3d-printable composition

Abstract

The present invention relates to compositions comprising pellets comprising vegetal fibres embedded in a network of hyphae of Pleurotus fungi, which can be extruded or printed. The extruded or printed products can be dried and rehydrated while retaining their shape and have advantageous nutritional properties.

Description

[0001] 3D-Printable composition

[0002] Technical field

[0003] The present invention relates to compositions comprising pellets comprising vegetal fibres embedded in a network of hyphae of Pleurotus fungi, which can be extruded or printed. The extruded or printed products can be dried and rehydrated while retaining their shape and have advantageous nutritional properties.

[0004] Background of the invention

[0005] Food waste represents a significant global challenge, particularly evident in industrialized nations where it is estimated that over 30% of all food produced is wasted at various stages from production to consumption. This waste not only represents a loss of economic value but also contributes substantially to environmental issues, including greenhouse gas emissions and unnecessary land use. According to the United Nations Environment Programme (UNEP), addressing these inefficiencies necessitates the development of scalable, profitable solutions for industries and agri-food sectors. This substantial waste calls for effective strategies that repurpose waste into valuable, functional materials, utilizing advancements in biotechnology and other sustainable practices.

[0006] To address this issue, a variety of strategies have been developed, including transforming food waste into biofuels and platform chemicals, composting, and use as animal feed. Fermentation, a well-established industrial process, has been employed to valorise food waste. Through these methods, bacteria, yeast, and fungi convert waste into valuable biochemicals, including enzymes, proteins, and functional sugars. Furthermore, fermentation of food waste offers an additional benefit by reintegrating such waste back into the food chain, enhancing their digestibility and nutritional values while reducing anti -nutritional factors.

[0007] A relatively new yet rapidly growing field involves using microorganisms as biobased materials rather than solely as agents of transformation. Fungal hyphae, which have cell walls primarily composed of chitin, P-glucans and various proteins, have been extensively studied in materials science over the last decade. Depending on the quantity of fungal biomass in the system, fungal mycelia can act as natural binders to bind waste materials into new, functional forms or serve as primary fibrous materials themselves. Different processing techniques can be used to shape these bio-based materials into 2D or 3D forms, such as high-density films and panels or low-density foams (Sun, W. Fungal mycelia: From innovative materials to promising products: Insights and challenges. Biointerphases 19, 018502 (2024)).Advancements in 3D printing technology, which allow for the precise fabrication of complex structures layer by layer, have opened up new possibilities for utilizing mycelial materials. Particularly, mycelium-bound composites have been explored through Direct Ink Writing (DIW), where inks are formulated with specific rheological properties to ensure smooth printing. Mycelium can be incorporated either before (Gantenbein, S. et al. Three-dimensional printing of mycelium hydrogels into living complex materials. Nat. Mater. 22, 128-134 (2023)) or after printing (C. Shen, S. et al. Robust myco-composites: a biocomposite platform for versatile hybrid-living materials. Materials Horizons 11, 1689-1703 (2024)), respectively using direct or indirect inoculation methods, primarily serving as a reinforcement material. Current applications include producing living or non-living structural elements (Biala, E. & Ostermann, M. Mycostructures - growth-driven fabrication processes for architectural elements from mycelium composites. Archit. Struct. Constr. 2, 509-519 (2022)) and soft skins for robotics (Gantenbein, S. et al. Three-dimensional printing of mycelium hydrogels into living complex materials. Nat. Mater. 22, 128-134 (2023)). However, the full potential of mycelium in 3D printing remains largely untapped and using mycelium as the main structural component remains unexplored. Prior studies typically required ink formulation with nutritional supplements and rheological modifiers, potentially limiting the scope of applications and increasing both costs and environmental impact.

[0008] The present invention aims at addressing the above-mentioned problem by providing compositions comprising mycelium that can be 3D-prined directly and that can be obtained by fermentation. It is further desired that the forms made by printing mycelium retain their shape (i.e. do not collapse fully) upon drying, such as air drying without the need of crosslinking. It is further desirable to make use of food waste in the preparation of such printable compositions.

[0009] Summary of the invention

[0010] In first aspect, the invention relates to a composition comprising pellets comprising vegetal fibres embedded in a network of hyphae of Pleurotus fungi,

[0011] wherein 50% or less of the pellets are characterized by a circularity of 0.8 or more; and wherein 30% or less of the pellets are characterized by a ferret diameter of 0.75 mm or more.

[0012] In a second aspect, the invention relates to an ink for 3D printing, comprising a composition of the invention.

[0013] In a third aspect, the invention relates to the use of a composition according to the invention as an ink for 3D printing.In a fourth aspect, the invention relates to a method for the manufacture of a three-dimensional object comprising

[0014] a. providing a composition according to the invention; and

[0015] b. forming the three-dimensional object by

[0016] i. printing the composition using a 3D printer or

[0017] ii. extruding the composition.

[0018] In a fifth aspect the invention relates to a three-dimensional object comprising pellets comprising vegetal fibres embedded in a network of fungal hyphae,

[0019] wherein less than 50% of the pellets are characterized by a circularity of 0.8 or more; and wherein 30% or less of the pellets are characterized by a ferret diameter of 0.75 mm or more.

[0020] In a sixth aspect, the invention relates to a three-dimensional object obtained or obtainable by the method of the invention.

