TECHNICAL FIELD

The present document is directed to a printed food product comprising fungal biomass, such as a meat analogue product. The present document is also directed to materials and methods for preparing such food products.

BACKGROUND

In recent years, the excessive use of animal-based dietary protein source has come under close scrutiny and received significant negative criticism. Several global factors are at play like food security, scarce resources, sustainability, animal welfare, and climate change, but the root cause of this movement can be narrowed down to the issue that production, distribution and consumption of meat leads to substantial negative environmental impact.

Livestock rearing not only emits massive quantities of greenhouse gases due to its excessive use of land, water and resources, but also contributes to deforestation, biodiversity loss, eutrophication, and a range of other climate-related issues. Furthermore, excessive consumption of animal-based protein is associated with a range of detriments to health and wellbeing that include but are not limited to higher prevalence of obesity, and elevated risks of cancer and cardiovascular disease. In addition, the unsustainable practices that prevail in many parts of meat and dairy manufacturing contribute to increased risks of zoonosis as well as antibiotic resistance. In recent years, these issues have led to a heavily increased demand for meat resembling food products (‘meat replacements’) comprised of protein sources of non-animal origin (‘alternative protein’). These forces have spilled over into the segment for fish as well.

Consumers are increasingly looking for fish replacements based on alternative protein and the food manufacturing industry has responded by innovating heavily within the area, outputting large quantities of products that are perceived as capable of meeting the emerging needs of the market. Typically, these products are made using plant-based protein sources. However, some plant protein sources lack some of the amino acids needed in human diet, or they contain antinutrients which can possibly make the absorption of other nutrients more difficult.

Another non-animal protein source are fungi, as the mycelium of fungi contain up to 60 % protein (dry weight) and 12 % fiber, making it a nutritious source of protein. Additionally, fungi can be easily grown in a liquid environment and opposed to traditional agriculture processes, fungal fermentation is independent of climate or geographic location and can be scaled vertically, which allows effective land use. Furthermore, the turnover of the fermentation process is very high, allowing quick production of protein.

However, the main issue with producing fungi-based protein meat analogues is the mouthfeel, visual appeal, and palatability of the consumer product. This problem is also known from plant-based protein sources. Overall, creating non-meat and nonfish products that have taste profiles similar to those of meat and fish is difficult. More pressing, however, is the issue of texture. Most raw materials of alternative protein are provided in non-texturized (e.g. as powder) form such as plant protein isolates or concentrates, meaning that several advanced processing steps and extensive use of additives is required to acquire a meat- or fish-like texture.

One suggested solution has been to use mycoprotein, i.e. protein derived from fungi that are produced for the purpose of human consumption. Mycoprotein and its various fermentation-based production methods have a range of advantages and characteristics that make them highly suitable for solving present challenges related to poor nutrition, food security and climate change. Consumption of mycoprotein is associated with a range of benefits to health and wellbeing, attributable to its beneficial nutritional composition. Furthermore, the turnover of the fermentation process is very high, allowing quick production of protein.

The potential of using biomass from filamentous fungi as a protein source has therefore garnered positive attention in recent years, as it offers high quality nutrition benefits, low allergenicity, and the biomass is naturally texturized. This texture is due partly to: (a) its morphology, growing as filaments in a highly structured network, and partly to; (b) its naturally high content of dietary fiber, which is located in the fungi cell walls and contributes to a resistant structure.

One alternative to solving one or more of the objects described above is thus to use species of filamentous fungi for the production of fungi biomass to be used in food production. Many species, however, have morphologies and rigid fiber levels that create an issue with regards to excessive toughness, chewiness, and pulpy mouthfeel, making products too chewy to the extent that they are not acceptable for consumers. Certain other fungi biomass products are mixed with egg albumin to achieve a meat- or fish-like structure, but this makes the product unsuitable for vegans and there have been complaints from consumers that such food products are “too soft”, “mushy”, and “lacking chewiness” instead.

There is thus still need for a non-animal based, high-nutritious and environmentally friendly food product which has a taste and texture resembling a wide variety of meat- and fish-based food product types.

SUMMARY

The above problems are solved or at least mitigated by the invention as disclosed herein.

The present document is thus directed to a method for producing a printable material, such as a 3D printable or extrudable material, comprising fungal biomass, said method comprising the steps of: a) providing a fungal biomass comprising or consisting of food-safe filamentous fungi; b) optionally washing and/or heat-treating and/or dewatering and/or grinding said fungal biomass; c) adjusting the pH of the fungal biomass of step a) or b) to a pH of at least 7 with a pH regulator; d) optionally grinding and/or high shear mixing the pH adjusted fungal biomass of step c). The printable material obtained from such method may be a food product for humans and/or other animals. The pH of the fungal biomass may be adjusted in step c) to a pH of from about 7 to about 10, such as from about 7.5 to about 9. The pH regulator may be an inorganic base such as sodium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, calcium bicarbonate, potassium bicarbonate, potassium hydroxide, ferrous hydroxide, lime, calcium carbonate, and/or trisodium phosphate, and/or an organic acid such as lactic acid, citric acid, acetic acid, hydrochloric acid, and/or ascorbic acid.

One or more of a stabilizing agent and/or food additive may be added before or after the pH adjustment step c). The stabilizing agent may be a thickening and/or gelling agent, such as native starch, modified starch, alginate, pectin, gelatin, agar, guar gum, gellan gum, xanthan gum, konjac gum, curdlan gum, carrageenan, kappa carrageenan, cellulose derivative (such as methylcellulose, carboxymethylcellulose, and/or hydroxypropyl methylcellulose), or any combination thereof. The stabilizing agent may preferably be kappa carrageenan, which may be blended to the fungal biomass for at least 5 minutes at a temperature of about 65 °C. The stabilizing agent may be alginate in an amount of from about 0.2 % to about 3 % dry weight, based on the total weight of the printable material, konjac gum in an amount of from about 2 % to about 6% dry weight and starch in an amount of from about 0 % to about 5 % dry weight, based on the total weight of the printable material, and/or kappa carrageenan in an amount of from about 0.5 % to about 5 % dry weight, such as about 0.5 % to about 2.5 % dry weight, based on the total weight of the printable material. In case only starch is used as a stabilizing agent, the amount of starch may be up to about 10% dry weight, based on the total weight of the printable material.

The stabilizing agent may preferably comprise kappa carrageenan in an amount of from about 0.5% to about 2.5% dry weight and a cellulose derivative, such as methylcellulose, carboxymethylcellulose, and/or hydroxypropyl methylcellulose, in an amount of from about 0% to about 5% dry weight, such as about 0.5% to about 2% dry weight, based on the total weight of the printable material.

The food additive may be a protein containing ingredient, an oil, a fat, a colorant, a flavorant, a salt; and/or a crosslinking agent, or any combination thereof. Any stabilizing agent stable at a pH of at least 7, preferably from 7 to 10, may be used. The skilled person knows which chemical and/or temperature treatment is required for each stabilizing agent and/or food additive in order to for instance obtain a fungal biomass in a gelled state.

The amount of fungal biomass in the printable material produced is from about 10 % to about 45 % dry weight, such as from about 30 % to about 40 % dry weight, based on the total weight of the printable material.

The printable material may comprise from about 55 % to about 90 % water based on the total weight of the printable material.

The fungal biomass provided in step a) may be a freeze-dried and/or a fresh fungal biomass.

