the dry mass. Even more preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) is characterized by the protein content of at least 40% w / w of the dry mass.

[0116] The fungal ingredient dough (or the compressed fungal ingredient dough) may also be characterized by the content of fibrous mycelium biomass, when referred to as dry mass. Preferably, the fungal ingredient dough (or the compressed fungal ingredient dough) is characterized by the content of fibrous mycelium biomass of at least 80 % w / w.

[0117] It is further preferred that, the fibrous mycelium biomass is characterized by dry matter content of between 5% w / w and 30% w / w, preferably between 5% w / w to 25% w / w, more preferably between 10% w / w to 20% w / w, while the dry matter content of the food product (such as whole-cut food product) is characterized by dry matter content of between 60% w / w to 100% w / w, preferably between 80% w / w to 100% w / w.

[0118] As understood herein, the food product of the invention is not particularly limited and the present invention encompasses embodiments wherein said food product is selected from steaks, breasts, fillets, chops,cutlets, chunks, ribs, loins, sticks and nuggets designed to imitate animal meats such as beef, chicken, fish, lamb, veal, duck, and others. It is to be understood that each of these products is meant as a replacement product that is vegetarian or vegan. It is further preferred, within the scope of the present invention, that the product of the present invention is not a meatball replacement product.

[0119] As further provided by the present invention, the food product of the present invention is such that there is no step of extrusion in the production of the product. As understood herein, an extrusion step in preparing a food product refers to a process wherein a mixture of ingredients is forced through a specialized die under controlled conditions of heat, pressure, and mechanical shear. During extrusion, the food mixture is subjected to intense conditions that cause it to undergo physical and chemical changes, such as gelatinization of starches, protein denaturation, and the formation of a specific texture. The material exits the extruder through the die, taking on the desired shape and texture. The extrusion process can be used to produce a wide variety of food products, including plant-based meat substitutes. This step is crucial for enhancing the texture, consistency, and mouthfeel of the final product while also allowing for the incorporation of functional ingredients. Additionally, the extrusion process can improve the digestibility and shelf-life of the food product.

[0120] As provided by the present invention, the fibrous mycelium biomass, as referred to herein, comprises a mycelium biomass of an edible fungus (or an edible fungal strain). It is to be understood that an edible fungus refers to a fungal organism that is safe for human consumption and provides nutritional value when consumed. The term "edible fungus" encompasses both wild and cultivated species that are traditionally used in cooking or as ingredients in processed food products. These fungi may be consumed fresh, dried, fermented, or processed into food additives or supplements. Examples include Agaricus bisporus (button mushrooms), Pleurotus ostreatus (oyster mushrooms), and various types of yeast used in baking and brewing. Further examples of edible fungus that can be used in the product and the methods of the present invention are provided herein. An edible fungus must meet regulatory safety standards to ensure it is free from toxic compounds and contaminants when used in food products.

[0121] Preferably, the edible fungal strain is selected from Basidiomycota, Ascomycota, Pezizomycotina, Agaromycotina, Pezizomycetes, Agaricomycetes, Sordariomycetes, Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Hypocreales, Morchellaceae, Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae,Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, and Fistulinacea.

[0122] According to the present invention, the edible fungal strain can be selected from the division Basidiomycota. Preferably, the fungal strain selected from Basidiomycota can be a fungal strain selected from the subdivision Agaromycotina. As defined herein, a fungal strain selected from the subdivision Agaromycotina can be a fungal strain selected from the class Agaricomycetes. Preferably, a fungal strain selected from Agaricomycetes can be a fungal strain selected from the order Agaricales, Auriculariales, Boletales, Cantharellales, Polyporales, and Russulales.

[0123] When the fungal strain is selected from the order Agaricales, the fungal strain is preferably selected from the family Agaricaceae, Fistulinaceae, Lyophyllaceae, Marasmiaceae, Omphalotaceae, Physalacriaceae, Pleurotaceae, Schizophyllaceae, Strophariaceae, and Tricholomataceae.

[0124] The fungal strain selected from Agaricaceae can be Agaricus bisporus or Agaricus blazei, more preferably Agaricus bisporus.

[0125] The fungal strain selected from Fistulinaceae is preferably Fistulina hepatica. The fungal strain selected from Lyophyllaceae is preferably Calocybe indica. The fungal strain selected from Marasmiaceae is preferably Lentinula edodes. The fungal strain selected from Omphalotaceae is preferably Calvatia gigantea. The fungal strain selected from Physalacriaceae is preferably Flammulina velutipes.

[0126] More preferably, the fungal strain selected from Agaricales can be a fungal strain selected from Pleurotaceae. Even more preferably, the edible fungal strain of the present invention is a fungal strain selected from Pleurotus pulmonarius, Pleurotus ostreatus, Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotus florida, Pleurotus eunosmus, Pleurotus columbinus, Pleurotus ferulae, Pleurotus salmoneo-stramineus, Pleurotus sapidus, and Pleurotus salmoneostramineus, still more preferably from Pleurotus pulmonarius, Pleurotus ostreatus, Pleurotus citrinopileatus, Pleurotus florida, Pleurotus eunosmus, Pleurotus columbinus, Pleurotus ferulae, Pleurotus salmoneo-stramineus and Pleurotus salmoneostramineus, even more preferably selected from Pleurotus pulmonarius or Pleurotus ostreatus, most preferably Pleurotus pulmonarius.

[0127] The fungal strain selected from Schizophyllaceae is preferably Schizophyllum commune.The fungal strain selected from Strop hariaceae is preferably a fungal strain selected from Agrocybe aegerita and Hypholoma capnoides.

[0128] The fungal strain selected from Tricholomataceae is preferably a fungal strain selected from Hypsizygus tesselatus and Clitocybe nuda.

[0129] Alternatively, a fungal strain selected from Agaricomycetes can be a fungal strain selected from the order Auriculariales, more preferably a fungal strain selected from the family Auriculariaceae. Preferably, a fungal strain selected from Auriculariaceae is Auricularia auricula-judae.

[0130] When the fungal strain is selected from the order Boletales, the fungal strain is preferably selected from the family Boletaceae and Sclerodermataceae. The fungal strain selected from Boletaceae is preferably Boletus edulis.

[0131] When the fungal strain is selected from the order Cantharellales, the fungal strain is preferably selected from the family Cantharellaceae and Hydnaceae. The fungal strain selected from Cantharellaceae can be Cantharell us cibarius. The fungal strain selected from Hydnaceae can be Hydnum repandum.

[0132] When the fungal strain is selected from the order Polyporales, the fungal strain is preferably selected from the family Ganodermataceae, Meripilaceae, Polyporaceae, and Sparassidaceae.

[0133] The fungal strain selected from Meripilaceae is preferably Grifola rondose. The fungal strain selected from Polyporaceae can be from Polyporus umbellatus and Laetiporus sulphureus (L. sulphureus). The fungal strain selected from Sparassidaceae can be Sparassis crispa. The fungal strain selected from Meruliaceae is preferably selected from Bjerkandera adusta and Bjerkandera fumosa.

[0134] When the fungal strain is selected from the order Russulales, the fungal strain can be selected from the family Bondarzewiaceae and Hericiaceae. Preferably, a fungal strain selected from Russulales is a fungal strain selected from Hericiaceae, preferably selected from Hericium erinaceus and Hericium coralloides. The fungal strain selected from Bondarzewiaceae can be Bondarzewia berkeleyi.

[0135] According to the present invention, the fungal strain can be selected from the division Ascomycota. Preferably, the fungal strain selected from Ascomycota can be a fungal strain selected from the subdivision Pezizomycotina.The fungal strain selected from Pezizomycotina can be selected from the class Pezizomycetes. Preferably, the fungal strain selected from Pezizomycetes can be a fungal strain selected from the order Pezizales. Preferably, the fungal strain selected from Pezizales can be selected from the family Morchellaceae and Tuberaceae.

[0136] Preferably, the fungal strain selected from Morchellaceae is Morchella esculenta, Morchella angusticeps, Morchella deliciosa, Morchella sceptrifomtis, Morchella steppicola, Morchella puncripes, Morchella rufobrunnea, Morchella importuna, Morchella Jaurentinaa, or Morchella purpumscens or Morchella elata, preferably Morchella esculenta, Morchella angusticeps or Morchella deliciosa.

[0137] Preferably, the fungal strain selected from from Tuberaceae is Tuber magnatum, T. estivum, T. uncinatum, T. indicum, T. rufum or T. melanosporum, more preferably T. melanosporum and T. magnatum.

[0138] Alternatively, the at least one fungal strain selected from Ascomycota can be a fungal strain selected from the class Sordariomycetes.

[0139] Preferably, the fungal strain selected from Sordariomycetes can be a fungal strain selected from the order Hypocreales.

[0140] The fungal strain selected from Hypocreales can be a fungal strain selected from the family Cordycipitaceae. The fungal strain selected from Cordycipitaceae can be a fungal strain selected from Cordyceps militaris and Cordyceps sinensis.

[0141] Alternatively, a fungal strain selected from Hypocreales can be a fungal strain selected from the family Nectriaceae. The fungal strain selected from Nectriaceae can be a Fusarium strain, for example Fusarium venenatum.

[0142] In another embodiment, the fungal strain selected from Sordariomycetes can be a fungal strain selected from the family Sordariaceae. The fungal strain selected from Sordariaceae can be a Neurospora strain, for example Neurospora crassa.It is particularly preferred that the edible fungal strain, as provided by the present invention, is selected from Agaricus spp., Aspergillus spp., Bjerkandera spp., Boletus spp., Calvatia spp., Cordyceps spp., Flammulina spp., Fusarium spp., Fomitopsis spp., Ganoderma spp., Grifola spp., Hericium spp., Hyphoroma spp., Inonotus spp., Laetiporus spp., Lentinula spp., Lepista spp., Morchella spp., Mycetinis spp., Neurospora spp., Paecilomyces spp., Panus spp., Pholiota spp., Pleurotus spp., Piptoporus spp., Polyporus spp., Rhizopus spp., Sparassis spp., Tremella spp., Trametes spp., Tricholoma spp., Tuber spp., and Wolfiporia spp..

[0143] In one embodiment of the present invention, the edible fungal strain is selected from mould species, preferably selected from Aspergillus spp., Fusarium spp., Neurospora spp., Paecilomyces spp., Rhizopus spp..

[0144] In an alternative embodiment, the edible fungal strain is selected from non-mould species, specifically selected from Agaricus spp., Bjerkandera spp., Boletus spp., Calvatia spp., Cordyceps spp., Flammulina spp., Fomitopsis spp., Ganoderma spp., Grifola spp., Hericium spp., Hyphoroma spp., Inonotus spp., Laetiporus spp., Lentinula spp., Lepista spp., Morchella spp., Mycetinis spp., Panus spp., Pholiota spp., Pleurotus spp., Piptoporus spp., Polyporus spp., Sparassis spp., Tremella spp., Trametes spp., Tricholoma spp., Tuber spp., Wolfiporia spp..

[0145] More preferably, the edible fungal strain is Agaricus spp., Bjerkandera spp., Boletus spp., Calvatia spp., Flammulina spp., Fomitopsis spp., Ganoderma spp., Hericium spp., Hyphoroma spp., Laetiporus spp., Lentinula spp., Morchella spp., Mycetinis spp., Pholiota spp., Pleurotus spp., Piptoporus spp., Polyporus spp., Sparassis spp., Tremella spp., Tuber spp., or Wolfiporia spp..

[0146] Even more preferably, the edible fungal strain is Agaricus spp., Bjerkandera spp., Boletus spp., Ganoderma spp., Hericium spp., Laetiporus spp., Lentinula spp., Morchella spp., Pleurotus spp. Polyporus spp., or Tremella spp..

[0147] In one particularly preferred embodiment, a fungal strain comprised in the fibrous mycelium biomass of the present invention may be a mould fungal species. Accordingly, as encompassed by the present invention, the fibrous mycelium biomass may comprise a fungal strain selected from Aspergillus niger, Aspergillus oryzae, Byssochlamys nivea, Fusarium venenatum, Neurospora crassa, Paecilomyces puntonii, Paecilomyces variotii, Rhizopus oligosporus, and Rhizopus oryzae. In one embodiment, said fungal strain is Aspergillus niger. In one embodiment, said fungal strain is Aspergillus oryzae. In oneembodiment, said fungal strain is Byssochlamys nivea. In one embodiment, said fungal strain is Fusarium venenatum. In one embodiment, said fungal strain is Neurospora crassa. In one embodiment, said fungal strain is Paecilomyces puntonii. In one embodiment, said fungal strain is Paecilomyces variotii. In one embodiment, said fungal strain is Rhizopus oligosporus. In one embodiment, said fungal strain is Rhizopus oryzae.