[0021] In a seventh aspect, the invention relates to a culture medium for growing mycelium or a filamentous fungus comprising a source of vegetal fibres, preferably a source of cellulosic vegetal fibres, preferably a source of cellulose or hemicellulose, preferably a source of crystalline cellulose or hemicellulose, preferably a source of microcrystalline cellulose or hemicellulose, preferably a source of cellulose nanocrystals, cellulose nanofibers, microcrystalline cellulose or combinations thereof, more preferably okara.

[0022] In a eighth aspect, the invention relates to a process for the preparation of a composition according to the invention, which comprises growing fungal mycelium or a filamentous fungus in a culture medium of the invention under conditions suitable for the growth of the mycelium or filamentous fungus.

[0023] Brief description of the figures

[0024] Fig- 1 : Schematic of okara production.

[0025] Fig. 2: Approximate nutritional value of okara (g / lOOg).

[0026] Fig- 3 : Polarized optical microscopy image of okara, showing plant cell debris and fibres. Fig. 4: Microscopy images using periodic acid schiffs (PAS) stained slides from Day 0 (A), Day 5 (B), Day 10 (C) and Day 25 (D).

[0027] Fig. 5: density peaks for Feret diameter (A), circularity (B) and aspect ratios (C) of the pellets after 0, 5, 10, 15, 20 and 25 days.

[0028] Fig. 6: Scanning electron microscopy (SEM) images showing that the branching hyphae encapsulated and embedded the soy pulp elements at 0 Days, 5 Days, 10 Days and 25 Days(EHT=2.00KV, WD= 4.9mm, Mag =1.0Kx, Signal A=HE-SE2, I Probe=100pA, Column Mode = Analytic).

[0029] Fig. 7: Graph representing the amount of solids in the sediment and in the supernatant after centrifugation of the growth medium after 0, 5, 10, 15, 20 and 25 days of incubation, as measured in Example 1.

[0030] Fig. 8: Graph representing the conductivity of the growth medium after 0, 5, 10, 15, 20 and 25 days of incubation, as measured in Example 1.

[0031] Fig. 9: FTIR spectra of Pleurotus ostreatus grown in the okara after 0, 5, 10, 15, 20 and 25 days of incubation and of a Pleurotus ostreatus control grown in synthetic liquid medium (PO LM).

[0032] Fig. 10: Amplitude sweeps representing the storage modulus (G1, in Pa) and the loss modulus (G" in Pa) of Pleurotus ostreatus grown in okara after 0 and 5 days (A), 10 and 15 days (B), 20 and 25 days (C) of incubation as a function of the oscillation strain (%) as measured in Example 1; graph representing G7G" of Pleurotus ostreatus grown in okara after 0, 5, 10, 15, 20 and 25 days of incubation as a function of time (D); graph representing the viscosity (in Pa.s) as a function of the oscillation strain rate in 1 / s (E).

[0033] Fig. 11: Graphs representing the flow point strain (A, in %), the flow point stress (B, in Pa), the storage modulus G' (C, in Pa), the yield stress (D, in Pa), and the yield strain ( E, in %) of Pleurotus ostreatus grown in okara after 0, 5, 10, 15, 20 and 25 days of incubation, as measured in Example 1.

[0034] Fig. 12: Graphs showing the printability of Pleurotus ostreatus grown in okara after 0, 5, 10, 15, 20 and 25 days of incubation, at different pressures and different gauge: 16G (A), 18G (B) and 20G (C) as measured in Example 1. Optimal printability with well-defined structures is indicated by circles, intermediate conditions where partial filament deformation or irregular extrusion was observed is indicated by triangles and non-printable conditions where the ink failed to form stable filaments is indicated by squares.

[0035] Fig. 13: Graph representing the spreading factor of Pleurotus ostreatus grown in okara after 0, and 25 days of incubation at two different Gauge (18G and 20G) for each time point.

[0036] Fig. 14: Nutritional composition of Pleurotus ostreatus grown in okara after 0, and 28 days of incubation, compared to other commonly consumed foods.

[0037] Fig. 15: Graph representing volume shrinkage (in %) of a sediment of Pleurotus ostreatus grown in okara after 0, and 25 days of incubation and of a Pleurotus ostreatus mushroom upon drying as described in Example 1.Fig. 16: Graph representing the water uptake (in %) of dried sediment of Pleurotus ostreatus grown in okara after 0, and 25 days of incubation and dried Pleurotus ostreatus mushroom upon soaking in water (A) and boiling (B).

[0038] Fig. 17: Graphs representing the volume swelling (in %) of dried sediment of Pleurotus ostreatus grown in okara after 0, and 25 days of incubation and dried Pleurotus ostreatus mushroom upon soaking in water (A) and boiling (B).

[0039] Fig. 18: Microscopy images of dried sediment of Pleurotus ostreatus grown in okara after 0 (A), and 25 days of incubation and dried Pleurotus ostreatus mushroom (C).

[0040] Fig. 19: Graph representing the stress (in kPa) of Pleurotus ostreatus grown in okara for 0, respectively 25 days of incubation followed by centrifugation, drying and boiling, as described in Example 1, compared to various commonly consumed boiled foods.

[0041] Fig. 20: Graph representing the stress at 20% strain (in kPa) of Pleurotus ostreatus grown in okara for 0, respectively 25 days of incubation followed by centrifugation, drying and boiling, as described in Example 1, compared to various commonly consumed boiled foods.

[0042] Fig. 21: Graph representing the solid content (in %) of Pleurotus ostreatus grown in okara for 0, respectively 25 days of incubation followed by centrifugation, drying and boiling, as described in Example 1, compared to various commonly consumed boiled foods.