The fungal biomass comprises or consists of food-safe filamentous fungi, such as food safe filamentous fungi of the Zygomycota and/or Ascomycota phylum, such as fungi of the genera Aspergillus, Cordyceps, Fusarium, Ganoderma, Inonotus, Neurospora, Pennicillium, Pleurotus, Rhizopus, Trametes, Trichoderma, Tuber, Ustilago, Xylaria, or any combination thereof. Examples of food safe filamentous fungal species include, but are not limited to, Aspergillus oryzae, Cordyceps militaris, Cordyceps sinensis, Fusarium graminareum, Fusarium venenatum, Lentinula edodes, Neurospora crassa, Neurospora intermedia, Neurospora sitophila, Pennicillium camemberti, Rhizomucor miehei, Rhizopus microsporus, Rhizopus oligosporus, Rhizopus oryzae, Tuber magnatum, Tuber melanosporum, Xylaria hypoxion or any combination thereof.

The fungal biomass is typically obtained through liquid fermentation or solid fermentation.

The present document is also directed to a printable material obtained or obtainable by the method for producing a printable material as disclosed herein.

The present document is also directed to a method for preparing a printed fungal biomass food product, such as a meat analogue product, said method comprising the steps of: a) providing a printable material as defined herein, such as preparing a printable material comprising fungal biomass as disclosed herein; b) printing said printable material comprising fungal biomass to prepare a printed material comprising one or more printed strands of fungal biomass; and c) optionally solidifying the printed material comprising one or more printed strands of fungal biomass obtained in step b) through heating and/or crosslinking, to provide said printed fungal biomass food product.

Step b) may comprise 3D printing or printing by extrusion. Furthermore, the printed fungal biomass food product may have from about 1 % to about 45 % of dry fungal biomass based on the total weight of the printed fungal biomass food product.

The printing may generate one or more layers of printed strands of fungal biomass, wherein each layer comprises multiple printed strands of fungal biomass arranged in substantially parallel direction. Some or all of the printed strands of fungal biomass may be in contact with at least one neighboring printed strand.

The method for preparing a printed fungal biomass food product may comprise i. feeding the printable material into a single or multiple print heads; ii. extruding the printable material through a nozzle or multiple nozzles.

The nozzle(s) may have a diameter between about 0.2 mm and about 5 mm, preferably between about 0.8 mm and about 1 .6 mm. However, depending on the desired shape of the printed strands, the nozzles may also have a larger diameter, such as on the centimeters’ scale.

The method for preparing a printed fungal biomass food product may further comprise a further step of lowering the pH of the printed material obtained in step b). Such as step of lowering the pH is preferably performed after step c), when a step c) is performed. The step of lowering the pH may e.g. be performed by adding a pH regulator to said printable material before printing and/or by washing the printed material with an acidic solution. The temperature in optional step c) typically ranges from about 60 °C to about 200 °C, such as from about 80 °C to about 120 °C.

The crosslinking in optional step c) may be performed by external crosslinking and/or by internal crosslinking. The crosslinking may e.g. be chemical, thermal and/or physical crosslinking.

The printed fungal biomass food product may have from about 1 % to about 45 % of dry fungal biomass based on the total weight of the printed fungal biomass food product.

The present document is also directed to a printed fungal biomass food product, such as a meat replacement product, such as a chicken, pork, beef, lamb, or seafood replacement product, said printed fungal biomass food product comprising one or more strands of printed fungal biomass. Such a printed fungal biomass food product may be obtained or obtainable by the method for preparing a printed fungal biomass food product described herein.

The printed fungal biomass food product comprises food-safe filamentous fungi as described elsewhere herein.

The printed strands of fungal biomass in the printed fungal biomass food product are arranged in a substantially parallel direction. The printed strands of fungal biomass may be arranged in one or more layers. One or more of the printed strands of fungal biomass may be in contact with at least one neighboring printed strand of fungal biomass.

The printed fungal biomass food product may be 3D printed or extruded.

The printed fungal biomass food product may comprise from about 1 % to about 45 % of dry fungal biomass based on the total weight of the printed fungal biomass food product.

The printed fungal biomass food product may comprise a pH regulator, a stabilizing agent, and/or a food additive as described elsewhere herein. The printed fungal biomass food product may comprise from about 70 to about 95 % of water based on the total weight of the printed fungal biomass food product.

The printed strands of fungal biomass in the printed fungal biomass food product may be aligned in a first direction, wherein the printed strands of fungal biomass have a Young’s modulus of about 0.03 MPa to about 0.5 MPa when measured in a direction perpendicular to the first direction, and wherein the printed strands of fungal biomass aligned in the first direction have a Young’s modulus of about 0.1 MPa to 0.8 MPa when measured in a direction parallel to the first direction.

The printed fungal biomass food product may e.g. be a meat replacement product, such as a chicken, pork, beef, lamb replacement product, or a seafood replacement product, such as fish or shrimp.

The present document is also directed to a printed fungal biomass food product comprising a filamentous fungal biomass, wherein said printed fungal biomass food product comprises: at least one first layer of printed strands; at least one second layer of printed strands arranged to be stacked on the at least one first layer, said at least one first layer of printed strands and said at least one second layer of printed strands being substantially aligned in a single direction; and optionally at least one layer of fat, preferably intercalated with the at least one first layer and the at least one second layer; wherein the printed fungal biomass food product has from about 1 % to about 45 % of dry fungal biomass based on the total weight of the printed fungal biomass food product.

The printed fungal biomass food product has preferably from about 1 % to about 45 % of dry filamentous fungal biomass based on the total weight of the printed filamentous fungal biomass food product.

The printed fungal biomass food product may be in a gelled state, for instance by said printed fungal biomass food product further comprising gelling agent as defined elsewhere herein.

The printed fungal biomass food product may be arranged such that one optional layer of fat is stacked on one first layer of printed strands, and one second layer of printed strands is stacked either on the layer of fat (if any) or on the first layer of printed strands. The printed fungal biomass food product may comprise a plurality of first layers, second layers and optional layers of fat stacked one over the other as described.

Said printed fungal biomass food product advantageously has an appearance and behaviour similar to meat fiber, such as chicken fibers. Furthermore, when pulled apart, said food product also behaves as a meat product, such as chicken meat fibers. By printing a fungal biomass food product, particularly by 3D printing, it is possible to create a food product having any meat shape, including chicken, pork, beef, lamb, or seafood replacement product. Therefore, it is possible to provide a meat analogue that has a similar appearance and behaviour of for example chicken fillet. Furthermore, the addition of at least one layer of fat, particularly when intercalated with the at least one first layer and the at least one second layer, advantageously provide a fungal food product with increased flavor and improved tenderness and/or moistness. The fungal biomass food product also has a visual appearance of meat due to the formation of marbling-like structures, i.e. , a structure similar to white flacks of intramuscular fat in a cut of meat.

The printed fungal biomass food product may have a hardness of from about 10,000 to about 40,000 g. Said hardness may be measured by a Texture Profile Analysis (TPA) as defined elsewhere herein. The hardness range of from about 10,000 to about 40,000 g advantageously provides a printed fungal biomass food product having an improved firmness for human and/or animal consumption.

The printed strands of fungal biomass in the printed fungal biomass food product may be aligned in a first direction, wherein the printed strands of fungal biomass have a Young’s modulus of about 0.03 MPa to about 0.5 MPa when measured in a direction perpendicular to the first direction, and wherein the printed strands of fungal biomass aligned in the first direction have a Young’s modulus of about 0.1 MPa to 0.8 MPa when measured in a direction parallel to the first direction.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims. DEFINITIONS

Singular references do not exclude a plurality, i.e. terms such as "a", "an", "first", "second" etc. do not preclude a plurality.