[0148] Further according to the present invention, a fungal strain comprised in the fibrous mycelium biomass of the present invention may be a non-mold fungal species. In particular, accordingly to the present invention, the fibrous mycelium biomass may comprise a fungal strain selected from Agaricus bisporus, Boletus edulis, Bjerkandera adusta, Bjerkandera fumosa, Ganoderma lucidum, Laetiporus sulphureus, Morchella angusticeps, Morchella deliciosa, Morchella esculenta, Morchella rufobrunnea, Morchella elata, Pleurotus. citrinopileatus, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus salmoneostramineus, Pleurotus sapidus, and Polyporus umbellatus. In one embodiment, said fungal strain is Agaricus bisporus, In one embodiment, said fungal strain is Boletus edulis. In one embodiment, said fungal strain is Bjerkandera adusta. In one embodiment, said fungal strain is Bjerkandera fumosa. In one embodiment, said fungal strain is Ganoderma lucidum. In one embodiment, said fungal strain is Laetiporus sulphureus. In one embodiment, said fungal strain is Morchella angusticeps. In one embodiment, said fungal strain is Morchella deliciosa. In one embodiment, said fungal strain is Morchella esculenta. In one embodiment, said fungal strain is Morchella rufobrunnea. In one embodiment, said fungal strain is Morchella elata. In one embodiment, said fungal strain is Pleurotus citrinopileatus. In one embodiment, said fungal strain is Pleurotus ostreatus. In one embodiment, said fungal strain is Pleurotus pulmonarius. In one embodiment, said fungal strain is Pleurotus salmoneostramineus. In one embodiment, said fungal strain is Pleurotus sapidus. In one embodiment, said fungal strain is Polyporus umbellatus

[0149] It is to be understood that the present invention, when referring to an edible fungal species, includes a direct reference to each and every single of the edible fungal species referred to herein.

[0150] According to the present invention, the food product (such as the whole-cut food product) of the present invention, or the compressed fungal ingredient dough, may further be characterized by calculated equivalent umami concentration (EUC) value. As apparent to the skilled person, glutamate (in particular in form of monosodium glutamate is a major contributor of umami, or savoury, taste in the food product. However, as further apparent to the skilled person, umami or savoury taste is also defined by the 5-ribonucleotides or 5’NMP, including the following 5'-nucleotides (5’NMP): 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), 5'-adenosine monophosphate (AMP) and xanthosine 5'-monophosphate (XMP) preferably expressed in g / kg of the food product (such as a whole-cut food product of the invention, or compressed fungal ingredient dough of the invention). Accordingly, calculation of EUC parameter requires measurement of the flavours in terms of the known flavour 5' nucleotides (5’NMP) content in g / kg. Said known flavour 5’ nucleotides include 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), 5'-adenosine monophosphate (AMP), and xanthosine 5'-monophosphate (XMP). Of note, 5'-GMP is known to give a meaty flavour and is considered to be a much stronger flavour enhancer than monosodium glutamate (MSG) (J.H. Litchfield, Morel mushroom mycelium as a food flavouring material Biotechnology and Bioengineering, 9 (1967), pp. 289-304).

[0151] As referred to herein, the EUC (equivalent umami concentration) is defined as:

[0152] EUC=Zaibi+1218(Zaibi)(Zajbj),

[0153] wherein the EUC of the sample is expressed in g monosodium glutamate (MSG) / 100 g, ai is the concentration (g / 100 g) of each umami amino acid Asp or Glu, aj is the concentration (g / 100 g) of each umami 5'-nucleotide 5-IMP, 5-GMP, 5’-XMP or 5-AMP, bi is the relative umami concentration (RUC) for each umami amino acid to MSG, defined as 1 for Glu and 0.077 for Asp and bj is the RUC for each umami 5'-nucleotide defined as 1 for 5-IMP, 2.3 for 5-GMP, 0.61 for 5’-XMP and 0.18 for 5-AMP. Preferably, EUC is provided per 100g of dry mass of the product. It is further to be understood that EUC value as referred to herein and as defined herein may also be referred to as calculated EUC.

[0154] This is a measurement that considers the relative taste intensity of the umami amino acids and 5’-nucleotides (Yamaguchi (1967) and Yamaguchi S, Yoshikawa T, Ikeda S, Ninomiya T. Measurement of the relative taste intensity of some a-amino acid and 5'-nucleotides. J Food Sci. 1971;36:846-49.).

[0155] Preferably, the product (compressed fungal ingredient dough) of the present invention is characterized by calculated EUC of not more than 1500 g MSG / 100g, more preferably not more than 1000 g MSG / 100g, even more preferably not more than 500 g MSG / 100g, again more preferably not more than 150 g MSG / 100g, still more preferably not more than 100 g MSG / 100g, even more preferably not more than 50 g MSG / 100g. The present invention is not limited with respect to any particular EUC value and encompasses products with high (e.g. > 500 g MSG / 100g) EUC values as the final EUC depends on the EUC of the initial raw material used (i.e. the fibrous mycelium biomass) as reported in WO 2024 / 084014, which is incorporated herein by reference in its entirety.The present invention encompasses the food product of the present invention (such as the whole-cut food product) in any form. For example, the whole-cut food product of the present invention may be fermented, frozen, hydrated, dehydrated, raw and / or thermally treated (i.e., for example cooked or boiled). In particular, the whole-cut food product of the present invention may be frozen, for instance frozen as directly obtained upon treatment under 75 bar while frozen, as described herein. It also applies to the compressed fungal ingredient dough of the present invention, which may be fermented, frozen, hydrated, dehydrated, raw and / or thermally treated (i.e., for example cooked or boiled). In particular, the compressed fungal ingredient dough of the present invention may be frozen, for instance frozen as directly obtained upon treatment under 75 bar while frozen, as described herein.

[0156] In one embodiment, the present invention further relates to a method of preparing a compressed fungal ingredient dough, as described herein. The method of the present invention comprises the steps of (a) providing a fibrous mycelium biomass;

[0157] (b) forming a fungal ingredient dough comprising the biomass of (a) and a binding agent;

[0158] (c) freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; and

[0159] (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a frozen compressed fungal ingredient dough.

[0160] As understood herein, in the method of the present invention, compressed fungal ingredient dough is formed. To this end, said compressed fungal ingredient dough may be present in its frozen form, as directly obtained in step (d). However, the compressed fungal ingredient dough may also be provided as a non-frozen product (or, in other words, thawed product). The skilled person is capable of transforming the frozen compressed fungal ingredient dough into compressed fungal ingredient dough. Accordingly, optionally the method of the present invention comprises a step of transforming the frozen fungal ingredient dough into compressed fungal ingredient dough (for example, by thawing).

[0161] Accordingly, in step (a) of the method of the present invention, a fibrous mycelium biomass is provided. The fibrous mycelium biomass is as defined herein, including any specific embodiments and preferred definitions thereof.

[0162] As understood herein, in the step (a) of the method the fibrous mycelium biomass for further processing is provided. The term providing herein refers to an act of supplying, making available or ensuring the presence of the biomass. Unless indicated to the contrary, the term providing as used in step (a) of themethod of the present invention does not necessarily require any step of cultivation or fermentation of said biomass.

[0163] Preferably, in certain embodiments of the present invention, the step (a) may involve a step of preparing the fibrous mycelium biomass, as defined herein. The skilled person is aware of many ways of preparing fibrous mycelium biomass. One particularly preferred method of preparing fibrous mycelium biomass is submerged fermentation. Submerged fermentation for fungal mycelium refers to a process in which fungal spores or mycelial fragments are cultured in a liquid nutrient medium, where the mycelium grows throughout the medium. Under controlled conditions of temperature, pH, oxygen, and agitation, the fungal mycelium proliferates, forming a dense network of hyphal structures. This method is commonly used for large-scale cultivation of fungal mycelium to produce biomass for applications in food, materials, or biotechnology. The submerged environment allows for efficient nutrient absorption and rapid growth of the mycelium, facilitating the production of high yields of fungal biomass.

[0164] The submerged fermentation can be operated as a batch, a fed-batch or a continuous process. These three main methods of fermentation are known to the skilled person and differ by outflow and inflow of material from / to the fermentation vessel. The fibrous mycelium biomass, as provided in the present invention, may for example be obtained as described in the following.

[0165] Accordingly, the mycelium can be sourced or purchased externally or cultivated via submerged fermentation or solid-state fermentation. For instance, Fusarium venenatum, is commercially available and can be procured to be used according to the present invention. There are many other commercially available strains that could be purchased and used according to the invention.

[0166] It is further to be understood that the fibrous mycelium biomass employed in this invention can be used in various physical forms or morphologies, including but not limited to wet biomass, gels, wet biomass or gels suspended in a liquid broth, or any other conceivable state.

[0167] For example, the fibrous mycelium biomass as in the invention is produced via submerged fermentation, wherein the fermentation medium comprises of 5 to 60 g / L of carbon source, 0.1 to 60 g / L nitrogen source, 0.01-15 g / L of minerals, and 0.01-50 mg / L of vitamins. The skilled person shall be able to extend the method by adjusting the carbon source (i.e. the total added sugars) and / or other parameters to meet the requirements of the reactor configuration used (batch mode versus fed-batch mode versus continuous mode).The fermentation medium preferably comprises of 5 to 60 g / L of carbon source, 0.1 to 60 g / L nitrogen source, 0.5-15 g / L of minerals, and 0.1-10 mg / L of vitamins. Preferably the carbon source is selected from Potato dextrose agar, Starch, Cellulose, Malt Extract, Beet molasses, Corn molasses, Sucrose, Glycerol, Glucose, Fructose, Lactose, Galactose, Xylose, Arabinose, and / or Maltose. Preferably, the nitrogen source is selected from Corn steep liquor (CSL), Yeast extract, Peptone, Ammonia, Urea, Potassium nitrate, Ammonium sulfate, Ammonium chloride and / or Ammonium carbonate. Preferably the minerals are selected from Sodium selenate, Magnesium sulfate, Magnesium chloride, Iron sulfate, Iron chloride, Manganese chloride, Manganese sulfate, Zinc sulfate, Calcium sulfate, Calcium chloride, Calcium carbonate, Copper chloride, Copper sulfate, Diammonium hydrogen phosphate, Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Sodium phosphate monobasic hydrate and / or Sodium chloride. Preferably the vitamins are selected from Biotin, Choline chloride, Folic acid, Myo-inositol, Niacinamide, Pantothenic acid, Pyridoxal, Riboflavin, Thiamine, Cobalamin and / or Ascorbic acid. pH control is preferably controlled via substances selected from, Sodium hydroxide, Potassium hydroxide, Sulfuric acid, Phosphoric acid, Hydrochloric acid, Citric acid, Acetic acid, and / or Hypochlorous acid. It is to be noted, that other components may be used like agar or food grade anti-foaming like edible oils or others. The skilled person shall be able to extend the method by including further substances, metabolites and further types of carbon, nitrogen, mineral, and vitamin sources.

[0168] The medium, as described herein, may be enriched with vitamin B12 (cobalamin), by adding B12 in its pure chemical form for the purpose of nutritionally supplementing the mycelium biomass. In a further embodiment, the medium is enriched with vitamin B12 between a range of 1 pg / L-200pg / L, preferably between a range of 10pg / L-1 OOpg / L. In certain embodiments, the accumulated vitamin B12 in the biomass is at least 10%, preferably at least 5% preferably at least 2.5 %, preferably at least 1 %, preferably at least 0.5% from the initial added amounts. In a preferred embodiment, the accumulation of vitamin B12 in the biomass is 10% at most, preferably 7.5% at most, more preferably 5% at most, more preferably 2.5% at most, preferably 1% at most, more preferably 0.5% at most.

[0169] Further methods disclosed in WO 2024 / 084014, which is incorporated herein by reference in its entirety, are used to produce the mycelium of this invention.

[0170] The fibrous mycelium biomass as provided in step (a) is as defined herein. Preferably, the fibrous mycelium biomass comprises at least one edible fungal strain. It is to be understood that in theembodiments wherein step (a) involves production of the fungal biomass through its cultivation, an edible fungal strain is cultured, as described hereinabove.

[0171] The edible fungal strain as in the fibrous mycelium biomass is as described herein, including any preferred definitions and specific embodiments thereof, when discussing the fungal ingredient dough of the present invention.

[0172] The method of the present invention further includes step (b) of forming a dough comprising the biomass of (a) and a binding agent. Said dough may also be referred to as fungal ingredient dough.

[0173] The binding agent used in step (b) is as defined hereinabove when discussing the binding agent being the component of the fungal ingredient dough (or the compressed fungal ingredient dough) according to the invention. Accordingly, the binding agent as used in making the dough according to the invention may be gluten. Alternatively, the binding agent as used in making the dough according to the invention may be egg white powder. Further according to the present invention, the binding agent as used in making the dough according to the present invention may include more than one binding agent. For example, the binding agent, as used in making the dough according to the present invention, may be gluten and egg white powder. In other words, in step (b) of the method of the present invention, both gluten and egg white powder may be used as binding agent(s).

[0174] The skilled person is capable of forming the dough, as required in step (b). The process of forming dough is as described herein in the context of the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention) Accordingly, the process of forming dough in step (b) begins typically with the mixing of ingredients, such as fibrous mycelium biomass, binding agent and optionally further ingredients, which are combined in a mixing apparatus under controlled conditions. The mechanical mixing applies shear and compressive forces, ensuring the uniform distribution of ingredients. The intensity and duration of mixing are adjusted to achieve the desired dough consistency, ensuring proper integration of all components. Following mixing, the dough may undergo kneading, for example mechanically, to further improve its mechanical properties. Kneading involves repetitive folding, stretching, and pressing of the dough, which strengthens its elasticity, smoothness, and workability, ultimately improving the dough's structure and preparing it for subsequent processing steps.