[0043] Detailed description of the invention

[0044] The present inventors have developed compositions comprising pellets comprising vegetal fibres embedded in a network of fungal hyphae, which can advantageously be extruded and / or printed using a 3D printer for various applications, including food applications, due to the high nutritional value of the vegetable fibres and the mycelium. The composition is obtained by growing mycelium or a filamentous fungus in a liquid culture medium with water and a source of vegetal fibres. The fibre is used as nutrient for the mycelium and is consumed over time, to generate fungal hyphae biomass and CO2. The properties of the composition change over time while the mycelium digests the fibre. In particular, the nutritional content, the rheological properties and the structure of the composition evolve during fermentation. Thus, in the preferred aspects described below, the composition is optimized for printing.Pellets

[0045] The pellets comprising vegetal fibres embedded in a network of fungal hyphae are characterized in that

[0046] 50% or less, preferably 45% or less, preferably 40% or less, preferably 35% or less, preferably 30% or less, preferably 25% or less of the pellets are characterized by a circularity of 0.8 or more; and

[0047] 30% or less, preferably 25% or less, preferably 20% or less, preferably 15% or less of the pellets are characterized by a ferret diameter of 0.75 mm or more.

[0048] The percentages of pellets are defined by number, based on the total number of the pellets in the composition. The pellet size and the circularity can be determined using any suitable method known to the person skilled in the art. Preferably these parameters are defined by microscopy image analysis, preferably using a suitable software, such as ImageJ (1.53a), preferably after conversion of the images to the 8-bit format.

[0049] The pellet size and circularity determine the printability of the mycelium. When the pellets are either more circular or larger, the compositions have significantly impaired printability using a 3D printer and extrudability. In particular, larger pellets would fail to adequately stick together during the printing process.

[0050] In particular, the limitation of the pellet size translates in a better cohesion of the pellets with each other, which is an important parameter of a printable / extrudable material.

[0051] Pellet circularity is a 2D shape factor that measures how closely a pellet's projected outline resembles a perfect circle. Defined as the ratio of the perimeter of a circle with the same area to the actual perimeter of the pellet, values range from 0 to 1, where 1 represents a perfect circle. Ferret diameter is defined as the longest distance between any two points along a pellet’s boundary, preferably excluding pellets having a ferret diameter smaller than 0.04mm. Circularity and ferret diameter are preferably measured by analysis of microscopy images of the composition, preferably converted to 8-bit format. Such analysis can be performed using available softwares, such as ImageJ (version 1.53a).

[0052] The structure of the pellets comprises vegetal fibres embedded in a network of fungal hyphae. The pellets can further comprise other components, such as sugars, oligosaccharides, proteins, adhered exopolysaccharides, and other molecules originating from the source of vegetal fibres.Vegetal fibres and source thereof

[0053] In a preferred aspect the vegetal fibres comprise cellulosic fibres, such as cellulose or hemicellulose. In a preferred aspect, the cellulose or hemicellulose is at least partially in crystalline form, more preferably in microcrystalline or nanocrystalline form. In an aspect the vegetal fibre comprises cellulose nanocrystals, cellulose nanofibers, microcrystalline cellulose or combinations thereof. These cellulose forms advantageously provide small pellet size (small ferret diameter).

[0054] In a preferred aspect the vegetal fibre, such as described above, is provided in the form of a vegetal fibre source, comprising the vegetal fibres together with other chemical components, such as proteins, carbohydrates, ash and / or lipids. In a preferred aspect, the vegetal fibres source is in the form of fibrous vegetal material or in the form of microbial cellulose. Examples of fibrous vegetal materials include wood pulp, cereal stalks, cereal grain hulls, fruit and seeds shells or hulls, vegetable peals and legumes. Advantageously, many of such sources of vegetal fibres are side streams of agricultural or food production. Preferred sources of vegetal fibres are legumes, preferably legume residues obtained after protein and fat extraction.

[0055] In a preferred aspect the source of vegetal fibre is selected from food leftovers or byproducts of the food or beverage industry. In a more preferred aspect, it is a side stream or waste obtained after extraction of proteins and fats from a vegetal source.

[0056] In a most preferred aspect, the source of vegetal fibres is okara. Okara is the material remaining after extraction of protein from soybeans to obtain soy milk. This vegetal fibre source is produced in large amounts in tofu and soymilk production facilities and is presently only used for low value applications, such as feed. The present invention advantageously provides a high-value use of this abundant side-stream and transforms this material into a composition that can be printed in the form of a large diversity of products, including high nutritional value food.

[0057] The presence of the fibre, in combination with the fungal hyphae, make the composition denser and more resistant to extrusion, printing and drying, compared to compositions comprising fungal hyphae in the absence of the fibre. This makes the composition of the invention particularly advantageous for printed and extruded applications.

[0058] Fungal hyphae

[0059] The composition of the invention comprises hyphae of Pleurotus fungi, preferably Pleurotus ostreatus (or oyster mushroom). A hypha is defined as a fungal tubular structurecomprising one or several cells surrounded by a tubular cell wall. Such hyphae can be hyphae of a filamentous fungus or hyphae forming mycelium. Filamentous fungi are defined as fungi that grow in the form of long, branching, tube-like filaments. Mycelium is defined as a dense network of hyphae forming the vegetative body of a fungus (which can be a filamentous fungus or a fungus forming fruiting bodies or non-tubular shape).

[0060] The hyphal pellets of Pleurotus fungi, preferably of Pleurotus ostreatus have proved effective in producing a printable composition, whereas other fungal species were not. This benefit is associated with the ability of this fungus to form smaller pellets than other species, in combination with vegetal fibers. In particular, the material obtained with Pleurotus fungi, in particular Pleurotus ostereatus exhibited an improved ability to retain its shape after being imparted a particular shape and dried, in contrast to material obtain under the same conditions with other fungi, which lost their shape upon drying.