In the context of the present document, “printed”, “printing” and the like refers to the forming of three-dimensional objects by pushing a material through a die, nozzle and the like. Such printing forms “strands” which may be used to build up three- dimensional objects. Examples of techniques for printing include but are not limited to extrusion and 3D printing. Whenever the term “printing” and the like is used in the present document, all such techniques are intended to be covered by the term unless something else is explicitly referred to.

By “extrusion” is intended a process for creating objects of fixed cross-sectional profiles by pushing material through a die, nozzle and the like, having the desired cross-section.

“3D printing”, “3D printed” and the like in the context of the present document refers to a process of making a physical object from a three-dimensional digital model by forming many thin layers of a material in succession.

“Meat analogue” is in the context of the present document intended to refer to products with a similar texture as commonly consumed protein sources of animal origin such as chicken, pork, beef, lamb, fish, or seafood (e.g. fish or shrimps).

“Fungal fibers” in the context of the present document intended to refer to the hyphae (e.g. single hyphae or groups of hyphae bundled together) of the filamentous fungi in the fungal biomass.

“In contact” in the context of the present document intended to refer to the proximity of the strands. By one strand being in contact with another strand it is understood that the printable material constituting each of the strands have at least one common point. The different ways of contacting the strands include such as attaching, arranging, fusing, layering and the like.

“Substantially parallel” in the context of the present document intended to refer to the overall direction of different parts such as the fungal fibers and/or the printed strands. Substantially means that the parts have a clear direction, but a certain degree of deviation is expected and acceptable.

“Food-safe” in the context of the present document intends to refer to that the product is safe for consumption by humans and/or animals.

“Fungal biomass” in the context of the present document refers to any fungal mycelium biomass obtained through e.g. liquid fermentation or solid fermentation. Fungal biomass may comprise filamentous fungal cells only or in combination with water, salts and other components from the fermentation process.

“Texture” as used in the context of the present document refers to the sensory profile of a product, such as the hardness, toughness, springiness and/or chewiness of the product. The term can also refer to the visual appearance of the product.

“Printable material” as used in the context of the present document refers to a fungal biomass adapted such that its smoothness allows the use of said fungal biomass as a printable material.

In the context of the present document, “% of dry weight”, “ % w/w” or the like refers to the percentage in dry weight of the substrate (e.g., fungal biomass or stabilizing agent) based on the total weight of the printable material and/or fungal biomass, unless otherwise provided. The total weight of the printable material and/or fungal biomass includes any water or liquid in said printable material and/or fungal biomass.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a schematic drawing representing strands aligning direction.

Fig. 2 shows a flow chart of a method for preparing a printed fungal biomass food product.

Fig. 3 shows a printable material comprising fungal biomass of (A) pH 6 and (B) pH 9.

Fig. 4 shows an example of (A) a 3D printing design, (B) a 3D printed meat analogue containing fungal biomass, and (C) a fibrous structure of obtained meat analogue upon manual tearing. Fig. 5 shows a 3D printed octopus analogue.

Fig. 6 shows (A) a piping bag with customized nozzle was used as simple extrusion device, (B) extruded paste crosslinked in 0.5 % CaC solution, (C) fibrous strings observed when final product being feared, and (D) alignment of filamentous fungi in a microscale observed with confocal laser scanning microscope.

Fig. 7A shows pictures of a printable material comprising fungal biomass at pH 9 and dry weight of 45.5%, Figure 7B shows pictures fungal biomass at pH 9 and dry weight of more than 50%.

Fig. 8A shows a picture of a 3D-printed paste comprising fungal biomass. Fig. 8B shows a Texture Profile Analysis (TPA) test result of the 3D-printed paste comprising fungal biomass and carrageenan (duplicate). Fig. 8C shows a TPA test result of the 3D-printed paste comprising fungal biomass and without gelling agents (duplicate).

Fig. 9A shows a picture of fungal biomass dispersed in water at a pH of 5.25 (left side), 7.5 (middle) and 9 (right side). Fig. 9B shows a microscopy picture of fungal biomass dispersed in water at a pH of 5.25 (top), 7.5 (middle) and 9 (lower).

Fig. 10 shows a picture of 3D-printed square shaped using a dog bone mold which was perpendicularly molded.

DETAILED DESCRIPTION

As already explained above, the recent demand for non-animal based, high-nutritious and environmentally friendly food has rapidly increased. Such food products may take the form of meat replacement products which have a texture, nutritional content and/or visual appearance resembling meat (e.g. beef, pork, seafood, or chicken). Obtaining such a meat replacement product is challenging e.g. due to consumers’ demand for a similar mouthfeel as known from traditional protein sources.

The texture of e.g. a muscle is characterized by having aligned fibers on multiple scales; myosin filaments, myofibril, muscle fibers, and fascicle.

The present inventors surprisingly found that by printing strands of fungal biomass, food products with meat-like appearance could be obtained. However, the preparation and the printing of fungal biomass gave rise to several technical challenges relating to e.g. the composition of the fungal biomass, the flowability during extrusion and/or printing process, the desired stability after deposition and how to mimic different textures in a more realistic way. For example, the fungal biomass tended to form clumps which made it hard to print and form into an appealing product.

Surprisingly the inventors found that the printabil ity of the fungal biomass could be improved by raising the pH value of the fungal biomass to a certain pH range before printing and maintaining this higher pH while printing. A smoother printable material, which did not form clumps, could in that way be obtained that could easily be printed and formed into appealable meat-replacement products. Once the printing is done, the pH may be adjusted back to a lower pH, e.g. by washing with an acidic solution (e.g. acidic water). Another way of lowering the pH after printing is to include a pH regulator, such as glucono-delta-lactone, in the printable material that slowly adjusts the pH back with time.

Additionally, the inventors surprisingly found that the fungal fibers (hyphae) align themselves within the fungal biomass at the same pH range which improves the printability. Thereby especially printed fungal biomass-based products can provide palatable meat replacement products with an appealing texture as they can exhibit a structure similar to the one of muscle with aligned hyphae being analogues to myofibril and printed strands being analogues to single muscle fibers.

Thereby it was possible to print finer and more homogenous strands of fungal biomass, which could be deliberately arranged, connected, fused, layered, and/or assembled into larger products mimicking meat products of different kinds.

The printable material comprising fungal biomass

The present document is thus also directed to a method for producing a printable material comprising a fungal biomass, the method comprising the steps of: a) providing a fungal biomass; b) optionally washing and/or heat-treating and/or dewatering and/or grinding said fungal biomass; c) adjusting the pH of the fungal biomass of step a) or b) to a pH of at least 7 with a pH regulator; d) optionally grinding and/or high shear mixing the pH adjusted fungal biomass of step c).

The printable material is suitable for use in any printing technique. The printable material may e.g. be a 3D printable material and/or an extrudable material. The extrudable material may be suitable for room temperature or high temperature extrusion.

The method for producing a printable material comprises a step for adjusting the pH of the fungal biomass of step to a pH of at least 7. The pH adjustment step may be performed on the provided biomass directly or with and/or after the optional washing and/or heat-treating and/or dewatering and/or grinding steps. The pH adjustment is critical to ensure a smooth printing.

The pH of the fungal biomass is adjusted in pH adjustment step to a pH of from about 7 to about 10, such as from about 7.5 to about 9, such as 8 to 8.5. At this pH range a smoother biomass material of dispersed filaments is advantageously obtained, see for instance data from Fig.3A and Fig 3B, Example 1 .

The pH may be regulated by adding a pH regulator. The pH regulator may be any substance that can be used for regulating pH, e.g. an inorganic base such as sodium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, calcium bicarbonate, potassium bicarbonate, potassium hydroxide, ferrous hydroxide, lime, calcium carbonate, trisodium phosphate. The pH regulator may also be lactic acid, citric acid, acetic acid, hydrochloric acid, ascorbic acid. It is possible to use two or more different pH regulators.