[0175] As apparent to the skilled person, various mixing equipment may be utilized in industrial processes to combine, homogenize, and disperse materials. The mixing equipment may include, but is not limited to:planetary mixers, rotary mixers, bowl cutters, high shear mixers, batch high shear mixers, inline high shear mixers, ultra-high shear inline mixers, powder / liquid mixers, central mixers, horizontal mixers, ribbon blenders, paddle mixers, double cone blenders, V-blenders, tumble blenders, fluidized bed mixers, static mixers, jet mixers, impeller mixers, kneading mixers, sigma blade mixers, double arm mixers, twin-screw extruders, and single-screw extruders. The aforementioned mixing equipment may be employed in various configurations and combinations to achieve desired mixing outcomes. For example, the mixing equipment may be equipped with multiple mixing tools or interchangeable components to enhance versatility. The selection of specific mixing equipment may depend on factors such as material properties, desired outcomes, and process requirements.

[0176] The composition of the dough in step (b) of the method of the present invention is as disclosed in the context of the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention, including any preferred definition and any particular embodiments of the product of the present invention.

[0177] Accordingly, as understood herein, the dough obtainable in step (b) of the method of the present invention, may further be characterized by its protein content. It is preferred that the dough obtainable in step (b) of the method of the present invention is characterized by the protein content of at least 20% w / w, preferably of at least 30% w / w. As understood herein, this value refers to a value with respect to the dry mass of the dough obtainable in step (b) of the method of the present invention. More preferably, the dough obtainable in step (b) of the method of the present invention is characterized by the protein content of at least 35% w / w of the dry mass. Even more preferably, the dough obtainable in step (b) of the method of the present invention is characterized by the protein content of at least 40% w / w of the dry mass.

[0178] The dough obtainable in step (b) of the method of the present invention may also be characterized by the content of fibrous mycelium biomass, when referred to as dry mass. Preferably, the dough obtainable in step (b) of the method of the present invention is characterized by the content of fibrous mycelium biomass of at least 80 % w / w.

[0179] It is further preferred that the dough obtainable in step (b) of the method of the present invention comprises at least 70% w / w of fibrous mycelium biomass and at least 10% w / w of binding agent as defined herein. Thus, the dough obtainable in step (b) of the method of the present invention may comprise at least 70% w / w, at least 71% w / w, at least 72% w / w, at least 73% w / w, at least 74% w / w, at least 75% w / w, at least 76% w / w, at least 77% w / w, at least 78% w / w, at least 79% w / w, at least 80% w / w, at least 81 % w / w, atleast 82% w / w, at least 83% w / w, at least 84% w / w, at least 85% w / w, at least 86% w / w, at least 87% w / w, at least 88% w / w, at least 89% w / w, or at least 90% w / w of fibrous mycelium biomass. Preferably, the dough obtainable in step (b) of the method of the present invention comprises at least 75 % w / w of fibrous mycelium biomass, more preferably the fungal ingredient dough of the present invention comprises at least 80% w / w of fibrous mycelium biomass, even more preferably the fungal ingredient dough of the present invention comprises at least 85% of fibrous mycelium biomass. Further to that, the dough obtainable in step (b) of the method of the present invention may comprise at least 10% w / w, at least 11% w / w, at least 12% w / w, at least 13% w / w, at least 14% w / w, at least 15% w / w, at least 16% w / w, at least 17% w / w, at least 18% w / w, at least 19% w / w, at least 20% w / w, at least 21 % w / w, at least 22% w / w, at least 23% w / w, at least 24% w / w, at least 25% w / w, at least 26% w / w, at least 27% w / w, at least 28% w / w, at least 29% w / w, at least 30% w / w of binding agent as defined herein. Preferably, the dough obtainable in step (b) of the method of the present invention comprises at least 15% w / w of the binding agent. More preferably, the dough obtainable in step (b) of the method of the present invention comprises at least 20% w / w of the binding agent. It is further to be understood that the % w / w amount of fibrous mycelium biomass and of the binding agent do not necessarily need to add up to 100% w / w, as other ingredients, as disclosed in the following, may also be present in the fungal ingredient dough of the present invention.

[0180] In certain embodiments of the present invention, these additional components may be included in the dough obtainable in step (b) of the method of the present invention. Accordingly, as mentioned before, the dough obtainable in step (b) of the method of the present invention, may comprise further components, beyond fibrous fungal mycelium and binding agent. Thus, the dough obtainable in step (b) of the method of the present invention may comprise at least one additional component, preferably selected from plantbased protein sources, mushrooms and mushroom concentrates, animal-based protein sources, plantbased fat and oil sources, carbohydrate sources and fiber sources. These components are as described herein in the context of fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention, including any preferred definition and any specific embodiment thereof.

[0181] As understood herein, the dough obtainable in step (b) of the method of the present invention may comprise up to 20% w / w of additional component(s), such as component selected from plant-based protein sources, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources. Thus, the dough obtainable in step (b) of the method of the present invention may comprise 0% w / w, 1 % w / w, 2% w / w, 3% w / w, 4% w / w, 5% w / w, 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11 % w / w, 12% w / w, 13% w / w, 14% w / w, 15% w / w, 16% w / w, 17% w / w, 18% w / w, 19% w / w, or 20%w / w of additional component(s), such as component selected from plant-based protein sources, animalbased protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources.

[0182] As encompassed by the present invention, optionally in step (b) a cross-linking enzyme is added to the fungal ingredient dough formed in step (b). As understood herein, when referring a cross-linking enzyme, at least one cross-linking enzyme is meant. As understood herein, a cross-linking enzyme catalyzes the formation ofcovalent bonds between molecules, typically proteins, resulting in the creation or stabilization of complex structures. Various cross-linking enzymes are used to enhance the structural and functional properties of food products. Preferred examples include transglutaminase, sortase, lysyl oxidase, amine oxidase, tyrosinase, laccase, peroxidase, glucose oxidase, polyphenol oxidase, sulfhydryl oxidase, protein disulfide isomerase, lipoxygenase, horseradish peroxidase, or a combination thereof.

[0183] Thus, optionally, in the step (b) further a cross-linking enzyme is added, preferably transglutaminase, sortase, lysyl oxidase, amine oxidase, tyrosinase, laccase, peroxidase, glucose oxidase, polyphenol oxidase or lipoxygenase or a combination thereof, is added to the fungal ingredient dough formed in step (b). Preferably, in the step (b) a transglutaminase is added to the fungal ingredient dough formed in step (b). Accordingly, in one embodiment, in in the step (b) further a transglutaminase is added to the dough formed in step (b). Preferably, the transglutaminase is added in an amount of 0.01 to 3 wt.%, preferably 0.1 to 2 wt%, more preferably 0.2 to 1.2 wt% of the dough weight.

[0184] Optionally, further additional enzyme may also be added to the dough formed in step (b), alone or in combination with the cross-linking enzymes, as defined herein. Preferred such enzymes to be added are selected from lipolytic enzymes, proteases, p-glucanases, xylanases, amyloglucosidases, hemicellulases, and digestive enzymes - such as lipase or amylase (a- and 0-amylase, trypsin).

[0185] As understood herein, transglutaminase is an enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues in proteins, facilitating protein cross-linking. This enzyme is used to modify the texture, structure, and functional properties of proteins, often in food processing to improve product consistency, elasticity, and binding. Transglutaminase can be derived from natural sources, such as microorganisms, or produced through fermentation, and is commonly employed in applications like meat, dairy, and vegan food products to enhance texture and improve processing characteristics.

[0186] The method of the present invention further includes the step (c) of freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough.The step of freezing is known to the skilled person. Preferably, the step of freezing is as described hereinabove. In step (b), preferably, a temperature of between -30°C and -4°C of the dough obtained in step (b) of the method of the present invention is achieved. Accordingly, the dough may be frozen to be at the temperature of -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21 °C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11 °C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, or -4°C. More preferably, the dough is frozen to be at the temperature of between -20°C and -15°C. Accordingly, the dough may be kept frozen at a temperature of -20° C, -19°C, -18°C, -17°C, -16°C, or -15°C. Freezing of dough in order to achieve such temperature can be performed in different ways known to the skilled person. It is particularly preferred that the freezing of the dough in step (c) of the method of the present invention is performed using a plate freezing assembly, as described herein.

[0187] As understood herein, and as disclosed herein in the context of the fungal ingredient dough (or the compressed fungal ingredient dough) of the present invention, the dough can be processed in two primary ways in step (c) of the method of the present invention. It can be crumbled and frozen before being placed into the mold, where high pressure is applied to achieve the desired shape. Alternatively, the fresh biomass or dough can be frozen as a block and then sawed (cut / separated) into smaller pieces for further processing under high pressure. The second method, which involves freezing the biomass or dough in a block before pressurization, is preferred over the first method due to its operational ease.

[0188] The method of the present invention further includes a step (d) of applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a frozen compressed fungal ingredient dough.

[0189] As understood herein, applying pressure may also be understood as treating at a pressure or pressing at a particular pressure. It is to be understood that said applying pressure is to be performed by using a pressure applying system, as defined herein.

[0190] According to the present invention, in (d) a pressure of at least 75 bar is applied. Unless specifically indicated to the contrary, the pressure recited herein as applied to the dough corresponds to an operating pressure of pressure applying system. Accordingly, in step (d) of the method of the present invention the pressure of at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 or more bar may be applied to frozen dough. Preferably, a pressure of at least 80 bar is applied to frozen dough in step (d) of the method of the present invention. More preferably, a pressure of at least 90 bar is applied to frozen dough in step (d) of the method of the present invention. Even more preferably, a pressure of at least 100 bar applied to frozen dough in step (d) of the method of the present invention. Again more preferably, a pressure of at least 110 bar (such as a pressure of at least 120 bar, at least 130 bar, at least 140 bar, or at least 150 bar) is applied to frozen dough in step (d) of the method of the present invention. Still more preferably, a pressure of at least 150 bar (such as a pressure of at least 160 bar, at least 170 bar, at least 180 bar, at least 190 bar or at least 200 bar) is applied to frozen dough in step (d) of the method of the present invention. Even more preferably, a pressure of at least 200 bar is applied to frozen dough in step (d) of the method of the present invention.

[0191] It is further preferred that in step (d) of the method of the present invention the pressure is applied unidirectionally. As shown by the present inventors, unidirectional application of pressure results in the plurality of mycelium fibers being aligned in a single direction. This can be achieved, for example, by application of an electro-mechanical press. Thus, preferably, according to the present invention, the pressure in step (d) of the method of the present invention is applied by an electro-mechanical press.

[0192] Further in step (d) of the method of the present invention, the pressure of at least 75 bar, as referred to herein and as defined herein, including any preferred definition of the pressure and any specific embodiment of the invention, is to be applied for a time of up to 10 s, as described herein. Accordingly, in step (d) of the method of the present invention, the pressure of at least 75 bar, as defined herein, may be applied for a time of 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s. More preferably, in step (d) of the method of the present invention, the pressure of at least 75 bar, as defined herein is applied for up to 5 s (such as for 1 s, for 2 s, for 3 s, for 4 s, or for 5 s). Even more preferably, in step (d) of the method of the present invention, the pressure of at least 75 bar is applied for up to 2 s. Again more preferably, in step (d) of the method of the present invention, the pressure of at least 75 bar is applied for up to 1 s.

[0193] It is preferred that step (d) of the method of the present invention is performed in a mold. A mold, as understood herein, refers to a hollow container or form designed to shape a material or substance into aspecific desired shape configuration. The mold typically includes one or more cavities that define the shape of the final product when the material is introduced and allowed to set. Molds can be made from a variety of materials, such as metal, plastic, rubber, or silicone, and are used in processes such as casting, molding, or shaping. Molds can be rigid or flexible, depending on the intended application.