[0061] In a preferred aspect, the cells of the fungal hyphae are intact, i.e. neither chemically nor physically disrupted. In particular the hyphae are preferably not disrupted during the production of the composition of the invention and preferably also during the printing process.

[0062] Composition of the invention

[0063] In preferred aspects, the composition of the invention is characterized by specific physical properties, preferably specific rheological properties, preferably selected from: i. a flow point stress of 5000 Pa or less, preferably 4500 Pa or less, more preferably 4000 Pa or less;

[0064] ii. a flow point strain of 20% or less, preferably 18% or less, preferably 15% or less, preferably 10% or less;

[0065] iii. a storage modulus G' of 30000 Pa or less, preferably 25000 Pa or less, most preferably 20000 Pa or less; and / or

[0066] iv. a yield stress of 25000 Pa or less, preferably 20000 Pa or less;

[0067] each preferably measured using a DHR-3 TA instrument, with a 8mm diameter parallel steel geometry. Measurements are preferably performed at 25°C, with a 1000 pm gap. Amplitude sweeps are preferably performed at 1.0 rad.s-1 for a 0.1% to 200% strain range. From this, the Linear Viscoelasticity (LVE) is determined by screening G7G” as a function of the strain. Frequency sweeps are preferably then performed at the LVE limit strain value, here set at 1%, for a range of 0.001-200 Hz. The elastic recovery behaviour of the samples is preferably carried out by a step-strain sweep where low strain (1%) and high strain (200%) are cycled every 100s with an angular frequency of 1.0 rad / s.Measurement of the structural strength is typically carried out with a frequency sweep. Amplitude sweeps are preferably performed at 1.0 rad. s'1for a 0.1% to 200% strain range. From this, the Linear Viscoelasticity (LVE) is preferably determined by screening G7G” as a function of the strain. Frequency sweeps are then preferably performed at the LVE limit strain value, preferably set at 1%, for a range of 0.001-200 Hz. The elastic recovery behaviour of the composition is preferably carried out by a step-strain sweep where low strain (1%) and high strain (200%) are cycled every 100s with an angular frequency of 1.0 rad / s. The rheometer software calculates the storage (elastic) modulus G’ and the loss (viscous) modulus G” as a function of the oscillation frequency. The frequency is typically indicated as angular frequency (in [s-1]).

[0068] In an aspect, the storage modulus of the composition of the invention is of up to 30000 Pa, up to 29000 Pa, up to 28000 Pa, up to 27000 Pa, up to 26000 Pa, up to 25000 Pa, up to 24000 Pa, up to 23000 Pa, up to 22000 Pa, up to 21000 Pa or up to 20000 Pa. In an aspect, the Flow point strain (%) is of up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 14, up to 13, up to 12, up to 11 or up to 10. In an aspect, the Flow point stress (Pa) is of up to 6500, up to 6000, up to 5000, up to 4900, up to 4800, up to 4700, up to 4600, up to 4500, up to 4400, up to 4300, up to 4200, up to 4100, or up to 4000. In an aspect the yield strain (%) is of at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.1, at least 3.2, at least 3.3, at least 3.4 or at least 3.5. In an aspect the yield strain (%) is of up to 6, up to 5.5 or up to 5.

[0069] In another particular aspect, the composition of the invention further comprises lower molecular weight carbohydrates, such as oligosaccharide and sugars. Preferably, the fibre to lower molecular weight carbohydrates ratio (by weight) in the composition of the invention is of at least 10, such as 10 to 50, 10 to 40, 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22 or 19 to 21, for example 19.5.

[0070] From a nutritional point of view. The total fibres content in the composition of the invention originates both from the remaining fibres from the vegetal fibre source and those present in the fungal hyphae. Preferably the composition of the invention comprises insoluble fibres, in a particular aspect, it comprises insoluble fibres, soluble fibres and optionally smaller carbohydrates, such as sugars. In a particular aspect the total fibre content in the composition of the invention is of at least 48wt%, preferably at least 50wt%, preferably at least 52wt%, preferably at least 54wt%, preferably at least 55w%, more preferably at least 56wt%, as defined by weight, based on total solids. In an aspect, the insoluble fibre content in the composition of the invention is of at least 20wt%, at least 25wt%, at least 30wt%, at least 35wt%, at least40wt%, at least 45wt% or at least 50wt% of insoluble fibres based on total solids. In a particular aspect the insoluble fibre content in the composition of the invention is of up to 90wt%, up to 85wt%, up to 80wt%, up to 75wt%, up to 70wt%, or up to 65wt%, up to 60wt%, up to 65wt%, up to 60wt%, up to 55wt%, up to 50wt%, up to 45wt%, up to 40wt%, or up to 35wt%, based on total solids.

[0071] In an aspect, the total carbohydrate content in the composition of the invention is of at least 50wt%, preferably at least 55wt%, more preferably at least 58wt%, most preferably at least 59wt%, based on total solids.

[0072] In an aspect, the amount of other carbohydrates in the composition of the invention is of less than 20wt%, less than 15wt%, less than 10wt%, less than 5wt% or less than 3wt%, based on total solids. In an aspect, the protein content in the composition of the invention is of at least 10wt%, at least 15wt%, at least 20wt%, at least 25wt% or at least 30wt% based on total solids.