The step for adjusting the pH of the fungal biomass may be followed by a mixing step, such as a high shear mixing step, in order to disperse the fungal fibers. Therefore, grinding the pH-adjusted fungal biomass afterwards is optional and may be performed in case such high shear mixing was not performed. Alternatively, the fungal biomass may be grinded, followed by pH adjustment and a subsequent high shear mixing step. The method for producing a printable material may comprise a step of optionally washing and/or heat-treating and/or dewatering and/or grinding the fungal biomass. Such washing and/or heat-treating and/or dewatering and/or grinding may be performed before and/or after adjusting the pH of the fungal biomass. However, typically such washing and/or heat-treating and/or dewatering and/or grinding is performed before adjusting the pH of the fungal biomass. Through washing the fungal biomass certain constituents of the fungal biomass may be removed, such as salts. Washing may also be applied to adjust the pH value of the fungal biomass. Hence, a washed biomass may have improved properties relating to edibility. Heat- treating and/or dewatering of, e.g. through filtering and/or mechanical pressing, the fungal biomass may be used to reduce the water content. A reduction of the water content results in an increase in the fungal fiber and hence protein content of the fungal biomass. By adjusting the amount of fungal fiber content, different nutritional compositions and textures may be achieved. Furthermore, a grinding step advantageously provide a fungal biomass having a more uniform average particle size, which facilitates further processing of said fungal biomass, including the pH- adjustment step.

Heat treating the fungal biomass after growth may e.g. also or alternatively be done to sterilize the fungal biomass, e.g. to prevent further growth and/or to inactive enzymes and the like in it. Such a heat treatment step may e.g. take place at a temperature of from about 50 °C to about 95 °C, such as from about 60 °C to about 85 °C, or it may take place using two different temperatures of the above range, one for RNA reduction and one for inactivation. Said heat treatment step may occur for about 1 minute to about 30 minutes, such as from about 5 minutes to about 15 minutes.

The method for producing a printable material may further comprise an optional grinding step of the pH adjusted fungal biomass. The fungal biomass may be ground down or applied as a whole body for constructing a printable material. Furthermore, said optional grinding step may occur before the pH adjustment of the fungal biomass. In such a case, the fungal biomass may be subject to a mixing step after the pH adjustment of the fungal biomass, which may be for instance a high shear mixing step. Alternatively, the pH of the provided fungal biomass may be adjusted to at least 7, followed by a mixing step, such as a high shear mixing step. Also, one or more of a stabilizing agent and/or food additive may be added to the fungal biomass before and/or after the pH adjustment step c). Typically, such a stabilizing agent and/or food additive is added after adjusting the pH.

Examples of stabilizing agents include, but are not limited to, a thickening and/or gelling agent, such as native starch, modified starch, alginate, guar gum, gellan gum, xanthan gum, konjac gum, curdlan gum, carrageenan, cellulose derivative (such as methylcellulose, carboxymethylcellulose, hydroxypropyl methylcellulose), or any combination thereof. The total amount of stabilizing agent in the printable material may e.g. be from about 0.1 % w/w to about 10 % w/w based on the total weight of the printable material. The stabilizing agent may e.g. help improving the consistency of the printable material and/or the final printed fungal biomass food product.

One or more food additive(s) may alternatively or additionally also be added to the printable material before printing. The food additive may e.g. be a protein containing ingredient such as egg albumin, dairy protein, protein extracted from fungi, insect protein, and/or a plant-based protein, e.g. added in a total amount of from about 0.5 % w/w to about 30 % w/w based on the total weight of the printed fungal biomass product. Another example of a food additive is an oil, such as rapeseed oil, sunflower oil, olive oil, and/or a fat such as shea butter, milk fat, coconut fat, and palm fat. Food additives in the form of oil(s) or a fat(s) may be added in a total amount of from about 0.5 % w/w to about 50 % w/w based on the total weight of the printable material. Further examples of suitable food additives are colorants, and/or a flavorants which may be added in a total amount of from about 1 % w/w to about 5 % w/w based on the total weight of the printable material. The printable material may further comprise a food additive in the form of a salt. Examples of salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride. The salt may be added in an amount of from about to 0.1 % w/w to about 10 % w/w based on the total weight of the printable material. Further, food additives in the form of a crosslinking agent, such as calcium carbonate, calcium sulfate dihydrate, and dicalcium phosphate may also be added. The crosslinking agent may be added in an amount of from about 0.02 % w/w to about 2 % w/w based on the of the total weight of the printable material. Crosslinking agents may be used to effect a connection of strands of the printable material after printing.

The printable material may comprise from about 55 % to about 90 % water based on the total weight of the printable material. The inventors have surprisingly found that said water range provides a particularly good fungal biomass printable material. A water content above said range does not have a consistency for use as a printable material since the material is too liquid. A water content below said range does not have a consistency for use as a printable material since the material is too crumbly and does not adequately extrude. A preferred water content for the printable material is from about 60 % to about 70 % water based on the total weight of the printable material.

The present document is also directed to printable material obtained or obtainable by the method for producing a printable material as described in the present document.

The present document is also directed to a printable material comprising fungal biomass, optionally one or more additives, stabilizers and/or pH regulators as disclosed herein, wherein said printable material has a pH of at least 7, such as about 7.5 to about 9.

The printable material may be used for producing a food product, such as a meat analogue product as disclosed elsewhere herein. The method for producing a printable material may therefore be a method for producing a printable material for preparing a food product.

The fungal biomass

The fungal biomass comprises food-safe fungi, such as food safe filamentous fungi. Food-safe filamentous fungi are well-known in the art and include, but are not limited to, fungi of the Zygomycota and/or Ascomycota phylum, excluding yeasts, such as fungi of the genera Aspergillus, Cordyceps, Fusarium, Ganoderma, Inonotus, Neurospora, Pennicillium, Pleurotus, Rhizopus, Trametes, Trichoderma, Tuber, Ustilago, Xylaria, or any combination thereof. Examples of fungal species that may be used according to the present document include, but are not limited to fungi of the species Aspergillus oryzae, Cordyceps militaris, Cordyceps sinensis, Fusarium graminareum, Fusarium venenatum, Lentinula edodes, Neurospora crassa, Neurospora intermedia, Neurospora sitophila, Pennicillium camemberti, Rhizomucor miehei, Rhizopus microsporus, Rhizopus oligosporus, Rhizopus oryzae, Tuber magnatum, Tuber melanosporum, Xylaria hypoxion, or any combination thereof. For example, fungi of the genus Rhizopus may be used.

The fungal mycelium may have a size ranging from few micrometers to few centimeters. The morphology of the fungal mycelium may be in filamentous or in any pellet forms. The fungal mycelium may e.g. be grinded down or applied as a whole body when preparing the printable material comprising fungal biomass.

Any method commonly used for growing fungi may be used to produce the fungal biomass used in the present document. The fungal biomass may e.g. be obtained by growing fungi by liquid or solid fermentation. For example, the fungi may be grown under aerobic submerged fermentation conditions in a closed fermentation vessel with a liquid substrate medium with stirring.

The culture media used for growing the fungi may advantageously contain a carbon source, nitrogen source, phosphates and sulphates, and a trace metal solution to enable growth of the fungi and obtaining of the biomass. The bioreactor conditions may be kept at e.g. a pH between 4.0 and 6.0, with an aeration of at least 0.1 vvm and stirred using propeller blades. The growth can e.g. be done in a batch mode, in which fungi are harvested from the production tank after a 24 h process or until less than 5 % of the remaining carbon source is present, or as a continuous process, in which biomass is removed at a constant rate that matches growth and nutrient feed rate, or a semi-batch mode where biomass is partially harvested from one or several reactor and then such reactor(s) are filled with new media to continue fungal growth.