[0194] According to the present invention, the mold can be available in a variety of shapes. For example, the mold may be characterized by a shape of a food product by a shape of a food product selected from steaks, breasts, fillets, chops, cutlets, chunks, ribs, loins, sticks and nuggets designed to imitate animal meats such as beef, chicken, fish, lamb, veal, duck, and others. Additionally, this food product may also be a dairy alternative, such as cheese, coming in sliced or block or molded form, or under confectionery or sweets, coming in a block or molded form

[0195] Preferably, according to the present invention, step (d) is performed in a mold which is filled with the frozen fungal ingredient dough to at least 60% of its volume. Accordingly, step (d) may be performed in a mold which is filled to 60% of its volume, 61% of its volume, 62% of its volume, 63% of its volume, 64% of its volume, 65% of its volume, 66% of its volume, 67% of its volume, 68% of its volume, 69% of its volume, 70% of its volume, 71% of its volume, 72% of its volume, 73% of its volume, 74% of its volume, 75% of its volume, 76% of its volume, 77% of its volume, 78% of its volume, 79% of its volume, 80% of its volume, 81 % of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91% of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume, 99% of its volume, or 100% of its volume. More preferably, step (d) is performed in a mold which is filled to at least 70% of its volume. Accordingly, step (d) may be performed in a mold which is filled to 70% of its volume, 71% of its volume, 72% of its volume, 73% of its volume, 74% of its volume, 75% of its volume, 76% of its volume, 77% of its volume, 78% of its volume, 79% of its volume, 80% of its volume, 81 % of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91 % of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume, 99% of its volume, or 100% of its volume. Even more preferably, step (d) is performed in a mold which is filled to at least 80% of its volume. Accordingly, step (d) may be performed in a mold which is filled to 80% of its volume, 81% of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91% of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume,99% of its volume, or 100% of its volume. This optimal texture should exhibit no visible gaps or cracks in the matrix and can be effectively removed from the mold during continuous operation. A higher fill level also strengthens the corners, which is particularly important when considering irregular three-dimensional shapes. The filling process ensures that the biomass or dough is compacted adequately, promoting structural integrity and enhancing the overall quality of the final product. Filling the mold to at least 60% of its operational volumetric capacity is crucial for maintaining structural integrity and minimizing gaps or cracks. This approach strengthens the corners of irregular three-dimensional shapes and facilitates effective removal from the mold during continuous operation, thereby improving the overall quality of the final product. It is further preferred that the mold is filled up to 90% of its volume. Thus, in some embodiment, the mold is filled to between 60% and 90% of its volume.

[0196] Step (d) of the method of the present invention, in which the pressure is applied, may also be referred to as the step of high-pressure texturizing the dough while frozen.

[0197] Preferably, the method of the present invention further comprises the step (e) of post-processing of the product of step (d). The product of step (d) may also be referred to as compacted dough, compacted fungal ingredient dough, or compressed fungal ingredient dough.

[0198] The compacted dough can be post-processed using various methods well known to the skilled person, in order to enhance its texture, flavour, and versatility, enabling its adaptation for a wide range of culinary applications. These techniques are preferably used alone. However, these techniques can also be used or in combination. As apparent to the skilled person, the post-processing step (e) of the method of the present invention not only refines the sensory attributes but also improves consumer acceptance, particularly in plant-based (or fungal-based) meat alternatives.

[0199] Accordingly, the step (e) of the method of the present invention may comprise coating the frozen compressed fungal ingredient dough, or coating the compressed fungal ingredient dough, as applicable. Coating is well known to the skilled person, Preferably, coating may refer to coating of the compacted fungal ingredient dough with starch-based ingredients, such as panko breadcrumbs. Coating may be done using frozen compacted fungal ingredient dough, thawed fungal ingredient dough, or cooked fungal ingredient dough. As understood herein, and as apparent to the skilled person, coating can create a desirable crispy exterior when cooked. This method is often preferred to replicate the texture of traditional fried foods in plant-based products. Binding agents or liquid solutions can be used to ensure uniform adherence of the coating material, resulting in consistent and appealing surface textures.Further accordingly, step (e) of the method of the present invention may comprise marinating the frozen compressed fungal ingredient dough of the present invention, or compressed fungal ingredient dough of the present invention, as applicable. Marinating is the process well known to the skilled person. Preferably, marinating is a process of soaking a food product, in a flavorful liquid mixture containing seasonings, acids, and / or enzymes to enhance its flavor, tenderness, and moisture before cooking. Marinating introduces deeper flavours and aromas by immersing the biomass in solutions containing oils, herbs, spices, or other flavouring agents. The marination process can vary in duration, preferably ranging from 30 minutes to overnight, depending on the desired intensity. Acidic ingredients, such as lemon juice or vinegar, can enhance flavour absorption, but non-reactive containers should be used to prevent interaction with metals that might alter the flavour. Herb-infused oils or umami-rich soy-based solutions are particularly effective in optimizing the sensory appeal of the biomass.

[0200] Further accordingly, step (e) of the method of the present invention may comprise cooking the frozen compressed fungal ingredient dough (or compressed fungal ingredient dough of the present invention). The process of cooking is well known to the skilled person. According to the present invention, cooking the compacted dough (which may also refer to cooking of the whole-cut food product) may be performed using a variety of methods, depending on the desired final texture and flavour. Pan-searing or grilling is often preferred to develop a flavourful crust and caramelized surface while preserving moisture. Oven baking provides consistent cooking throughout, resulting in a firm and even texture. Sous vide cooking maintains moisture and ensures uniform cooking at controlled temperatures, making it ideal for plantbased proteins, as well as fungal-based proteins. These methods can be combined or followed by additional processes, such as roasting or grilling, to further refine the product's sensory qualities. As further understood herein, the reference to cooking the frozen compressed fungal ingredient dough (or compressed fungal ingredient dough) may include direct cooking of the frozen product, or a process involving first thawing or defrosting of the frozen product, followed by cooking, as disclosed herein.

[0201] Further accordingly, step (e) of the method of the present invention may comprise reshaping of the frozen compressed fungal ingredient dough (or the compressed fungal ingredient dough, as applicable). Reshaping the compacted biomass offers flexibility in its application, allowing it to mimic traditional meat structures. Slicing, preferably at specific angles, replicates the appearance of meat grain alignment, enhancing the visual and textural realism of the product. Dicing or crumbling makes the biomass suitable for salads, stir-fries, or fillings, while portioning ensures consistent sizes for products such as patties,nuggets, or steaks. These reshaping methods allow for customization of the biomass’s structure to meet diverse culinary and manufacturing requirements.

[0202] As mentioned before the methods disclosed as optionally comprised in step (e) of the method of the present invention may be used alone or may be combined with each other. By combining coating, marinating, cooking, and / or reshaping techniques, the compacted dough can be transformed into highly appealing products with customized flavours, textures, and applications, meeting the needs of both consumers and food manufacturers. For example, coating according to the invention can be done using the frozen compressed fungal ingredient dough, or said dough may have been post-processed, for example marinated, and then frozen upon coating.

[0203] In one embodiment, the present invention further relates to a compressed fungal ingredient dough obtainable according to the method of the present invention. Likewise, the present invention further relates to whole-cut food product comprising the compressed fungal ingredient dough obtainable to the method of the present invention (wherein, in a preferred embodiment, the whole-cut food product is the compressed fungal ingredient dough of the present invention). As understood herein, the present invention encompasses the compressed fungal ingredient dough obtainable according to the method of the present invention and the food product of the invention in any form. For example, the compressed fungal ingredient dough obtainable according to the method of the present invention may be fermented, frozen, hydrated, dehydrated, raw and / or thermally treated. Preferably, the compressed fungal ingredient dough obtainable according to the method of the present invention is frozen. However, the skilled person is in position to thaw the frozen compressed fungal ingredient dough which is directly obtainable according to the method of the present invention. Thus, in one embodiment, the method of the present invention further includes a step of thawing the frozen compressed fungal ingredient dough. Thereby, the compressed fungal ingredient dough is obtained. This can be achieved, for example, by resting said frozen compressed fungal ingredient dough at room temperature for certain amount of time, for example for a time of up to 48 hours, preferably between 24 and 48 hours.

[0204] In one embodiment, the present invention further relates to a method for reducing RNA content in a mycelium. It is to be understood that said method is a method for mechanically reducing RNA content in a mycelium. Said method may also be referred to as a method for mechanically reducing RNA content in a mycelium.Accordingly, the method for reducing RNA content in mycelium according to the present invention comprises the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

[0205] The mycelium, as referred to herein, is a fibrous mycelium biomass, as defined herein. It is to be understood that said mycelium may be a pure (or substantially pure) fibrous mycelium biomass, as defined herein. However, the present invention also encompasses the embodiments wherein the mycelium, i.e., the fibrous mycelium biomass, is comprised within a dough comprising mycelium biomass and a binding agent, as defined herein. Accordingly, the mycelium may be comprised in the fungal ingredient dough, as described herein.

[0206] As understood herein, applying pressure may also be understood as treating at a pressure or pressing at a particular pressure. It is to be understood that said applying pressure is to be performed by using a pressure applying system, as defined herein.

[0207] According to the present invention, in the method for reducing RNA content in a mycelium a pressure of at least 75 bar is applied. Unless specifically indicated to the contrary, the pressure recited herein as applied to the dough corresponds to an operating pressure of pressure applying system. Accordingly, in the method for reducing RNA content in a mycelium of the present invention the pressure of at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 or more bar may be applied to said mycelium (or frozen fungal ingredient dough comprising the same). Preferably, a pressure of at least 80 bar is applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium of the present invention. More preferably, a pressure of at least 90 bar is applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium of the present invention. Even more preferably, a pressure of at least 100 bar applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium ofthe present invention. Again more preferably, a pressure of at least 110 bar (such as a pressure of at least 120 bar, at least 130 bar, at least 140 bar, or at least 150 bar) is applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium of the present invention. Still more preferably, a pressure of at least 150 bar (such as a pressure of at least 160 bar, at least 170 bar, at least 180 bar, at least 190 bar or at least 200 bar) is applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium of the present invention. Even more preferably, a pressure of at least 200 bar is applied to said mycelium (or frozen fungal ingredient dough comprising the same) in the method for reducing RNA content in a mycelium of the present invention.

[0208] It is further preferred that in the method for reducing RNA content in a mycelium of the present invention the pressure is applied unidirectionally. This can be achieved, for example, by application of an electromechanical press. Thus, preferably, according to the present invention, the pressure in the method for reducing RNA content in a mycelium of the present invention is applied by an electro-mechanical press.

[0209] Further in the method for reducing RNA content in a mycelium of the present invention, the pressure of at least 75 bar, as referred to herein and as defined herein, including any preferred definition of the pressure and any specific embodiment of the invention, is to be applied for a time of up to 10 s. Accordingly, in the method for reducing RNA content in a mycelium of the present invention, the pressure of at least 75 bar, as defined herein, may be applied for a time of 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s. More preferably, in the method for reducing RNA content in a mycelium of the present invention, the pressure of at least 75 bar, as defined herein is applied for up to 5 s (such as for 1 s, for 2 s, for 3 s, for 4 s, or for 5 s). Even more preferably, in the method for reducing RNA content in a mycelium of the present invention, the pressure of at least 75 bar is applied for up to 2 s. Again more preferably, in the method for reducing RNA content in a mycelium of the present invention, the pressure of at least 75 bar is applied for up to 1 s.

[0210] As encompassed by the present invention, it is preferred that the method for reducing RNA content in a mycelium of the present invention is performed in a mold. The mold is as disclosed herein, including any preferred definitions and any particular embodiments of the present invention.

[0211] Preferably, according to the present invention, the method for reducing RNA content in a mycelium of the present invention is performed in a mold which is filled to at least 60% of its volume. Accordingly, the method for reducing RNA content in a mycelium of the present invention may be performed in a moldwhich is filled to 60% of its volume, 61 % of its volume, 62% of its volume, 63% of its volume, 64% of its volume, 65% of its volume, 66% of its volume, 67% of its volume, 68% of its volume, 69% of its volume, 70% of its volume, 71% of its volume, 72% of its volume, 73% of its volume, 74% of its volume, 75% of its volume, 76% of its volume, 77% of its volume, 78% of its volume, 79% of its volume, 80% of its volume, 81% of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91 % of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume, 99% of its volume, or 100% of its volume. More preferably, the method for reducing RNA content in a mycelium of the present invention is performed in a mold which is filled to at least 70% of its volume. Accordingly, the method for reducing RNA content in a mycelium of the present invention may be performed in a mold which is filled to 70% of its volume, 71% of its volume, 72% of its volume, 73% of its volume, 74% of its volume, 75% of its volume, 76% of its volume, 77% of its volume, 78% of its volume, 79% of its volume, 80% of its volume, 81% of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91% of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume, 99% of its volume, or 100% of its volume. Even more preferably, the method for reducing RNA content in a mycelium of the present invention is performed in a mold which is filled to at least 80% of its volume. Accordingly, the method for reducing RNA content in a mycelium of the present invention may be performed in a mold which is filled to 80% of its volume, 81 % of its volume, 82% of its volume, 83% of its volume, 84% of its volume, 85% of its volume, 86% of its volume, 87% of its volume, 88% of its volume, 89% of its volume, 90% of its volume, 91% of its volume, 92% of its volume, 93% of its volume, 94% of its volume, 95% of its volume, 96% of its volume, 97% of its volume, 98% of its volume, 99% of its volume, or 100% of its volume.

[0212] As understood herein, the method for reducing RNA content in a mycelium of the present invention results in up to 90% reduction of RNA content. In certain embodiments of the invention, reduction of RNA content of 95% is achieved. Accordingly, the method for reducing RNA content in a mycelium of the present invention results in a reduction of RNA content ranging between 50 and 99%, preferably between 75 and 95%. The reduction of RNA content can also be illustrated in absolute terms. Accordingly, the mycelium (or the frozen dough comprising the same) upon reducing the RNA content according to the present invention is preferably characterized by RNA content of not more than 2.0% RNA by weight. Accordingly, said mycelium (or the frozen dough comprising the same) may be characterized by RNA content of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 % by weight.Preferably, the mycelium (or the frozen dough comprising the same) is characterized by RNA content of not more than 1.0 % RNA by weight. More preferably, the mycelium (or the frozen dough comprising the same) is characterized by RNA content of not more than 0.8 % RNA by weight. Even more preferably, the mycelium (or the frozen dough comprising the same) is characterized by RNA content of not more than 0.5 % RNA by weight.