[0073] In a particular aspect the protein content in the composition of the invention is of at least 10wt%, at least 15wt%, at least 20wt%, at least 25wt% or at least 30wt%, based on total solids. In a particular aspect the protein content in the composition of the invention is of up to 90wt%, up to 85wt%, up to 80wt%, up to 75wt%, up to 70wt%, or up to 65wt%, up to 60wt%, up to 65wt%, up to 60wt%, up to 55wt%, up to 50wt%, up to 45wt%, up to 40wt%, or up to 35wt%, based on total solids.

[0074] In a particular aspect the total content of fibres, such as insoluble fibres and protein in the composition of the invention is of at least 50wt%, at least 55wt%, at least 60wt%, at least 65wt% or at least 70wt%, at least 75wt%, at least 80wt%, at least 85wt%, at least 90wt%, based on total solids.

[0075] Printing and extruding the composition

[0076] The composition of the invention is advantageously printable and / or extrudable. Accordingly, it can be used as an ink for printing, preferably for 3D printing. The invention therefore also encompasses an ink comprising the composition of the invention.

[0077] Advantageously, the composition of the invention does not require any additive for being printable and / or extrudable. Thus, in a preferred aspect, the composition of the invention or the ink comprising the composition of the invention is free from stiffness modifiers. In another aspect, the composition of the invention or the ink comprising the composition of the invention is free from mineral particles. Examples of such mineral pellets include clay and ceramic. In a further aspect, the composition of the invention or the ink comprising the composition of the invention is free from gelling agents. Examples of such gelling agentsinclude gums and agar agar. In the frame of the present disclosure, “free from” means completely or substantially free from the mentioned substance(s). A composition according to the present disclosure which is “substantially free” from a substance is a composition where the substance is present in less than 10% of the total weight of the composition, preferably less than 5% of the total weight of the composition, more preferably less than 4%, 3%, 2% or 1% of the total weight of the composition. Preferably, the composition is made up of at least 90% of fungal hyphae and vegetal fibres, preferably 95% of fungal hyphae and vegetal fibres, even more preferably 96%, 97%, 98%, 99% or 100% of fungal hyphae and vegetal fibres.

[0078] The composition of the invention can be used to shape a three-dimensional object, preferably by 3D printing or extrusion. The three-dimensional object can be any solid item that can be formed by 3D printing or extrusion. In a particular aspect, the three-dimensional object is a food product. In an aspect it is an extruded food product. In an aspect it is a 3D printed food product. The composition of the invention can advantageously be printed using standard 3D printers, for example at a gauge of 10G to 30G, such as 12G to 28G, 14G to 26G, 16G to 24G, 16G to 22G or 16G to 10G and at pressures of 10 to 50 Pa, such as 15 to 45 Pa.

[0079] In an aspect, the spreading factor of the composition of the invention at 18G is of up to 3, up to 2.9, up to 2.8, up to 2.7, up to 2.6, up to 2.5, up to 2.4, up to 2.3, up to 2.2, up to 2.1, up to 2.0, up to 1.9, up to 1.8, up to 1.7, up to 1.6, up tol.5, up to 1.4, or up to 1.3. In an aspect, the spreading factor at 18G is of at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1. Spreading factor is a measure of the wetting behaviour of a liquid on a solid surface. It is defined as the difference between the interfacial free energy of the liquid-solid interface and the sum of the interfacial free energies of the liquid-vapor and solid-vapor interfaces. A positive spreading coefficient indicates that the liquid tends to wet the solid surface, meaning that it spreads out over the surface and forms a thin film.

[0080] The ink comprising the composition of the present invention can contain additional ingredients, such as colorants, odorant, flavours, nutrients (such as minerals or vitamin) and the like. In an aspect, the ink comprises a texturizer, such as polysaccharide gums (such as gum Arabic or locust bean gum), gellan, cellulose derivatives (such as hydroxyethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC) or Hydroxypropyl cellulose (HPC)), proteinbased thickeners such as albumin, starches and the like.Process for producing a composition of the invention and method for the manufacture of a three-dimensional object.

[0081] These processes comprise a step of growing the fungal hyphae in a liquid medium comprising water and vegetal fibres, such as described above. In an aspect, the medium consists in vegetal fibres (optionally in the form of a fibre source as described above) and water, i.e. the medium does not substantially contain any further nutrient, in addition to what is contained in the vegetal fibre source.

[0082] In an aspect, the fungal hyphae are grown for up to 30 days. In aspect, they are grown for up to 5 days. In another aspect the fungal hyphae are grown for more than 15 days, preferably 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more or 25 days or more. Preferably, the composition is grown for up to 30 days, up to 29 days, up to 28 days, up to 27 days, up to 26 days or up to 25 days. In preferred aspect, the fungal hyphae are grown for 20 to 30 days, such as 21 to 29 days, 22 to 28 days, 23 to 28 days, 24 to 28 days, 20 to 25 days, 21 to 25 days, 22 to 25 days, 23 to 25 days, 24 to 25 days, preferably 25 days. Such growth periods result in the best printability of the composition. Most of the fibre source has been consumed and most of the composition is made of mycelium. The network is less stiff and has adequate rheological behaviour making it optimal for printing and extrusion.

[0083] In an aspect, the fungal hyphae are grown until the growth medium becomes a composition of the invention, as described above.

[0084] Product-by-process

[0085] The invention provides a composition obtained or obtainable by the process of the present invention.

[0086] The invention provides a three-dimensional object obtained or obtainable by the method of the present invention.