The fungal biomass used for producing the printable material may be a fresh fungal biomass and/or heat-treated fungal biomass. The fungal biomass may alternatively be a freeze-dried fungal biomass and/or a freeze-dried heat-treated fungal biomass. In any case, the water content of the fungal biomass may need to be adjusted for obtaining a fungal biomass as a printable material. Thus, the water content may be adjusted to from about 55% to about 90%, such as from 60% to 70%, based on the total weight of the printable material. The water content of the fungal biomass may be adjusted during the step of adjusting the pH of the fungal biomass or after said step. After the water content adjustment, said fungal biomass printable material may have a consistency of a paste.

Heat treating the fungal biomass after growth may e.g. be done to sterilize and/or to reduce the RNA content of the fungal biomass, e.g. to prevent further growth and/or to inactive enzymes and the like in it. Depending on the fermentation process the fungal biomass provided in the beginning of the production of the printable material may differ and hence certain optional steps may be chosen or omitted to generate a printable material with good flowability in the printing and/or extrusion process. A heat-treatment step is usually mandatory to obtain an edible product that can be stored for longer periods. It is preferred that the heat treatment of the fungal biomass occurs before pH adjustment of step c) and/or after the printable material is printed into a printed fungal biomass food product, as described elsewhere herein.

The method for preparing a printed fungal biomass food product

The present document is also directed to a method for preparing a printed fungal biomass food product. The method for preparing a printed fungal biomass food product, such as a meat analogue product, comprises the steps of: a) providing a printable material as defined herein, e.g. by preparing a printable material comprising fungal biomass as disclosed elsewhere herein; b) printing said printable material comprising fungal biomass to prepare a printed material comprising one or more printed strands of fungal biomass; and c) optionally solidifying the printed material comprising one or more printed strands of fungal biomass obtained in step b) through heating and/or crosslinking, to provide said printed fungal biomass food product. An exemplary method for preparing a printed fungal biomass food product is outlined in Fig. 2. The method for preparing a printed fungal biomass food product is particularly suitable for preparing a meat analogue product even though it can be used for preparing any type of food product of a specific three-dimensional shape.

The contents and preparation of the printable material comprising fungal biomass is disclosed elsewhere herein. The contents of the printable material may be adjusted so that a printed fungal biomass food product as disclosed elsewhere herein may be obtained, e.g. as regards the amount of fungal biomass and/or water.

The printing may e.g. be performed by 3D printing and/or extrusion. Such techniques are well-known even if they previously have not been used for forming three- dimensional objects of fungal biomass.

The printing, such as when performed by additive manufacturing (as known as 3D printing), may generate one or more printed strands of fungal biomass, i.e. strands prepared by printing the printable material comprising fungal biomass disclosed elsewhere herein. The printed strands may be arranged in layers wherein each layer comprises a single or multiple printed strands of fungal biomass (as illustrated in Fig. 1 ). The printed strands may be arranged in substantially parallel direction. This means that the strands exhibit a common orientation along an axis. A certain deviation from this order is expected and even desirable to mimic the texture of typical animal-based protein sources. One or more of the printed strands of fungal biomass may be in contact with at least one neighboring printed strand. The arrangement of these contact points between the different strands may be used to generate different textures. The printed strands may also be printed as single strands, such as single extruded strands.

If the printing is performed by extrusion, the extrusion may generate bundles of clustered fungi oriented substantially parallel to one another.

The method for preparing a printed fungal biomass food product may comprise feeding said printable material into a single or multiple (i.e. two or more) print heads. The shape of the print heads is generally not critical but may have e.g. a tapered or straight shape. The nozzles may form part of a single or multiple print heads. Said nozzles may have a preferred diameter such as between about 0.2 mm and about 5 mm, preferably between about 0.8 mm and about 1 .6 mm. However, a nozzle with a larger diameter, such as up to centimeters in diameter, may also be used, depending on the desired shape of the printed strand. It is also possible to use two or more print heads where at least one print head prints fungal biomass while at least one print head prints another material, such as fat. Depending on the shape of the print head, different forms of the printed strands may be achieved, such as round, oval, rectangular etc.

The printed material may be processed further after being printed. The further processing may include mechanical and/or chemical processing.

For example, the printed material may be solidified. Such solidification may be achieved by heating the printed food product to temperatures ranges from about 60 °C to about 200 °C, such as from about 80 °C to about 120 °C. Such heating may e.g. effect denaturation of proteins present in the printed material and/or gelatination of food additives, such as of starch. Alternatively, or additionally, such solidification may be affected by crosslinking, such as chemical, thermal and/or physical crosslinking. Chemical crosslinking may be performed by external crosslinking through interaction of hydrocolloids present in the printable material with externally added crosslinking chemical(s). Alternatively, or additionally, crosslinking may be affected by internal crosslinking though interaction of hydrocolloids present in the printable material with crosslinking agents that are also present in the printable material. For example, when alginate is as a hydrocolloid, this may be crosslinked by the addition of Ca ²⁺ to the printable material (internal crosslinking) or externally to the printed material after printing (external crosslinking).

Also, the method for preparing a printed fungal biomass food product may comprise a further step of lowering the pH after the printing is performed. This may be advantageous if the pH of the printable material is too high for an edible product. Typically, the pH of the food product should be 8 or less, such as between about 6.5 and about 7.5. A printed fungal biomass food product having an acidic pH, for instance between 5 and 6, has an improved taste. Such lowering of the pH after printing may e.g. be performed by washing the printed fungal biomass product with an acidic solution (e.g. acidic water, e.g. acidified by citric acid, lactic acid, calcium acetate, acetic acid, malic acid, fumaric acid or tartaric acid) or by including a pH regulator, such as glucono-delta-lactone, that gradually will lower the pH in the printed fungal biomass food product. By adjusting the amount of such a pH regulator, the desired pH in the printed fungal biomass food product can be achieved. The step of lowering the pH of the printed material obtained in step b), is preferably performed after solidification step c (when a step c) is performed)). Alternatively, a pH regulator is added to the printable material before printing which will gradually lower the pH.

The present document is also directed to a printed fungal biomass food product obtained or obtainable by the method described in the present document.

The printed fungal biomass food product

The present document is also directed to a printed fungal biomass food product, wherein the printed fungal biomass food product comprises one or more strands of printed fungal biomass. Such a printed fungal biomass food product may e.g. be obtained or obtainable by the method for preparing a printed fungal biomass food product as disclosed elsewhere herein.

The printed fungal biomass food product may a meat analogue product, such as a e.g. a chicken, pork, beef, lamb, or seafood (e.g. fish or shrimp) replacement product.

The arrangement and possible connections of the strands of printed fungal biomass generate the desired structure of the printed fungal biomass food product. The strands of fungal biomass may be printed so that they become arranged in a substantially parallel direction. This means that the printed strands exhibit a common orientation along an axis. A certain deviation from this order is expected and even desirable to mimic the texture of typical animal-based protein sources, such as different kinds of meat.