[0213] It is to be understood that the method for reducing RNA content in a mycelium can also be understood as a method for obtaining a mycelium with reduced RNA content. The present invention relates, likewise, to a mycelium, as described herein, obtainable according to said method, as well as to a food product comprising the same.

[0214] The present invention further relates to a method for reducing (preferably, for mechanically reducing) EUC in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen. The method for mechanically reducing EUC in a mycelium encompasses the same step(s) as the method for mechanically reducing nucleic acid content in a mycelium of the present invention, including any specific embodiments and any preferred definitions of said methods. As apparent to the skilled person, the method for mechanically reducing EUC in a mycelium allows for controlling EUC of an available mycelium. Thus, in other words, the method of the present invention for mechanically reducing EUC of the mycelium may also be referred to as a method for controlling EUC of the mycelium. In particular, EUC of mycelium subjected to the step(s) of the method can be reduced in accordance with the present invention. For example, EUC of mycelium, as referred to herein, may be reduced by at least 50%, and up to 99%. Preferably, EUC of mycelium may be reduced by between 55% and 95%. Accordingly, as provided herein, the EUC of mycelium may be reduced by 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, or 95%. According to the present inventors, particular degree of reduction of EUC in the method disclosed herein may be controlled by controlling the pressure applied to said mycelium. In particular, it is to be understood that the EUC upon applying the method of the present invention is preferably not more than 1500 g MSG / 100g.

[0215] Further in the method for reducing EUC in a mycelium of the present invention, the pressure of at least 75 bar, as referred to herein and as defined herein, including any preferred definition of the pressure and any specific embodiment of the invention, is to be applied for a time of up to 10 s. Accordingly, in the method for reducing EUC in a mycelium of the present invention, the pressure of at least 75 bar, asdefined herein, may be applied for a time of 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s. More preferably, in the method for reducing EUC in a mycelium of the present invention, the pressure of at least 75 bar, as defined herein is applied for up to 5 s (such as for 1 s, for 2 s, for 3 s, for 4 s, or for 5 s). Even more preferably, in the method for reducing EUC in a mycelium of the present invention, the pressure of at least 75 bar is applied for up to 2 s. Again more preferably, in the method for reducing EUC in a mycelium of the present invention, the pressure of at least 75 bar is applied for up to 1 s. According to the present inventors, particular degree of reduction of EUC in the method disclosed herein may be controlled by controlling for how long the pressure is applied to said mycelium.

[0216] The mycelium, as referred to herein, is a fibrous mycelium biomass, as defined herein. It is to be understood that said mycelium may be a pure (or substantially pure) fibrous mycelium biomass, as defined herein. However, the present invention also encompasses the embodiments wherein the mycelium, i.e., the fibrous mycelium biomass, is comprised within a dough comprising mycelium biomass and a binding agent, as defined herein. Accordingly, the mycelium may be comprised in the fungal ingredient dough, as described herein.

[0217] It is to be understood that the method for reducing EUC in a mycelium can also be understood as a method for obtaining a mycelium with reduced EUC. The present invention relates, likewise, to a mycelium, as described herein, obtainable according to said method, as well as to a food product comprising the same.

[0218] As further provided by the present invention, the fibrous mycelium biomass may be replaced in the fungal ingredient dough of the present invention with a chopped fruiting body of an edible mushroom. As referred to herein, the term "chopped" refers to the process of cutting or reducing the fruiting body of a mushroom into smaller, uniform pieces using mechanical or manual methods. The chopping process results in mushroom fragments that vary in size but are generally consistent in shape and texture, making them suitable for incorporation into food products or further processing. The size of the chopped pieces may be adjusted based on the intended application, such as for use in cooking, packaging, or as an ingredient in prepared foods.

[0219] Accordingly, in one embodiment, the present invention relates to a compressed fungal ingredient dough wherein said fungal ingredient dough comprises a chopped fruiting body of an edible mushroom and a binding agent, wherein said dough is characterized by RNA content of not more than 2.0% RNA by weight. It is preferred that said dough has been treated at a pressure of at least 75 bar while frozen,The present invention likewise relates to an embodiment, wherein, in steps (a) and (b), a partially separated fermentation broth is provided instead of a fungal ingredient dough, as disclosed herein. Such a combined step (a) and (b) is herein referred to as step (a / b). It is to be understood that said broth contains fibrous mycelium biomass, as provided herein, and supernatant from the culture (which may include extracellular polysaccharides, or EPS, as provided herein). However, said partially separated fermentation broth is not limited to these components, and may also include additional components. Preferably, said partially separated fermentation broth includes at least one additional component selected from plant-based protein sources, mushrooms and mushroom concentrates, animal-based protein sources, plant-based fat and oil sources, carbohydrate sources and fiber sources (which are as described herein). Said partially separated fermentation broth is frozen in step (c), to obtain a frozen, partially separated fermentation broth, and further in step (d) a pressure of at least 75 bar for a time of up to 10 s is applied to the frozen partially separated fermentation broth. As understood herein, steps (c) and (d) of the method are as described herein for the embodiment in which fungal ingredient dough is obtained in step (b), including any specific embodiments and preferred definitions of said method. As also understood herein, partially separated fermentation broth refers to a fluid (pumpable) composition obtained from a fermentation process, wherein selective removal of undesired components has been performed while retaining a portion of the liquid phase and soluble extracellular compounds along with the produced biomass. The partial separation of the fermentation broth may be achieved through one or more of the following techniques, such as centrifugation, filtration or sedimentation. The resulting partially separated fermentation broth serves as an intermediate material suitable for further downstream processing. Selective removal of certain further components can be performed by dialysis, solid phase extraction, treatment with activated charcoal and precipitation, for example using ammonium sulfate. These techniques are well known to the skilled person.

[0220] In one embodiment, the partially separated mycelium broth is achieved by reducing the broth volume by 10 to 50% v / v, more preferably by 15 to 40% v / v, even more preferably by 20 to 30% v / v. This results in an increase of biomass concentration in the broth by at least 0.1 % w / w, more preferably by at least 0.3% w / w.

[0221] In one embodiment, the EPS concentration of the broth before separation ranges between 1 to 20 g / L, more preferably 1 to 10 g / L, even more preferably 1 to 5 g / L, while the partially separated mycelium broth comprises EPS ranging between 1 to 23 g / L, more preferably 1 to 13 g / L, even more preferably 1 to 7 g / L.The present invention likewise relates to a compressed fungal ingredient dough obtainable in a method of the present invention, the method comprising the step (a / b), as defined herein. The present invention also relates to a food product, in particular whole-cut food product, comprising such compressed fungal ingredient dough, as provided herein.

[0222] The present invention further provides a method of obtaining a compressed fungal ingredient dough, relying on fruiting body of an edible mushroom as a starting material. Accordingly, in one embodiment, the present invention relates to a method for preparing a compressed fungal ingredient dough, the method comprising the steps of

[0223] (a) providing a chopped fruiting body of an edible mushroom;

[0224] (b) forming a fungal ingredient dough comprising the chopped fruiting body of (a) and a binding agent;

[0225] (c) freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; and

[0226] (d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a frozen compressed fungal ingredient dough.

[0227] Preferably, the edible mushroom is as described hereinabove, including any preferred definition thereof and any specific embodiment thereof. As it is to be understood herein, the edible mushroom in step (a) is preferably Agaricus spp., Bjerkandera spp., Boletus spp., Hericium spp., Laetiporus spp., Lentinula spp., Morchella spp., Pleurotus spp. Polyporus spp., orTremella spp..

[0228] As understood herein, the fruiting body provided in (a) is chopped, or in other words, has been subjected to chopping. In the context of chopping a mushroom fruiting body, "chopping" refers to the process of cutting or dividing the mushroom fruiting body into smaller, uniform pieces using a sharp instrument. This operation involves mechanically severing the mushroom into discrete segments to facilitate further processing. For example, chopping can be used to facilitate process such as drying, cooking, or extraction of compounds. The chopping process may vary in size and shape of the resulting pieces depending on the intended application and the nature of the mushroom species.

[0229] It is further to be understood that step (b) of the method described herein of forming a fungal ingredient dough comprising the chopped fruiting body of (a) and a binding agent can be performed as in the case of step (b) of the method using mycelium biomass, as described herein, including any preferred definitionsand specific embodiments of said method. The same applies to steps (c) and (d) of the method of preparing a compressed fungal ingredient dough, starting from chopped fruiting body, as described herein. The method may include a further step (e), as described in the context of the method starting from fibrous mycelium biomass.

[0230] It is to be understood that the present invention further relates to compressed fungal ingredient dough (or frozen compressed ingredient dough) obtainable according to the method for preparing a frozen compressed fungal ingredient dough of the present invention, the method comprising the step (a) of providing a chopped fruiting body of an edible mushroom. The present invention relates likewise to a food product, in particular whole-cut food product, comprising the compressed fungal ingredient dough (or frozen compressed fungal ingredient dough) of the present invention. It is to be understood that in certain embodiments, said food product may be the compressed fungal ingredient dough, as provided herein.

[0231] As understood herein, the component contents, described as % w / w, % by weight or % wt, refer to the wet weight of a particular component, unless specifically indicated to the contrary. For example, the EUC and RNA contents, as referred to herein are reported in respect to dry weight.

[0232] Examples

[0233] Example 1

[0234] The experiments herein as summarized in the following Table 1.

[0235] Table 1. Summary of the experiments performed herein.

[0236]

[0237] As referred to herein, "very low” can be understood as a value ranging from 0 to 2 N.

[0238] The broth of Pleurotus pulmonarius biomass produced in Example 3 was diluted with water in the ratio of 3:1 v / v (water to broth) and then separated under vacuum via a screw with 4 to 6 fluid channels. This separated biomass was then used for further processing.

[0239] Equipment and Processes: In this section, the key processing equipment used in treating the biomass is described. In terms of pressuring systems i.e. presses, a mechanical hydraulic press is used with a manual function of loading the pressure and releasing it depending on the process time. This leads to higher operational times. An automatic electro-mechanical pressing system is also used which can apply pressures to a high degree within an ultrashort time duration of around 0.9 to 1 s per stroke / mold. To freeze the samples a plate freezing assembly is used to restrict the ice expansion beyond the mold thereby making the frozen block more compact and enhancing the fibre entanglement. These plate freezers are kept between -30°C and -4°C ideally between -20°C and -15°C. The doughs for the recipes were mixed in a planetary mixer (Thermomix / Kenwood mixer) for 5 minutes to ensure a homogeneous mixture. The product was heated or cooked using different time and temperature combinations, for thawing, the samples were rested at room temperature for 24 - 48 hours, for sous vide, the samples were packed in a vacuum bag and cooked in water between 70 and 90°C ideally at 80°C for 5 to 15 min, preferably for 10 mins. For pan frying the samples were cooked for 5 -to 10 mins at a temperature of 160 - 180°C on a non-stick pan.The samples were analysed for cutting strength or shear forces were determined using a TA.XTpluslOO Texture Analyzer (Stable Micro System, Surrey, United Kingdom). A 50 kg load cell was used for the tests. Frozen samples were analysed when cooled down at room temperature (= 20 °C). The results were given as an average of the maximum forces (N) required to cut through the samples. In addition to the force, the areas under the curve were calculated (N s) as well. Force-time curves were recorded with the maximum force being taken as maximum cutting strength. The area under the positive curve (N.s) represented the total amount of force.

[0240] Description of samples

[0241] Sample 1 (Control):

[0242] The harvested mycelium biomass was freeze-dried to approx. 100% dry matter (DM) and referred to as the "Control" sample. It was used primarily for nutrition and EUC / RNA measurements and provided a baseline for comparative analysis for the below samples. The freeze drying is performed in a "Christ Alpha 1-2" freeze dryer connected to an external vacuum pump, where the respective sample is freeze-dried at minus 50°C with a vacuum of 6 mbar. All samples were pre-frozen at - 80°C. The samples were freeze-dried for a duration of 72 hours.

[0243] Sample 2:

[0244] The mycelium biomass was separated to 15% DM using a hydraulic press. The pressing process applied a final pressure of 17 bar for a processing time of 300 seconds, achieved via manual pressuring and depressuring of the chamber. The biomass was compressed in a stainless-steel cylindrical mold with 8.5 cm diameter, 0.2 mm wall thickness, and perforations for water drainage. This compacted biomass was analyzed using Texture Profile Analysis (TPA) to measure cutting strength.

[0245] Sample 3:

[0246] Prepared similarly to Sample 2, the mycelium biomass was separated to 15% DM and pressed to 17 bar with a hydraulic press for 300 seconds. After pressing, the biomass was stored at -20°C overnight (frozen), thawed at room temperature for 24 hours, and then analyzed for cutting strength using TPA.

[0247] Sample 4:

[0248] The biomass was separated to 15% DM via screw press, shaped into blocks using a plate freezer, and subjected to ultra-high pressure (200 bar) for 1 second. The compacted biomass blocks were storedovernight at -20°C, thawed at room temperature for 24 hours, and analyzed using TPA for both uncooked and cooked samples (pan frying).