[0087] Examples

[0088] Example 1

[0089] Study objective

[0090] The present example aims at directly 3D-printing mycelial material obtained by cultivating the edible fungus Pleurotus ostreatus in okara (soy pulp obtained as a by-product ofsoy milk production, see Fig. 1) to directly 3D print mycelial materials without any extra nutrient, rheological modifiers or other additives.

[0091] From soy waste to mycelial pellets, dynamic changes

[0092] Soy pulp obtained from soymilk making was utilized as the sole nutrient source in our submerged fermentation system, without any additives. Microscopy images using periodic acid schiffs (PAS) stained slides from Day 0 to Day 25 (Fig. 4) reveal the various structures formed by the soy pulp components in Okara. Notably, the cellulose fibers displayed structural colors under polarized light. At Day 0 (Fig. 4A) these soy pulp pellets exhibited small diameters, low circularity, and varied aspect ratios as shown in the pellet size analysis results. By Day 5, small mycelial pellets began to form (Fig. 4B) with soy pulp elements both surrounding the pellets (Fig. 4B) and becoming encapsulated within the pellets (Fig. 6B), leading to increases in Feret diameter and circularity. As the fermentation progressed, (Fig. 4C at 10 Days and Fig. 4D at 25 Days) the pellets became larger and more spherical, as indicated by increased Feret diameter, circularity, and decreased aspect ratios. Additionally, the media surrounding the pellets becomes increasingly depleted in soy pulp residues (Fig. 4). Fig. 5 provides density peaks for Feret diameter (Fig. 5A), circularity (Fig. 5B) and aspect ratio (Fig. 5C) of the pellets at 0, 5, 10, 15, 20 and 25 days of fermentation. All three parameters reached their maximum on Day 10, with subsequent measurements from Day 10 to Day 25 showing almost overlapping curves, except for a decrease and further broadening of the circularity peak on Days 20 and 25. The morphology of individual hyphae also evolved, becoming thicker and more compact in later growth stages (Fig. 6). These changes signify a transition from a rapid growth phase to the maturation and depletion phases (Veiter, L., Rajamanickam, V. & Herwig, C. The filamentous fungal pellet — relationship between morphology and productivity. Appl Microbiol Biotechnol 102, 2997-3006 (2018)). Corresponding physical parameters were also monitored, including the solid mass in the sediment and supernatant of the growth mixture after centrifugation (Fig. 7). Initially, the total solid content was 4.74 (±0.35) g and it decreased consistently overtime, reaching 3.21 (±0.18) g on Day 25. This decline reflects the metabolic activity of the fungi, which degrades the solid soy pulp into smaller molecules, thereby reducing the mass in the sediment and increasing it in the supernatant. In parallel, the solid content of the sediment decreased from -10% to -6% (Table in Supporting), indicating a higher proportion of water retained within the mycelial network compared to the original soy pulp. The pH and conductivity also reflected those dynamic changes. They both increased throughout the fermentation (see Fig. 8), with no significant differences observed between Day 20 and Day25. The rise in pH could be attributed to the release of ammonia from protein degradation, the consumption of acids, or the metabolic production of alkaline compounds. The continuous increase in conductivity can be attributed to the accumulation of soluble degradation products, including sugars, organic acids, and nitrogenous metabolites, which together increased the ionic strength of the medium.

[0093] Rheological behavior and printability of the materials

[0094] The rheological properties of the sediments were evaluated to understand the dynamic changes occurring within the growth system and to assess their suitability as potential inks for 3D printing. All sediments were directly used after centrifugation under identical parameters, yet they displayed varying capacities to retain water. The solid content of the sediment decreased from approximately 10% at Day 0 to about 6% at Day 25, suggesting that the developing mycelial network retains more water than the original soy pulp.

[0095] Amplitude sweeps (Fig. 10) revealed that, irrespective of incubation time, the storage modulus (G’) and loss modulus (G”) for all sediments maintain a clear linear viscoelasticity (LVE) plateau at oscillation strains of around 1%. In addition, all samples showed shearthinning behavior in both amplitude and frequency sweeps, with rapid recovery from solid to liquid-like state and vice versa. These characteristics highlight their potential as shear-responsive materials. Notably, the amplitude and frequency sweep curves, along with derived parameters such as flow point stress (Fig. 1 IB), flow point strain (Fig. 11 A), and infinite shear viscosity differed substantially across the growth stages and could be broadly divided into three regimes based on the growth day.

[0096] At Day 0, in the absence of mycelium, the sediment shows the lowest G’ overall, consistent with a loose paste formed primarily of soy pulp fragments. The amplitude sweep indicates that as strain increases, G’ gradually transitions into the non-linear domain, exhibits significant yielding before reaching the flow state. Consequently, the yield strain is minimal, indicating that only a small deformation is needed before the flow initiates. Correspondingly, the flow point strain (where G’= G”) remains low (Fig. 11 A), showing that the Day 0 sample readily shifts to a liquid-like behavior under modest strain. Such behavior aligns well with its paste-like form.

[0097] By Day 5, the formation of small mycelial pellets becomes evident in a rise in G’ and yield strain (Fig. 10A). This stiffening peaks at Day 10, where G’ surpasses 105 Pa. As circularity and aspect ratio reach their maximum, the pellets are expected to behave as granular hydrogels, meaning their rheological properties are governed by their individual properties andcontact deformation. The observed increase in both flow point stress (Fig. 1 IB) and flow point strain (Fig. 11 A) at Day 10 indicates a notably stiffer material that nevertheless permits large deformations prior to flow. This could be explained by the newfound granularity of the system compared to day 0, in which the additional yielding observed is due to subpopulations of pellets flowing past each other, without yet triggering macroscopic liquid-like behaviour. Large error bars in the measurements also suggest some degree of inhomogeneity within these samples. The high flow point stress further confirms that the developing mycelial network is dense and well-entangled, making it resistant to deformation.