Further, the printed strands of fungal biomass may be arranged in layers. Each layer may comprise one or more strands of printed fungal biomass. The layers may be oriented in any plane. The layers may be bent or tilted. The layers may be arranged in stacks. The layers comprising the stack may vary in size and orientation. One or more of the printed strands of fungal biomass may be in contact with at least one neighboring printed strand of fungal biomass. The arrangement of these contact points between the different strands may be used to generate different textures, such as textures of different kinds of meat. By printing a fungal biomass food product having different arrangements of printed strands of fungal biomass, it is possible to prepare food products having virtually any shape. Also, this arrangement of printed strands of fungal biomass allows the preparation of food products having a texture, mouthfeel and/or visual appearance mimicking meat. In this way a nutritious as well as visually appealing and well-tasting food product may be prepared.

In the printed fungal biomass food product, the printed strands of fungal biomass may for example be aligned in a first direction, wherein the printed strands of fungal biomass may have a Young’s modulus of about 0.03 MPa to about 0.5 MPa when measured in a direction perpendicular to the first direction, and wherein the printed strands of fungal biomass aligned in the first direction may have a Young’s modulus of about 0.1 MPa to about 0.8 MPa when measured in a direction parallel to the first direction. Printed strands with a Youngs’s modulus as described above is advantageously perceived by consumers as having a similar chewiness, mouthfeel and visually similar tearing behavior as common animal-based protein sources.

One can increase Young’s modulus of printed samples in a parallel direction to the printed strings by for instance increasing the calcium content in the crosslinking solution, increasing crosslinking incubation time, and/or modifying the paste composition such as by increasing concentration of gelling agent.

Furthermore, one can increase Young’s modulus of printed samples in a perpendicular direction to the printed strings by for instance, increasing the calcium content in crosslinking solution, increasing crosslinking time, modifying the paste composition such as by increasing concentration of gelling agent, and/or by increasing the percentage of overlapping neighboring printed filaments.

Furthermore, a ratio can be calculated by taking the Young's modulus measured parallel to the first direction and dividing said value by the Young's modulus measured perpendicular to the first direction. Preferably, the printed fungal biomass food product has a ratio of from about 2 to about 6. Said ratio advantageously provides a printed fungal biomass food product having a similar chewiness and mouthfeel as common animal-based protein sources, such as chicken. The fungal fiber strands can also be printed in bundles, with varying a group of connected strands, intercalated with a larger space to the next group of connected strands. The bundle of fibers may advantageously be stretched in a single direction, which contributes to the improved mouthfeel and chewiness of the printed fungal biomass food product. This effect in a 3-dimensional structure mimics the effect of fiber bundles similar to meat fiber structures when tearing.

The desired three-dimensional structure of the printed fungal biomass food product may be obtained by 3D printing or extruding the printable material comprising fungal biomass. Other methods may also be used for obtaining the desired arrangement of printed strands of fungal biomass.

The printed fungal biomass food product comprises fungal biomass as described in further detail elsewhere herein.

The printed fungal biomass food product may also comprise one or more of a pH regulator(s), stabilizer(s) and/or food additive(s) as described herein in the context of the printable material.

The printed fungal biomass food product may comprise from about 1 % to about 45 % of dry fungal biomass based on the total weight of the printed fungal biomass food product. It is also possible to increase the amount of protein in the printed fungal biomass food product by adding extra protein, such as fungal protein and/or plant protein. Preferably, at least 70 % of the protein in the printed fungal biomass food product comes from fungi, such as filamentous fungi. For example, the majority, or all, of the proteinaceous material in the printable material comes from fungi, such as filamentous fungi.

The printed fungal biomass food product may comprise from about 70% to about 95 % water based on the total weight of the printed meat replacement product. As a comparison, meat typically contains about 75 % water and about 20 % protein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1. fungi biomass pastes with different pH

Production of fungal biomass

A fungal spore suspension of Rhizopus oligosporus was prepared by flooding a PDA plate culture with 10-20 mL of sterile water and spores scraped off the surface with a disposable, sterile spreader. Spores were counted in a hemocytometer under a light microscope and used directly as inoculum for liquid cultivations. Fungi cultures were cultivated in Erlenmeyer flasks (volumes 100-2000 mL) with or without baffles, filled with liquid growth medium to a maximum of 20 % of the total flask volume. 1 mL of spore suspension (10 ^A 7 spores/mL) per 100 mL of growth media was added to each flask, followed by incubation at 30-35 °C for 18-24 h under shaking (100-150 rpm).

Sterilization of the liquid in the bioreactor was done by heating up the liquid with steam (via the bioreactor’s double jacket) to 121 °C and 1 bar overpressure for 20 min. Upon sterilization, a volume of 30 L of fungi culture obtained from a 16-24 h rich media preculture was used to inoculate 300 L of media in a 400 L stirred-tank bioreactor using the media composition described previously. The pH was adjusted to 4.0-5.5 with 5M NaOH. Fermentation conditions were kept at pH 4.0 using NH3 as a base for pH titration, an air flow of 120 L/min (0.6 vvm) and a temperature of 30-35 °C were kept constant with a stirring of 200 rpm. The fermentation process was carried for 24 h and biomass was harvested after this period. 50 L from this culture was used to inoculate a volume of 500 L in a 600 L bioreactor and the process was repeated for an additional 24 h.

The obtained biomass was filtered and washed with tap water. Heat treatment of biomass was done by keeping biomass in 70 °C hot water for 15 min. After heat treatment, biomass was washed again with cold water and dewatered using a belt press. The final water content of dewatered biomass was between 65 and 70 %. The dewatered biomass was kept in freezer for further application use. pH adjustment of fungal biomass 50 g of frozen heat-treated biomass was defrosted at ambient temperature. Defrosted biomass was grinded using a kitchen blender (Bosch Multitalent 8) for 1 minute at lowest speed to eliminate big pieces for further handling. The size of the grinded biomass pieces was roughly 2 mm.

For sample 1 , 50 g distilled water was added to the grinded biomass and mixed with a kitchen blender for 2 min to obtain a paste. The pH of the obtained paste was 6, and the obtained paste appeared to be crumbly (Figure 3A). Chunks of biomass were clearly visible, and the prepared paste were not able to be extruded from a nozzle size of 1 .6 mm.

For sample 2, 50 g distilled water was added to the grinded biomass and mixed with a kitchen blender for 2 min to obtain a paste. The paste was adjusted to pH 9 using 1 M NaOH and further blended using a kitchen mixer for 30 s. The obtained paste had a smooth texture comparable to caramel (Figure 3B). Both the crumbly paste at pH 6 and the prepared smooth paste which was adjusted to pH 9 were pushed through a syringe system with an attached round nozzle of 0.8 mm diameter. The smooth pH 9 paste was able to be extruded through this nozzle in a fluid, non-interrupted way, while the crumbly material at pH 6 offered a high resistance and was not able to be pushed through in a continuous way. Printing applications and additive manufacturing require materials that are extrudable with a continuous resistance and flow. In this way, the shift to a high pH and mixing afterwards was shown to be essential to obtain an extrudable paste suitable for printing applications.

Example 2. 3D printed meat analogues containing fungi biomass and texture characterization

Preparation of fungal paste used for 3D printing

Frozen heat-treated biomass prepared as described above was defrosted at ambient temperature. Defrosted biomass was grinded using a kitchen blender (Bosch Multitalent 8) for 1 minute at lowest speed to eliminate big pieces for further handling. 4 % alginate solution was prepared by dissolving 4 g alginate into 96 g distilled water at ambient temperature.

Paste 1 : 40 g of grinded biomass was mixed with 20 g distilled water and blended using a kitchen blender (Bosch Multitalent 8). 1 M NaOH was used to adjust the paste to reach a stable pH of 7.5. 10 g of above prepared alginate solution was blended into the fungi paste to achieve a well-mixed paste.