[0249] Sample 5:

[0250] The mycelium biomass was separated to 15% DM via screw press and mixed with additional ingredients in a planetary mixer. The formulation of the additional ingredients is provided in the accompanying Table 1 above. The resulting dough was pressed in a hydraulic press to 17 bar for 300 seconds using a perforated stainless steel cylindrical mold (8.5 cm diameter, 0.2 mm wall thickness). The compacted dough was stored overnight at -20°C (frozen), thawed for 24 hours at room temperature, and analyzed for cutting strength using TPA, both in uncooked and cooked states.

[0251] Sample 6:

[0252] The biomass was separated to 15% DM via screw press and combined with additional ingredients using a planetary mixer. This dough was formed into blocks using a plate freezer and subjected to ultra-high pressure (200 bar) for 1 second. The compacted dough blocks were stored overnight at -20°C, thawed for 24 hours, and analyzed using TPA for both uncooked and cooked states.

[0253] Pre-Processing Steps:

[0254] Samples 4 and 6 underwent pre-processing in cuboidal plate freezers and were sawed into smaller portions before the application of 200 bar pressure. Similar pre-processing (freezing) was conducted for Samples 3 and 5, although they were pressed at 17 bar for 300 seconds.

[0255] Mold Types and Applications:

[0256] A variety of molds were utilized for pressing and shaping mycelium biomass: Three-dimensional molds replicating specific food shapes, such as chicken breast fillets, for applications targeting plant-based / fungal-based whole-cut products. Cylindrical molds featuring 204 perforations (2.45 mm diameter), constructed from stainless steel with an internal diameter of 84.0 mm and wall thickness of 2.7 mm. These molds facilitated uniform water drainage during pressing. Additional mold geometries, including spherical, cuboidal, conical, and custom designs, could be employed. The choice of mold geometry depended on the intended product characteristics, processing conditions, and analytical requirements.

[0257] DISCUSSION

[0258] Effect of Pressure on Mycelium Structure and TextureThe results indicate that pressure is a critical factor influencing the structural and textural properties of mycelium biomass and mycelium-containing doughs. Samples treated with ultra-high pressure (200 bar) showed markedly improved characteristics compared to those treated with low pressure (17 bar). The ultra-high pressure compacted the biomass matrix more effectively by removing air pockets and redistributing them uniformly. This process led to:

[0259] • Increased Density: The higher pressure resulted in a denser matrix, which created a firmer texture resembling animal muscle fibers.

[0260] • Enhanced Fiber Entanglement: The compacting action aligned and intertwined fibers within the mycelium structure, further stabilizing the matrix.

[0261] • Elimination of Air Pockets: Air pockets, typically trapped during lower-pressure treatments, were redistributed or dissolved, leading to a smoother and more uniform texture.

[0262] In contrast, samples treated at low pressure (17 bar) demonstrated localized densification, particularly on the exterior surface, due to gradual water removal. This effect caused a "caking" phenomenon, where the exterior hardened while the interior remained less dense, resulting in a less uniform texture.

[0263] Role of Freezing and Thawing (Frozen) on Texture Integrity

[0264] Samples subjected to frozen followed by thawing exhibited significant structural changes. The freezing process caused the formation of ice crystals, which mechanically punctured cell walls. Upon thawing, these punctures led to leakage of cellular material, compromising the overall structural integrity.

[0265] Sample 3: After thawing, the structure was too weak to retain its shape during cutting strength measurements, highlighting the disruptive effect of freezing on pure biomass samples without added binding agents or further components / ingredients.

[0266] Sample 6: Despite undergoing similar freezing and thawing, the addition of ingredients stabilized the matrix. The high pressure (200 bar) compacted the dough more effectively, mitigating the structural degradation caused by freezing.

[0267] Impact of Ingredients on Texture and Structural Stability

[0268] The incorporation of additional ingredients, such as proteins and hydrocolloids, significantly improved the structural and textural properties of mycelium-based products. Ingredients contributed by:• Water Binding: Ingredients absorbed available water, reducing leakage and increasing cohesiveness.

[0269] • Gap Filling: Ingredients filled intracellular gaps created during pressure application or freezing, stabilizing the structure.

[0270] • Matrix Reinforcement: Chemical and physical interactions between the ingredients and mycelium fibers enhanced the mechanical strength of the dough.

[0271] For instance, Sample 5, prepared with ingredients and subjected to 17 bar, exhibited cutting strength of 19.7 N after cooking. In contrast, Sample 6, treated with 200 bar, achieved 30.8 N after cooking, highlighting the synergistic effect of high pressure and additional ingredients on texture enhancement.

[0272] Effect of Mold Design on Product Features

[0273] The selection of mold geometry played a role in shaping and texturing the mycelium biomass:

[0274] • Three-dimensional molds replicating specific food structures, such as chicken breast fillets, allowed the products to mimic the shape and fibrous texture of traditional animalbased products.

[0275] • Cylindrical molds with perforations facilitated uniform water drainage during pressing, ensuring consistent compaction.

[0276] • Custom geometries (e.g., spherical, cuboidal, conical) enabled tailored designs based on specific applications, enhancing versatility in product development.

[0277] Mold design directly influenced fiber alignment and density distribution. In closed molds, pressure forced fibers to align linearly, creating a more realistic muscle-like texture. Open molds resulted in less controlled fiber orientation, producing less structured textures. The choice of mold really depends on the end application desired.

[0278] Effect of Pre-Processing Steps on Fiber Orientation and Texture

[0279] Freezing steps, including freezing, thawing, and block formation, influenced fiber alignment and orientation:

[0280] • Uniform Fiber Distribution: Pre-pressing treatments aligned fibers more uniformly, especially when followed by high-pressure application.

[0281] • Mechanical Strength Variation: Samples without freezing exhibited firmer textures due to undisturbed membranes, while those subjected to freezing showed softer textures due to punctured cell walls from ice crystal formation.Enhanced Texture Replication: Freezing combined with mold design allowed the creation of structured products with specific textural properties, such as those mimicking chicken fillet.

[0282] Comparison of Cooking States (Uncooked vs. Cooked)

[0283] Cooking further revealed the effects of processing conditions:

[0284] Sample 5 (17 bar): Cooking increased cutting strength to 19.7 N, indicating moderate structural stabilization by ingredients.

[0285] Sample 6 (200 bar): Cooking amplified cutting strength to 30.8 N, showcasing the superior stabilization achieved under high-pressure treatment. Resulting texture has a good firm bite with a chewy, fibrous texture; well-defined fibers and structures, making it denser.

[0286] This sample is juicier and more textured compared to Sample 5, which contains more free water and distinct layers; these characteristics result in Sample 5 having a less desirable bite and texture than Sample 6.

[0287] Pure Biomass Samples (2 and 3): Cooking softened textures, particularly in frozen and thawed samples, where structural integrity was already compromised.

[0288] Flash Pressurization vs. Gradual Pressurization

[0289] Flash pressurization at 200 bar for 1 second (as seen in Samples 4 and 6) resulted in:

[0290] Uniform Density Distribution: The rapid application of pressure compressed the entire matrix uniformly, preventing localized densification.

[0291] Core Strengthening: The sudden reduction in volume reinforced the core structure, enhancing mechanical resistance.

[0292] Fibrous Texture Formation: The rapid redistribution of fibers mimicked the structure of muscle tissue more effectively than gradual pressurization.

[0293] In contrast, gradual pressurization at 17 bar for 300 seconds led to uneven water removal and surface densification, limiting the formation of a homogeneously dense matrix.In both cases, the addition of additional ingredients helps further improve the texture of the food product, but this improvement is prominent when applying flash pressurization, i.e. high pressures on frozen fungal doughs as shown when comparing sample 5 and 6 for example.

[0294] Summary of Findings

[0295] • The combination of high-pressure treatment, freezing / thawing, and ingredient addition allows precise tailoring of texture, density, and structural properties in fungal or mycelium-based products. These processing variables can be optimized to create fungal-based alternatives with characteristics comparable to traditional animal-based foods. The application of ultra-high pressure (200 bar) for ultra-short durations (1 second) significantly enhances the texture of mycelium-based products, such as enhanced texture and density, resulting in a fibrous, juicy good bite, and muscle-like textures due to fibre entanglement and removal of air pockets. In combination with these operational conditions, additional ingredients like proteins and hydrocolloids amplified the structural integrity and cutting strength of compacted dough. When comparing high-pressure (200 bar) and low-pressure (17 bar) treatments, the former produced products with superior cutting strength, structural stability, and resistance to processing effects such as freezing. These findings highlight the transformative potential of tailored high pressure applied on frozen mycelium doughs in developing high-quality, plant-based food products. • It can be concluded that the application of ultra-high pressure (200 bar) for an ultra-short duration of 1s has a positive effect on the mycelium cake texture when the mycelium is pressed frozen compared to low pressure (17 bar) for a higher duration of 300s. Higher pressure application in a confined mold compresses the biomass, removing air and effectively distributing the intracellular liquids. This results in the entanglement of fibres creating randomized small and long fibres within the matrix mimicking an actual animal muscle fibres constitution. The effect of pressure on texture is significantly enhanced when ingredients such as proteins and hydrocolloids are added to supplement the biomass in combination with the operational conditions.

[0296] Example 2

[0297] Free bases, nucleosides, 5'NMP nucleotides and RNA quantification

[0298] The freeze-dried sample is dissolved in water 25 mg / mL, heated at 100°C for 10 min and homogenized. For protein precipitation, TCA was added to a 5% final concentration and incubated at-20°C for 16h. 5pL of deproteinized extract was then injected and analyzed by ion-paired HPLC.To quantify RNA, nuclease was added to a solubilized sample (25 mg / mL) and incubated for 16h at 60°C to convert RNA to 5'NMP For protein precipitation, TCA was added to a 5% final concentration, incubated at -20°C for 16h. 5 L of deproteinized extract was then injected and analyzed by ion-paired HPLC.

[0299] Table 2.

[0300]

[0301] The RNA content of the Pleurotus species in this example (Sample 1 , control sample of raw mycelium) is below 2% meeting the regulatory requirements. However, the method of this invention discussed in Example 1 was successfully used to reduce RNA further across samples, with Sample 4 (0.47%) and Sample 6 (0.37%) showing significant RNA reductions. This suggests that the processing effectively lowered RNA levels, particularly in Sample 4 (raw mycelium) or 6 (mycelium dough). The decrease in RNA correlates with lower 5'-nucleotide concentrations. When the RNA content of the mycelium present in sample 6 is back calculated based on the composition and dry matter, the RNA content of said mycelium is found to be 0.48% which correlates well with the findings of sample 4 when mycelium is treated alone compared to when mycelium is treated while present in a dough.

[0302] This method is particularly useful for species, like mold species or others, where the RNA level of the raw mycelium (similar to Sample 1) surpasses the regulatory limit of 2%. The RNA level of the currently available mycelium ingredients, mainly mold species, are usually actively reduced via a process involving a final treating step comprising of a heating step at a certain temperature and / or at an adjusted pH for a certain time to actively reduce the RNA content to less than 2% on dry matter basis, thus reducingunwanted related health risks and bitter tastes and meeting the regulatory requirements. Therefore, the method of this invention provides an efficient method to reduce RNA of edible mycelium species including mold species (see strains listed in the description) or of doughs comprising said mycelium species.

[0303] The EUC was reduced across samples, with Sample 1 having the highest umami potential (122 g MSG / 100g), followed by Sample 4 (45 g MSG / 100g) and Sample 6 (8.22 g MSG / 100g) (see Table 2). The reduction in EUC correlates with lower levels of glutamic acid and 5'-nucleotides, particularly in Sample 6, where nucleotide content is minimal. This indicates that the processing effectively diminished umami intensity, likely by targeting RNA degradation, which directly impacts the availability of 5’-nucleotides. This method allows to reduce the EUC accordingly of mycelium or mycelium doughs targeting food applications, where very high umaminess is to be avoided.

[0304] Example 3

[0305] The mycelium production is separated into two different lines. The seed line and the specific main fermentation in the respective media. The seed line is split into three distinct steps. First, mycelium is grown on a petri dish on a PDA medium. Then, it is cultured for 7 days in one medium at 28 °C. The whole culture is then cultivated at ambient temperature. For each respective main fermentation one culture from the seed line is used and cultivated aerobically in a fermentation system cultivated in a fermenter for about 5 to 6 days until complete carbon source consumption. The fermentation is done aerobically at ambient temperatures (25 °C) and at a pH ranging from 4 to 6, preferably 5. In fermentation, it is also usual to control dissolved oxygen. The ingredients are then harvested and ready to be used for further processing after washing them with acidic water at a pH 3.5.