[0098] Beyond Day 10, G’ decreases steadily (Fig. 10B), indicating a partial breakdown or reorganization of the pellet network. One clear observation is the notable shifts in G’-G” amplitude sweep curves, where the LVE regime ends more abruptly, transitioning into a shorter plastic domain (Fig. 10B). This leads to a more distinct yield stress and shows a reduction in plastic deformation before the transition to liquid-like flow. Meanwhile, the yield strain continues to rise through Day 15 and Day 20, suggesting that although the overall rigidity (G’) diminishes, the sediment still retains a relatively elastic structure capable of absorbing more strain before yielding. Additionally, the G” overshoot prior to flow, which is characteristic of granular and soft materials, becomes increasingly apparent. This is tied to viscous energy dissipation, where softer pellets are expected to dissipate more energy per volume via deformation.

[0099] Ultimately, the system’s maturation seems to dictate the rheological properties, and therefore, the potential printability of the mycelium. However, printability is not guaranteed. Even though all the samples demonstrated shear-thinning behavior, not all of them were printable during our 3D printing trials. Notably, the Pleurotus ostreatus hyphae compositions grown in okara at Days 10 and 15 are not printable under any of the parameters we have investigated, including nozzle sizes between 0.6, 0.84, and 1.2mm, and pressures ranging from 15 to 45 Pa (fig. 12). The high stiffness, flow point strain, and larger size of the Pleurotus ostreatus hyphae composition grown in okara at Day 10 pellets lead to an ink that is difficult to print. Upon pressure increase, water loss was observed prior to the ink sputtering. This water loss increases the contact area between each pellet, leading to a pressure increase, explaining the lack of continuous filament formation for this intermediate fermentation stage.

[0100] Assessment of the suitability of printed materials as food products

[0101] It appears that food waste has been transformed into a new material composed of mycelial hyphae. Since these hyphae originated from a common edible mushroom (oystermushroom), they are theoretically safe to eat. However, the following experiments aim at determining whether they share similar nutritional values with the fungus' fruiting body.

[0102] We first compared the nutritional values of this myceliated material (the Pleurotus ostreatus hyphae composition grown in okara at Day 28 was used instead of that at Day 25 in this analysis due to limited availability of the composition at Day 25 samples) with its fruiting Cii D 0ttomposon aay

[0103] body counterpart (oyster mushrooms), and other common or related foods (data taken from ()lso ppyu

[0104] USDA FOOD DATA CENTRAL). All samples were assessed in the fresh state, with their l Pttteurous osraus gronw

[0105] water content ranging from 60% to 93% (Table 1). To provide a fair comparison of nutritional i ()kD 28n oaraay

[0106] distribution independent of water content, the dry-weight composition of major components (carbohydrates, protein, fat and ash) was also visualized (Fig. 14).

[0107] Ohtser msroomyu

[0108] Table 1: nutritional values of diverse nutritional compositions

[0109] f Tou

[0110] Thempe

[0111] i Ehtgge w

[0112] ii Zhccnu Energy value

[0113] 129.82 73.64 139.00 317.00 803.00 216.00 88.00 79.00 (kJ)

[0114] Energy value

[0115] 31.24 17.81 33.00 76.00 192.00 52.00 21.00 77 S Pttt.eeoaow00 (kcal)

[0116] Water (g) 90.44 93.30 89.20 84.60 59.60 87.60 92.70 79.50 Total fat (g) 0.61 0.10 0.19 4.78 10.80 0.17 0.40 0.38 Carbohydrates

[0117] 5.52 3.99 6.94 1.87 7.64 0.73 3.11 17.30 (g)

[0118] Fiber (g) 4.32 3.79 2.80 0.30 N / A 0.00 1.10 4.44 Protein (g) 3.07 2.14 2.90 8.08 20.30 10.90 2.71 1.58

[0119]

[0120] Ash (g) 0.36 0.45 0.73 0.72 1.62 0.63 1.05 1.18

[0121] The nutritional content of the starting soy pulp (Day 0) and of the composition obtained after 28 days is provided on a dry weight basis in Table 2 below:Table 1: nutritional values (based on dry matter) of the composition at day 0 and of the composition at day 28

[0122] Content (wt%) Content (wt%) Total fat 6.4 1.5

[0123] Fiber 45.2 56.8

[0124] Lower molecular weight carbohydrates 12.5 2.9

[0125] Fiber 45.2 56.8

[0126] Protein 32.2 32.0

[0127] Moisture 0.0 0.0

[0128] Ash 3.7 6.8

[0129] Total 100 100

[0130]

[0131] The starting soy pulp (Day 0) is the insoluble residue left after straining soymilk and therefore retains more insoluble fibrous material while losing much of the protein and fat that stay in the serum phase. Nutritionally, the composition at Day 0 presents modest protein, low fat, and a carbohydrate fraction with a substantial fiber contribution. Tofu, on the other hand, is formed by coagulating the protein-rich fraction of soymilk, resulting in a product with higher protein and fat but considerably less fiber than the composition at Day 0.