Paste 2:

50 g of grinded biomass was mixed with 30 g distilled water and blended using a kitchen blender (Bosch Multitalent 8). 1 M NaOH was used to adjust the paste to reach a stable pH of 9. 10 g of above prepared alginate solution was blended into the fungi paste to achieve a well-mixed paste.

3D printing design and process

A ByFlow Focus 3D food printer (byFlow B.V., the Netherlands) was used to perform printing experiments. Printing was carried out at ambient temperature. A printing design of a cuboid geometry with a dimension of 100 x 50 x 5 mm (I x w x h) was created using OpenSCAD and translate into geode using Slic3r software (Figure 4A). Dispensing tip with an opening of 1.2 mm diameter was used as extrusion nozzle. Layer height of printed filaments was set at 0.8 mm, and printing speed was set at 60 mm/s. Infill pattern was set as “aligned rectilinear” and infill density was set at 90 %.

Post processing of printed objects and texture characterization

After printing, the printed objects were immersed in 1 % CaCI2 solution for 2 hours to solidify printed structures. The fixed printed pieces were boiled for 1 min and afterwards rinsed with cold water. The samples were then cut with a clone of a standard ASTMD- 638-V in both parallel and perpendicular direction to the printed aligned filaments. Tensile test was performed to the cut specimens using a texture analyzer (Stable Micro Systems, TA.XT.plusC) equipped with miniature tensile grips (A/MTG). Specimens were stretched at a speed of 2 mm/s until breakage. The Young’s modulus was defined as the slope of the initial linear portion of the stress-strain curve. Duplicates were done for all samples.

Figure 4 shows an example of a 3D printing design, a 3D printed meat analogues containing fungal biomass, and a fibrous structure of obtained meat analogues upon manual tearing. To compare the texture of above obtained 3D printed meat analogues with a real meat product, a boiled chicken breast cut with the same dimensions as the test sample was used as the reference sample.

Texture analysis results is shown in Table 1. As can be seen from the table, a wide range of textures can be achieved with 3D printing using fungal biomass paste by adjusting paste formulation, pH and crosslinking conditions. The instrumental measured texture revealed good match with real chicken, and possibly other meat types. It is also possible to achieve different textures by using different printing designs.

Table 1. Young’s modulus of boiled chicken breast and 3D printed meat analogues containing fungal biomass.

Example 3. Use of fungal biomass in printing of vertically-stable organic geometries

To evaluate suitability of the fungal biomass treatment and printing process for the creation of more advanced and natural-looking 3D structures, an animal 3D model of an octopus was used as the printing geometry.

Preparation of fungal paste used for 3D printing

Frozen heat-treated biomass prepared as described above was defrosted at ambient temperature. Defrosted biomass was grinded using a kitchen blender (Bosch Multitalent 8) for 1 minute at lowest speed to eliminate big pieces for further handling. 15 g of grinded biomass was mixed with 15 g distilled water and blended using a kitchen blender (Bosch Multitalent 8) and adjusted to pH 9 with the 1 M NaOH. 2 % alginate solution was prepared by dissolving 2 g alginate into 98 g distilled water at ambient temperature. 10 g above prepared alginate solution was blended into the fungi paste to achieve a well-mixed paste.

3D printing design and process A ByFlow Focus 3D food printer (byFlow B.V., the Netherlands) was used to perform printing experiments. Printing was carried out at ambient temperature. A printing design of an octopus with a dimension of 100 x 100 x 28 mm (I x w x h) was used. The geode was preinstalled in the 3D food printer, and a similar model can be found online (https://www.thingiverse.eom/thing:7900). The model used was hollow inside with a wall thickness of around 5 mm, which allowed for testing of printing vertically-stacking thin structures. Dispensing tip with an opening of 1.2 mm diameter was used as extrusion nozzle.

After printing, the printed objects were immersed in 1 % CaCI2 solution overnight to solidify printed structures. The solidified 3D printed octopus was washed with cold water before boiling. The resulting 3D printed octopus is shown in Fig. 5.

The printed octopus analogue had a chewy and pleasant texture and the pH of the boiled octopus analogue was around 8. Fibrous strings were observed when the printed octopus analogue was manually feared, showing alignment of fungal fibers. The structure was stable along all vertically-printed segments showing suitability of the paste and material to be used in the creation of a variety of foodstuffs.

Example 4. Extruded meat analogues containing fungi biomass Preparation of fungal paste used for extrusion

Frozen heat-treated biomass prepared as described above was defrosted at ambient temperature. Defrosted biomass was grinded using a kitchen blender (Bosch Multitalent 8) for 1 minute at lowest speed to eliminate big pieces for further handling. 2 % alginate solution was prepared by dissolving 2 g alginate into 98 g distilled water at ambient temperature.

40 g of grinded biomass was mixed with 40 g distilled water and 2 g baking powder. The mixture was blended using a kitchen blender (Bosch Multitalent 8) until a smooth paste was formed. 27 g of above prepared alginate solution was blended into the fungi paste to achieve a well-mixed paste. pH of the well-mixed paste was around 8.

Extrusion process and post processing

The well-mixed fungi containing paste was loaded in a piping bag with an attached customized tip (Figure 6A). The tip has an opening of 15 x 3 mm. The paste was manually extruded into a container filled with 0.5 % CaCI2 solution (Figure 6B). The extruded sample was left in a fridge for 4 hours to solidify the printed structures. The fixed printed pieces were boiled for 1 min and afterwards rinsed with cold water. The final boiled extruded fungi-based meat analogue had a pH around 7.5.

The manually extruded fungi-based meat analogue is chewy. Macroscale (>1 mm) fibrous strings were observed when the sample was feared (Figure 6C). Alignment of filamentous fungi can be observed in a microscale when observed with confocal laser scanning microscope (Figure 6D).

Example 5 pH shift for obtaining a printable paste

The fungal biomass was dried to 100% dry weight by dehydrating at 60°C for 12 hours using a dehydrator, Klarstein fruit jerky plus (Germany). Afterwards, the pH was shifted to 9 using NaOH 1M and water was added until achieving a printable paste. This process was also validated at pH 7.5 with similar results.

54.5% of water (total amount, also counting the water in the NaOH solution added) was added to the dried fungal biomass at pH 9 to achieve a printable paste (Figure 7A). Below that percentage the matrix is too crumbly to be considered a paste (Figure 7B). Up to about 88.8% of water can be added to the dried fungal biomass at pH 9 to achieve a printable paste. After that amount the paste becomes too liquid and does not hold the structure anymore.

In conclusion, this experiment shows that the pH shift method (pH range 7.5-9) is suitable for a printable fungal biomass paste with a dry matter in the range of 11 .2 and 45.5 %.

Example 6 - Fungal biomass gel matrix

Three different printable matrixes were prepared with the purpose of proving that a gelling agent is needed to create a stable printed matrix and explore different gel firmness space.

The first one did not include any gelling agent. 50 g of fungal biomass was mixed with 30 g of water and the pH was shifted to 9 using 1 M NaOH. The mixture was blended using a spice grinder, Bosch Multitalent 8 (Germany) and transferred to a Byflow Focus 3D food printer (The Netherlands). The paste was extruded using a 1 .2 mm nozzle into a layered square shape of 50x50x10 mm (figure 8A).

The second matrix contained an addition of kappa carrageenan. 1 g of kappa carrageenan (The kitchen lab, Sweden) was dissolved in 30g of water. The mixture was heated to 80 °C for 5 minutes. 50 g of fungal biomass were ground using a spice grinder, Braun Multitalent 8 (Germany) and pH was shifted to 9 using 1 M NaOH. The solution of kappa carrageenan was transferred to the fungal biomass and ground for another 5 minutes at a temperature of 65 °C. The paste was transferred to a Byflow Focus 3D food printer (The Netherlands) and extruded using a 1 .2 mm nozzle into a layered square shape of 50x50x10 mm (figure 8A).