[0306] Biomass production was conducted under submerged fermentation conditions, wherein the culture media is composed of glucose monohydrate 20 gL1, Yeast extract 3 gL1, magnesium sulfate heptahydrate 1.1 gL1, and mono-potassium hydrogen phosphate 1 gL1. To further enhance the growth environment, a trace solution was injected at the beginning of fermentation, comprising ferric chloride hexahydrate, manganese sulfate monohydrate, zinc sulfate heptahydrate, calcium chloride dihydrate, and copper sulfate pentahydrate, in such a way that the medium comprises iron (0.001 g / l), copper (0.01 g / l), magnesium (0.09 g / l), calcium (0.009 g / l), manganese (0.09 g / l), zinc (0.05 g / l).The airflow was controlled at around 0.5 vvm to ensure adequate oxygen supply, crucial for aerobic fermentation, while maintaining a robust agitation profile set between 100 and 1200 RPM to enhance mass transfer and homogeneity of the culture.The same methodology can be used to also produce Agaricus bisporus, Agaricus campestris, Bjerkandera adusta, Boletus reticulatus, Flammulina velutipes, Ganoderma lucidum, Hericium abietis, Hericium erinaceus, Laetiporus sulphureus, Lentinula edodes, Morchella deliciosa, Morchella elata, Morchella esculenta, Pleurotus eryngii, and Pleurotus ostreatus to test the performance of other strains using the method of this invention. It is to be understood that the mycelium of any of these strains behaves, in the context of the present invention, where the product comprising of said respective mycelium is characterized by cutting strength increased by at least 15% compared to a product texturized at atmospheric pressure instead of being treated at a pressure of at 110 bar while frozen.

[0307] Example 4

[0308] The mycelium biomass of Ganoderma lucidum was cultivated using a methodology consistent with that outlined in Example 3, utilizing both the seed line and primary submerged fermentation processes with the same culture media previously described. To effectively concentrate the biomass within the fermentation broth, a partial separation process was implemented to reduce the broth volume by 25-35%. This was achieved through the decantation of the liquid phase, or supernatant, allowing for the retention of valuable biochemical components, including proteins and polysaccharides, that contribute to the final product's texture and nutritional profile. As a result of the volume reduction the concentration of extracellular polysaccharides (EPS) within the broth was at levels between 3 and 10 g / L. These polysaccharides play a vital role as a binding agent, significantly enhancing the stability, mouthfeel, and texture of the whole cut product. To further enrich the properties of the partially separated broth, additional binding agents could be introduced, allowing for modifications in texture, taste, and nutritional profile based on desired specifications.

[0309] The partially separated fermentation broth was frozen at -30°C then a controlled pressure of 120 bar was applied for a brief duration of 1 second. This innovative pressing technique at frozen conditions led to a uniform whole cut with texture characteristics that were notably consistent with those typically found in whole cuts produced from other mushroom species, such as Pleurotus pulmonanus, as discussed in Example 2.

[0310] In terms of quality assessment, the whole cut obtained from the Ganoderma lucidum partially separated broth exhibited significantly higher EUC values in comparison to those produced in Example 2. This enhancement is largely attributed to the nuanced variation in pressing pressures; while the whole cut in Example 2 was subjected to a more intense pressure of 200 bar, the milder 120 bar pressure in the currentprocess allowed for a more considerable retention of essential 5’-nucleotides within the Ganoderma lucidum whole cut. The resulting increase in umami flavor complexity can be effectively adjusted by varying the process conditions, particularly the pressing pressure, to meet specific product requirements related to desired umami. A similar correlation is evident in the RNA content, further enhancing the nutritional value and sensory attributes of the final product.

[0311] Example 5

[0312] Table 2 below illustrates exemplary combinations of mold surface areas and applied compressive forces resulting in specific pressures during the production of the compressed fungal ingredient dough. As shown, a force of 90 kN applied to a mold area of 120 cm2results in a pressure of 75 bar, while smaller areas or higher forces lead to significantly increased pressures. These examples demonstrate how force and area may be adjusted to achieve target pressure ranges suitable for different mold geometries (e.g., blocks, pieces, 2D or 3D shapes). It is understood that the listed values are illustrative; actual force and area parameters can vary depending on the desired pressure and product design.

[0313] Table 3.

[0314] < > > >

[0315]

Claims

New PCT-Patent ApplicationMushlabs GmbHVossius Ref.: AJ3269 PCT-0 BSCLAIMS1. A compressed fungal ingredient dough, comprising fibrous mycelium biomass and a binding agent, wherein said compressed fungal ingredient dough is characterized by an RNA content of not more than 2.0% RNA by weight, and wherein said dough is characterized by a homogeneous cutting strength of at least 14 N.

2. The compressed fungal ingredient dough of claim 1 , characterized by an RNA content of not more than 1.0 % RNA by weight.

3. The compressed fungal ingredient dough of claim 2, characterized by an RNA content of not more than 0.8 % RNA by weight.

4. The compressed fungal ingredient dough of claim 3, characterized by an RNA content of not more than 0.5 % RNA by weight.

5. The compressed fungal ingredient dough of any one of claims 1 to 4, characterized by a homogeneous cutting strength of at least 14 N if measured when the compressed fungal ingredient dough is cooked sous vide in water at a temperature of between 70 and 90°C, preferably at 80°C, for a time of between 5 to 15 minutes, preferably for a time of 10 minutes, or when the compressed fungal ingredient dough is cooked by pan-frying for a time of 5 to 10 minutes at a temperature of between 160 and 180°C, wherein, if the compressed fungal ingredient dough is frozen, it is thawed prior to cooking.

6. The compressed fungal ingredient dough of any one of claims 1 to 5, characterized by a homogeneous cutting strength of at least 30 N, preferably if measured when the compressed fungal ingredient dough is cooked sous vide in water at a temperature of between 70 and 90°C, preferably at 80°C, for a time of between 5 to 15 minutes, preferably for a time of 10 minutes, or when the compressed fungal ingredient dough is cooked by pan-frying for a time of 5 to 10 minutes at a temperature of between 160 and 180°C, wherein, if the compressed fungal ingredient dough is frozen, it is thawed prior to cooking.

7. The compressed fungal ingredient dough of any one of claims 1 to 6, wherein the compressed fungal ingredient dough comprises a plurality of aligned mycelium fibers.

8. The compressed fungal ingredient dough of any one of claims 1 to 7, wherein the compressed fungal ingredient dough is substantially free of air pockets.

9. The compressed fungal ingredient dough of any one of claims 1 to 8, wherein the compressed fungal ingredient dough is characterized by a protein content of at least 20 wt. % of the dry mass.

10. The compressed fungal ingredient dough of any one of claims 1 to 9, wherein the dry matter of the fibrous mycelium biomass is between 5 and 30 wt%.

11. The compressed fungal ingredient dough of any one of claims 1 to 10, wherein the compressed fungal ingredient dough has been obtained by pressing the fungal ingredient dough while frozen at a pressure of at least 75 bar,and / orwherein the compressed fungal ingredient dough is frozen, wherein the cutting strength is to be determined on the compressed fungal ingredient dough that is thawed,and / orwherein the compressed fungal ingredient dough is not frozen.

12. The compressed fungal ingredient dough of claim 11, wherein the compressed fungal ingredient biomass has been obtained by pressing the fungal ingredient dough at a pressure of at least 75 bar while block-frozen.

13. The compressed fungal ingredient dough of any one of claims 1 to 12, wherein the fibrous mycelium biomass is a fibrous mycelium biomass of an edible fungus.

14. The compressed fungal ingredient dough of any one of claims 1 to 13, wherein the fibrous mycelium biomass comprises a fungal strain selected from Basidiomycota, Ascomycota, Pezizomycotina, Agaromycotina, Pezizomycetes, Agaricomycetes, Sordariomycetes, Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Hypocreales, Morchellaceae, Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilaceae, Polyporaceae, Strophariaceae, Lyophyllaceae, Tricholomataceae,Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, and Fistulinacea.

15. The compressed fungal ingredient dough of claim 13 , wherein the edible fungus is selected from Agaricus spp., Aspergillus spp., Bjerkandera spp., Boletus spp., Calvatia spp., Cordyceps spp., Flammulina spp., Fusarium spp., Fomitopsis spp., Ganoderma spp., Grifola spp., Hericium spp., Hyphoroma spp., Inonotus spp., Laetiporus spp., Lentinula spp., Lepista spp., Morchella spp., Mycetinis spp., Neurospora spp., Paecilomyces spp., Panus spp., Pholiota spp., Pleurotus spp., Piptoporus spp., Polyporus spp., Rhizopus spp., Sparassis spp., Tremella spp., Trametes spp., Tricholoma spp., Tuber spp., and Wolfiporia spp.

16. The compressed fungal ingredient dough of any one of claims 1 to 15, wherein the binding agent is selected from and additive binding agent and / or nutrient binding agent.

17. The compressed fungal ingredient dough of claim 16, wherein the additive binding agent is selected from alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, processed euchema seaweed, locust bean gum, guar gum, tragacanth, gum arabic (acacia gum), xanthan gum, tara gum, gellan gum, sorbitol, mannitol, glycerol, konjac, pectins, cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl methyl cellulose, sodium carboxy methyl cellulose, cellulose gum, enzymatically hydrolysed carboxy methyl cellulose, sodium- potassium and calcium salts of fatty acids, magnesium salts of fatty acids, mono-and diglycerides of fatty acids, acetic acid esters of mono- and diglycerides of fatty acids, lactic acid esters of mono-and diglycerides of fatty acids, citric acid esters of mono-and diglycerides of fatty acids, tartaric acid esters of mono-and diglycerides of fatty acids, microcrystalline cellulose-cellulose Gel, mono and diacetyl tartaric acid esters of mono-and diglycerides of fatty acids, mixed acetic and tartaric acid esters of mono-and diglycerides of fatty acids, sorbitol mannitol, psylium husk, polydextrose, oxidized starch, monostarch phosphate, distarch phosphate, phosphate distarch phosphate, acetylated distarch phosphate, acetylated starch, acetylated distarch adipate, hydroxy propyl starch, hydroxy propyl distarch phosphate, starch sodium octenyl succinate, starch-based ingredients, and acetylated oxidized starch, and / or wherein the nutrient binding agent is selected from protein rich ingredients (preferably selected from pea protein isolate, chickpea protein isolate, wheat gluten, egg white powder, potato protein and / or mungbean protein isolate), carbohydrate / dietary fiber rich ingredients (preferably selectedfrom grain-based flours, grain-based starches, legume-based starches, fruit-based fibers, polysaccharides, starch-based ingredients, psylium husk, inulin, wheat starch, corn starch, potato starch), vitamins / mineral-rich ingredients, and lipid rich ingredients (such as edible oils and butters).

18. The compressed fungal ingredient dough of any one of claims 1 to 15, wherein the binding agent is selected from gluten, methylcellulose, xanthan gum, alginate, locust bean gum, agar-agar, gum arabic, carrageenan, pectin, gellan gum, cellulose gum, egg white protein, konjac, and guar gum, preferably wherein the binding agent comprises a binding agent selected from gluten and egg white powder.

19. The compressed fungal ingredient dough of any one of claims 1 to 18, wherein the compressed fungal ingredient dough further comprises at least one additional component selected from plantbased protein sources, preferably selected from legumes (such as soybean, pea, chickpea, mung bean, fava bean, lupin bean, lentils, edamame, adzuki beans), cereals (such as wheat, rice, oat, quinoa, amaranth, spelt, barley, rye, teff), tubers (such as potato, sweet potato, cassava), nuts and seeds (such as almonds, walnuts, pumpkin seeds, sunflower seeds, chia seeds, hemp seeds, cashews, pistachios, and flaxseeds), vegetables (such as spinach, broccoli, Brussels sprouts, asparagus, kale, artichoke), fruits (e.g., guava, jackfruit, durian), algae (such as algae selected from Spirulina and Chlorella), mushrooms and mushroom concentrates, preferably selected from Agaricus spp. (Button mushroom), Honey mushroom, Boletus, Truffle, Morel, Chantarelle, Saffron milk-cap, Oyster mushroom, King oyster mushroom, Shiitake, Straw mushroom, and Enoki, animal sources, preferably selected from egg, dairy, meat (such as chicken, turkey, beef, pork, bison, lamb, duck), and seafood (such as fish, prawns, crab, lobster, mussels, oysters, scallops, clams, squid, and octopus); plant-based fat and oil sources, (preferably selected from soybean, rapeseed, canola, coconut, sunflower, avocado, olive, flaxseed, corn, palm, shea butter, macadamia, walnut, and grapeseed), carbohydrate sources (such as starch, wheat flour, potato flour, rice flour, oat flour, corn flour, tapioca flour, buckwheat flour, millet flour, sorghum flour, or fruit extracts) and fiber sources (such as pea, sugarcane, wheat, cellulose, oats, apple, psyllium husk, flaxseed, chia seeds, inulin, beet fiber, bamboo fiber, acacia fiber, or resistant starch).

20. The compressed fungal ingredient dough of any one of claims 1 to 19, further comprising at least one additional component selected from algae, preservatives, antioxidants and acidity regulators, pH regulators and anticaking agents, flavor enhancers, colours, and glazing agents and sweeteners, or further comprising at least one additional component selected from vitamin B12,vitamin B6, vitamin B2, vitamin B3 (also referred to as niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salt (NaCI, further comprising iodine salts in an amount of up to 5% w / w), enzymes (e.g. transglutaminase, amylase), minerals (e.g. salts comprising calcium, iron, and / or potassium, etc.), flavors or flavor components (salt, pepper, garlic, onions, mushroom fruiting body pieces, vegetable pieces, ginger, turmeric, curry, sugars (i.e., sucrose, glucose, mono- or disaccharide), oils, lemon juice, orange juice, herbs and spices, yeast flakes), texturized vegetable proteins, and natural aromatic compounds.