The third matrix was composed of Konjac gum and starch. 10 g of Konjac gum and 4 g starch were mixed with 200mL of water then heated at 70 °C for 60 min, then allowed to cool down. 200 g of fungal biomass were brought to pH 7.5 with 5 g of sodium bicarbonate, then ground to a dough and mixed with the Konjac + tapioca solution. The paste was then transferred to a Byflow Focus 3D food printer (The Netherlands) and extruded using a 1 .2 mm nozzle into a layered square shape of 50x50x10 mm. Finally, the square was heated for 15 minutes at 75 °C to gel completely.

To compare them a standard TPA test was carried out using a texture analyzer, Stable micro systems, (United Kingdom) with the following settings: Pre-test speed: 1 .Omm/sec, Test speed: 5.00mm/sec, Post-test speed: 5.00mm/sec, Strain: 75%, Time: 5.00sec, Trigger force: 5.0g. The dimensions of the 3D printed sample were a 50x50x10 mm square.

Table 2. TPA test. Firmness results

Only the paste with the gelling agents has a peak in the first compression (Example of Kapa carrageenan gel behavior in Figure 8B) as opposed to the matrix without it (Figure 8C), meaning that the square was gelled before the TPA test. This evidence suggests that a gelling agent may be needed to achieve a gelled matrix and only the fungal biomass with a pH shift may not be sufficient to create a stable product after printing.

Example 7 - pH optimization of printable paste

To explain the need for a pH shift to obtain a printable paste the agglomeration of the fungal biomass at different pH was studied. 50 g of fungal biomass (dry weight 24.04 %) was diluted adding 17.92 g of water. For two of the samples the pH was adjusted to 7.5 or 9 using NaOH 1 M. One sample was kept unadjusted (pH 5.25, left petri dish in Figure 9A) and was used as a reference. The petri dish in the middle shows a fungal biomass dispersed in water with a pH 7.5, while the petri dish on the right shows a fungal biomass dispersed in water with a pH 9. It can be clearly seen (Figure 9A) how the fungal biomass with non-adjusted pH has a crumblier texture and, therefore, is not printable.

The samples were studied using a stereo microscope, Olympus SZX7 (Japan) and the result is displayed in (Figure 9B). The top, middle and lower images in Figure 9B refer respectively to a fungal biomass dispersed in water with a pH of 5.25, 7.5 and 9. It can be clearly seen from the images that the pH affects the morphology and disperses the fungal biomass more evenly throughout the sample.

Example 8 - Texture analysis

The tensile strength of a 3D printed paste in different fiber directions was evaluated and compared to a paste which had been molded instead.

275 g of fungal biomass and 165 g of water were added to a Thermomix (Vorwerk, Germany). The pH was shifted to 9 using NaOH 1 M. While grinding the paste, 4.5 g of kappa carrageenan was added to the mixture, which was heated at 85 °C for 5 minutes while shearing at 2500 rpm. Then, the paste was either molded in a dogbone shape or 3D printed in a square shape (50x50x5 mm) and then cut to a dogbone shape (Figure 10). The 3D printed square shape of Figure 10 was perpendicular molded using a dog bone mold.

The samples were tested using a tensile force test using a texture analyzer, Stable micro systems, (United Kingdom) using the following settings: Pre-test speed: 1 mm/sec, Test speed: 2mm/sec, Post-test speed: 5 mm/sec, Target mode: distance, Distance: 15 mm, Trigger force: 5 g.

Triplicates were made and the result is displayed in Table 3. The results clearly show how the tensile force changes dramatically depending on the alignment of the fibers. The molded tensile force is higher since it consists of a more compact matrix and has no fiber alignment, which means that a similar firmness value is obtained in any direction. On the other hand, the different firmness values of the 3D printed fungal biomass food product, when molded perpendicularly or parallelly, are a clear indication of fiber alignment.

Table 3. Tensile force test. Firmness study

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

ITEMS

1 . A method for producing a printable material, such as a 3D printable or extrudable material, comprising fungal biomass, said method comprising the steps of: a) providing a fungal biomass; b) optionally washing and/or heat-treating and/or dewatering said fungal biomass; c) adjusting the pH of the fungal biomass of step a) or b) to a pH of at least 7 with a pH regulator; d) optionally grinding the pH adjusted fungal biomass of step c).

2. The method according to item 1 , wherein said fungal biomass comprises foodsafe filamentous fungi, such as food safe filamentous fungi of the Zygomycota and/or Ascomycota phylum, excluding yeasts, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof.

3. The method according to item 1 or 2, wherein said fungal biomass comprises food-safe filamentous fungi of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus and Rhizopus microsporus, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or any combination thereof.

4. The method according to any one of the preceding items, wherein the pH of the fungal biomass is adjusted in step c) to a pH of from about 7 to about 10, such as from about 7.5 to about 9.

5. The method according to any one of the preceding items wherein the pH regulator is an inorganic base such as sodium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, calcium bicarbonate, potassium bicarbonate, potassium hydroxide, ferrous hydroxide, lime, calcium carbonate, and/or trisodium phosphate, and/or an organic acid such as lactic acid, citric acid, acetic acid, hydrochloric acid, and/or ascorbic acid. The method according to any one of the preceding items, wherein a stabilizing agent and/or food additive is added before or after the pH adjustment step c). A printable material obtained or obtainable by the method according to any one of items 1 to 6. A method for preparing a printed fungal biomass food product, such as a meat analogue product, said method comprising the steps of: a) preparing a printable material comprising fungal biomass according to any one of items 1-6; b) printing said printable material comprising fungal biomass to prepare a printed material comprising one or more printed strands of fungal biomass; and c) optionally solidifying the printed material comprising one or more printed strands of fungal biomass obtained in step b) through heating and/or crosslinking, to provide said printed fungal biomass food product. The method for preparing a printed fungal biomass food product according to item 8, wherein step b) comprises 3D printing or printing by extrusion. The method for preparing a printed fungal biomass food product according to item 8 or 9, wherein said printing generates one or more layers of printed strands of fungal biomass, wherein each layer comprises multiple printed strands of fungal biomass arranged in substantially parallel direction, wherein one or more of the printed strands of fungal biomass is in contact with at least one neighboring printed strand. The method for preparing a printed fungal biomass food product according to any of items 8-10, said method comprising a further step of lowering the pH of the printed material obtained in step b). The method for preparing a printed fungal biomass food product according to any of items 8-11 , wherein the temperature in optional step c) ranges from about 60 °C to about 200 °C, such as from about 80 °C to about 120 °C. The method for preparing a printed fungal biomass food product according to any of items 8-12, wherein the chemical crosslinking in optional step c) is performed by external crosslinking and/or by internal crosslinking. A printed fungal biomass food product, such as a meat replacement product, such as a chicken, pork, beef, lamb, or seafood replacement product, said printed fungal biomass food product comprising one or more strands of printed fungal biomass. A printed fungal biomass food product according to item 14, which is obtained or obtainable by the method according to any one of items 8-13. The printed fungal biomass food product according to item 14 or 15, wherein said printed fungal biomass food product is 3D printed or extruded. The printed fungal biomass food product according to any one of items 14-16, wherein the printed strands of fungal biomass comprise from about 1 wt% to about 30 wt% of dry fungal biomass based on the total weight of the printed fungal biomass food product. The printed fungal biomass food product according to any one of items 14-17, wherein said printed food product comprises from about 70 to about 95 wt% water based on the total weight of the printed fungal biomass food product.