21. The compressed fungal ingredient dough of any one of claims 1 to 20, wherein the compressed fungal ingredient dough is characterized by a calculated EUC of not more than 1500 g MSG / 100g, preferably not more than 150 g MSG / 100g, more preferably not more than 50 g MSG / 100g, wherein the EUC is defined as:EUC=£aibi+1218(£aibi)(£ajbj),wherein the EUC of the sample is expressed in g monosodium glutamate (MSG) / 100 g, ai is the concentration (g / 100 g) of each umami amino acid Asp or Glu, aj is the concentration (g / 100 g) of each umami 5'-n ucleotide 5-IMP, 5-GMP, 5’-XMP or 5-AMP, bi is the relative umami concentration (RUC) for each umami amino acid to MSG, defined as 1 for Glu and 0.077 for Asp and bj is the RUC for each umami 5'-nucleotide defined as 1 for 5-IMP, 2.3 for 5-GMP, 0.61 for 5’-XMP and 0.18 for 5-AMP.

22. A method for preparing a compressed fungal ingredient dough, the method comprising the steps of(a) providing a fibrous mycelium biomass;(b) forming a fungal ingredient dough comprising the biomass of (a) and a binding agent; (c) freezing the fungal ingredient dough obtained in (b), thereby obtaining a frozen fungal ingredient dough; and(d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen fungal ingredient dough of (c) to yield a frozen compressed fungal ingredient dough.

23. The method of claim 22, wherein the fibrous mycelium biomass is a fibrous mycelium biomass of an edible fungus.

24. The method of claim 22 or 23, wherein the fibrous mycelium biomass comprises a fungal strain selected from Basidiomycota, Ascomycota, Pezizomycotina, Agaromycotina, Pezizomycetes, Agaricomycetes, Sordariomycetes, Pezizales, Boletales, Cantharellales, Agaricales, Polyporales, Russulales, Auriculariales, Hypocreales, Morchellaceae, Tuberaceae, Pleurotaceae, Agaricaceae, Marasmiaceae, Cantharellaceae, Hydnaceae, Boletaceae, Meripilaceae, Polyporaceae, Strop hariaceae, Lyophyllaceae, Tricholomataceae, Omphalotaceae, Physalacriaceae, Schizophyllaceae, Sclerodermataceae, Ganodermataceae, Sparassidaceae, Hericiaceae, Bondarzewiaceae, Cordycipitaceae, Auriculariaceae, and Fistulinacea.

25. The method of claim 23 or 24, wherein the edible fungus is selected from Agaricus spp ., Aspergillus spp., Bjerkandera spp., Boletus spp., Calvatia spp., Cordyceps spp., Flammulina spp., Fusarium spp., Fomitopsis spp., Ganoderma spp., Grifola spp., Hericium spp., Hyphoroma spp., Inonotus spp., Laetiporus spp., Lentinula spp., Lepista spp., Morchella spp., Mycetinis spp., Neurospora spp., Paecilomyces spp., Panus spp., Pholiota spp., Pleurotus spp., Piptoporus spp., Polyporus spp., Rhizopus spp., Sparassis spp., Tremella spp., Trametes spp., Tricholoma spp., Tuber spp., and Wolfiporia spp.

26. The method of any one of claims 22 to 25, wherein the binding agent is selected from additive an binding agent and / or a nutrient binding agent.

27. The method of claim 26, wherein the additive binding agent is selected from alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, processed euchema seaweed, locust bean gum, guar gum, tragacanth, gum arabic (acacia gum), xanthan gum, tara gum, gellan gum, sorbitol, mannitol, glycerol, konjac, pectins, cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl methyl cellulose, sodium carboxy methyl cellulose, cellulose gum, enzymatically hydrolysed carboxy methyl cellulose, sodium- potassium and calcium salts of fatty acids, magnesium salts of fatty acids, mono- and diglycerides of fatty acids, acetic acid esters of mono- and diglycerides of fatty acids, lactic acid esters of mono-and diglycerides of fatty acids, citric acid esters of mono-and diglycerides of fatty acids, tartaric acid esters of mono-and diglycerides of fatty acids, microcrystalline cellulosecellulose Gel, mono and diacetyl tartaric acid esters of mono-and diglycerides of fatty acids, mixed acetic and tartaric acid esters of mono-and diglycerides of fatty acids, sorbitol mannitol, psylium husk, polydextrose, oxidized starch, monostarch phosphate, distarch phosphate, phosphate distarch phosphate, acetylated distarch phosphate, acetylated starch, acetylated distarch adipate,hydroxy propyl starch, hydroxy propyl distarch phosphate, starch sodium octenyl succinate, starch- based ingredients, and acetylated oxidized starch, and / or wherein the nutrient binding agent is selected from protein rich ingredients (preferably selected from pea protein isolate, chickpea protein isolate, wheat gluten, egg white powder, potato protein and / or mungbean protein isolate), carbohydrate / dietary fiber rich ingredients (preferably selected from grain-based flours, grainbased starches, legume-based starches, fruit-based fibers, polysaccharides, starch-based ingredients, psylium husk, inulin, wheat starch, corn starch, potato starch), vitamins / mineral-rich ingredients, and lipid rich ingredients (such as edible oils and butters).

28. The method of any one of claims 22 to 25, wherein the binding agent is selected from from methylcellulose, konjac, carrageenan, pectin, gellan gum, guar gum, gluten, egg white powder, starch, and psyllium husk, or selected from gluten, methylcellulose, xanthan gum, alginate, locust bean gum, agar-agar, gum arabic, carrageenan, pectin, gellan gum, cellulose gum, egg white protein, konjac, and guar gum, preferably wherein the binding agent comprises a binding agent selected from gluten and egg white powder.

29. The method of any one of claims 22 to 28, wherein the fungal ingredient dough formed in step (b) further comprises at least one additional component selected from plant-based protein sources, preferably selected from legumes (such as soybean, pea, chickpea, mung bean, fava bean, lupin bean, lentils, edamame, adzuki beans), cereals (such as wheat, rice, oat, quinoa, amaranth, spelt, barley, rye, teff), tubers (such as potato, sweet potato, cassava), nuts and seeds (such as almonds, walnuts, pumpkin seeds, sunflower seeds, chia seeds, hemp seeds, cashews, pistachios, and flaxseeds), vegetables (such as spinach, broccoli, Brussels sprouts, asparagus, kale, artichoke), fruits (e.g., guava, jackfruit, durian), algae (such as algae selected from Spirulina and Chlorella), mushrooms and mushroom concentrates, preferably selected from Agaricus spp (Button mushroom), Honey mushroom, Boletus, Truffle, Morel, Chantarelle, Saffron milk-cap, Oyster mushroom, King oyster mushroom, Shiitake, Straw mushroom, and Enoki, animal sources, preferably selected from egg, dairy, meat (such as chicken, turkey, beef, pork, bison, lamb, duck), and seafood (such as fish, prawns, crab, lobster, mussels, oysters, scallops, clams, squid, and octopus); plant-based fat and oil sources, (preferably selected from soybean, rapeseed, canola, coconut, sunflower, avocado, olive, flaxseed, corn, palm, shea butter, macadamia, walnut, and grapeseed), carbohydrate sources (such as starch, wheat flour, potato flour, rice flour, oat flour, corn flour, tapioca flour, buckwheat flour, millet flour, sorghum flour, or fruit extracts) and fibersources (such as pea, sugarcane, wheat, cellulose, oats, apple, psyllium husk, flaxseed, chia seeds, inulin, beet fiber, bamboo fiber, acacia fiber, or resistant starch).

30. The method of any one of claims 22 to 29, wherein the fungal ingredient dough formed in step (b) further comprises at least one additional component selected from algae, preservatives, antioxidants and acidity regulators, pH regulators and anticaking agents, flavor enhancers, colours, and glazing agents and sweeteners, or wherein the fungal ingredient dough formed in step (b) further comprises at least one additional component selected from vitamin B12, vitamin B6, vitamin B2, vitamin B3 (also referred to as niacin), riboflavin, thiamine, vitamin A, vitamin E, omega-3 fatty acids, vitamin D2, folic acid, iodized salt (NaCI, further comprising iodine salts in an amount of up to 5% w / w), enzymes (e.g. transglutaminase, amylase), minerals (e.g. salts comprising calcium, iron, and / or potassium, etc.), flavors or flavor components (salt, pepper, garlic, onions, mushroom fruiting body pieces, vegetable pieces, ginger, turmeric, curry, sugars (i.e., sucrose, glucose, mono- or disaccharide), oils, lemon juice, orange juice, herbs and spices, yeast flakes), texturized vegetable proteins, and natural aromatic compounds.

31. The method of any one of claims 22 to 30, wherein step (c) is performed using a plate freezing assembly, optionally wherein the freezing is performed at a temperature of between -80°C and - 4°C, preferably at a temperature of between -20°C and -15°C.

32. The method of any one of claims 22 to 31 , wherein the pressure in step (d) is applied for up to 5 s, preferably for up to 2 s, more preferably for up to 1s.

33. The method of any one of claims 22 to 32, wherein step (d) is performed in a mold.

34. The method of claim 33,wherein the mold in step (d) is filled with the frozen fungal ingredient dough to at least 60% of its volume,and / orwherein the mold is characterized by a shape of a food product, preferably wherein said food product is selected from steaks, breasts, fillets, chops, cutlets, chunks, ribs, loins, sticks and nuggets designed to imitate animal meats such as beef, chicken, fish, lamb, veal, and duck,and / ormolding process involves applying a force of between 50 kN and 200 kN to shape the biomass material.

35. The method of any one of claims 22 to 34, wherein the pressure is applied unidirectionally, which preferably results in the plurality of mycelium fibers being aligned in a single direction, preferably wherein step (d) is performed by using electro-mechanical press.

36. The method of any one of claims 22 to 35, further comprising the step (e) of post-processing of the product of step (d).

37. The method of claim 36, wherein step (e) comprises coating or marinating or cooking or reshaping the compressed fungal ingredient dough.

38. The method of any one of claims 22 to 37, wherein further in step (b) at least one cross-linking enzyme is added to the fungal ingredient dough formed in step (b), preferably wherein in step (b) transglutaminase, sortase, lysyl oxidase, amine oxidase, tyrosinase, laccase, peroxidase, glucose oxidase, polyphenol oxidase or lipoxygenase, preferably transglutaminase is added to the fungal ingredient dough formed in step (b).

39. The method of any one of claims 22 to 38, wherein in step (b) at least one further enzyme selected from lipolytic enzymes, proteases, p-glucanases, xylanases, amyloglucosidases, hemicellulases, and digestive enzymes is added to the fungal ingredient dough formed in step (b).

40. The method of any one of claims 22 to 39, wherein in step (d) a pressure of at least 80 bar is applied.

41. The method of any one of claims 22 to 39, wherein in step (d) a pressure of at least 100 bar is applied.

42. A compressed fungal ingredient dough obtainable according to the method of any one of claims 22 to 41, optionally wherein the compressed fungal ingredient dough is frozen.

43. A method for mechanically reducing RNA content in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen.

44. The method of claim 43, wherein the reduction in RNA content of up to 99% is achieved.

45. A food product comprising the compressed fungal ingredient dough of any one of claims 1 to 21 or 42.

46. A method for preparing a compressed fungal ingredient dough, the method comprising the steps of(a / b) providing a partially separated fermentation broth;(c) freezing the partially separated fermentation broth of (a / b), thereby obtaining a frozen partially separated fermentation broth; and(d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen partially separated fermentation broth of (c) to yield a frozen compressed fungal ingredient dough.

47. A method for mechanically reducing a calculated EUC in a mycelium, the method comprising the step of applying pressure of at least 75 bar for a time of up to 10 s to the mycelium while frozen, wherein the EUC is defined as defined in claim 21.

48. The method of claim 47, wherein the reduction in calculated EUC of at least 50% is achieved.

49. A method for preparing a compressed fungal ingredient dough, the method comprising the steps of(a) providing a chopped fruiting body of an edible mushroom;(b) forming a dough comprising the biomass of (a) and a binding agent;(c) freezing the dough obtained in (b), thereby obtaining a frozen dough; and(d) applying pressure of at least 75 bar for a time of up to 10 s to the frozen dough of (c) to yield a frozen compressed fungal ingredient dough.

50. The method of claim 49, wherein the edible mushroom in step (a) is preferably Agaricus spp., Bjerkandera spp., Boletus spp., Hericium spp., Laetiporus spp., Lentinula spp., Morchella spp., Pleurotus spp. Polyporus spp., orTremella spp..

51. A compressed fungal ingredient dough obtainable according to the method of claim 46, 49 or 50.

52. A food product comprising a compressed fungal ingredient dough of claim 51.

53. A food product comprising the mycelium obtainable according to the method of claim 43 or 44.

54. A food product comprising the mycelium obtainable according to the method of claim 47 or 48.

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