[0132] After fungal colonization and transformation into the Pleurotus ostreatus hyphae composition grown in okara at Day 28, the apparent amounts of carbohydrates and protein decrease on a fresh-weight basis (Table 1), primarily due to increased water content (from 90.4% to 93.3%) and possibly metabolic consumption during fungal growth. However, when expressed on a dry -weight basis, the relative proportions of major components remain comparable to the composition at Day 0 (Fig. 14), suggesting that while the fungus metabolized part of the substrate to sustain growth, the overall macronutrient balance of the remaining solid matrix was largely preserved. The reduction in energy value (from 129.8 kJ to 73.6 kJ) reflects the utilization of energy-rich components during metabolism, while the slightly higher water content likely results from the development of a porous, hydrated hyphal network that retains more moisture than the original soy pulp.

[0133] When compared with its fungal counterpart, the oyster mushroom, the Pleurotus ostreatus hyphae composition grown in okara at Day 28 shows a distinct nutritional profile on a fresh-weight basis, containing less energy, fat, total carbohydrates, protein, and ash but more fiber and water (Table 1). However, when expressed on a dry-weight basis (Fig. 14), bothmaterials display broadly similar macronutrient proportions, with carbohydrates and protein as major components and very low fat content.

[0134] Tempeh provides another relevant comparison, as it also represents a fungus-bound soy product. On a fresh-weight basis, it contains substantially higher protein (20.3 g) and fat (10.8g) and lower water content (59.6%) compared with the Pleurotus ostreatus hyphae composition grown in okara at Day 28 (Table 1). When normalized to dry weight (Fig. 14), tempeh still shows higher proportions of protein and fat and lower carbohydrate content, indicating a more nutrient-dense composition. These differences reflect the distinct starting materials and processing conditions: whole soybeans in tempeh retain more concentrated macronutrients, whereas the myceliated material of the invention originates from soy pulp, a byproduct naturally lower in protein and fat.

[0135] When positioned among other foods (Fig. 14), the Pleurotus ostreatus hyphae composition grown in okara at Day 28 occupies an intermediate nutritional space. On a freshweight basis, it is less energy-dense than animal -derived foods such as egg white and is highly hydrated, with a water content (93.3%) similar to zucchini (92.7%) and considerably higher than sweet potato (79.5%). On a dry-weight basis, its macronutrient distribution, characterized by comparable proportions of carbohydrates and protein and very low fat, more closely resembles that of fungal materials such as oyster mushrooms than its soy-based starting substrate. Overall, the Pleurotus ostreatus hyphae composition grown in okara at Day 28 represents a low-calorie, high-fiber and water-retaining matrix that compositionally, on a dry basis, is distinct from its soy-pulp origin and very similar to its fungal counterpart.

[0136] Despite the interesting nutritional profile, an essential consideration for any food product is its storability. Much like mushrooms, vegetables, or pasta, drying effectively reduces water activity, prolongs shelf life, and improves stability. To investigate how drying and subsequent rehydration affect the printed materials, we examined the composition at Day 0 and the Pleurotus ostreatus hyphae composition grown in okara at Day 25 alongside oyster mushrooms. All samples were dried in a fume hood under ambient conditions. Given that their initial moisture content exceeded 85% (Table 1), they each experienced pronounced water loss. Shrinkage surpassed 50% for all, with the composition at Day 0 and the Pleurotus ostreatus hyphae composition grown in okara at Day 25 shrinking over 75%, approximately 23% more than oyster mushrooms (Fig. 15). This reflects microstructural differences in the printed soy-and hyphae-based matrix and the natural fungal fruiting body. The cap of the oyster mushroom has distinct gills and cell layers that form a porous, interwoven network, while the 3D-printed composition at Day 0 and the 3D printed Pleurotus ostreatus hyphae composition grown inokara at Day 25 samples are more homogeneous, lacking such a stratified structure and instead featuring a relatively uniform dispersion of soy particles and fungal mycelium.

[0137] We evaluated reconstitution by soaking and boiling, simulating common preparation and cooking methods. When soaked in water, each sample rapidly absorbed moisture and approached equilibrium by about 400 minutes (Fig. 16). When boiled, oyster mushrooms fully rehydrated in approximately 5 minutes, the Pleurotus ostreatus hyphae composition grown in okara at Day 25 in about 10 minutes, and the composition at Day 0 in roughly 20 minutes (Fig.

[0138] 16). As they reabsorbed water, their volumes also swelled, with the composition at Day O showing the greatest swelling capacity and oyster mushrooms the least (Fig. 17). Notably, because mushrooms underwent the least shrinkage, they demonstrated relatively consistent volume recovery overall.

[0139] These findings highlight the material's significant transformation and its potential as a storable food product. While its pronounced shrinkage is similar to the original soy pulp, its rehydration behavior is distinct. Critically, the Pleurotus ostreatus hyphae composition grown in okara at Day 25 reaches its rehydration equilibrium much faster than the composition at Day 0, particularly when boiled (approx. 10 vs. 20 minutes), which is a practical benefit for food preparation. However, this faster rehydration is linked to a lower overall water uptake and swelling capacity compared to the composition at Day 0. This suggests the myceliation creates a denser, more integrated matrix that, while highly compressible upon drying, rehydrates more readily but is also less porous or expansive than the unstructured original pulp.

[0140] We also compared the mechanical properties of the printed and boiled samples, as well as other common food to understand how they position texture-wise in the common food spectrum.

[0141] As shown in the stress-strain curves (Fig. 19), the transition from Day 0 to Day 25 demonstrates a substantial enhancement in strength and stiffness. The Pleurotus ostreatus hyphae composition grown in okara at Day 25 exhibits a markedly steeper stress-strain curve and roughly an order of magnitude higher stress at 20% strain than the initial material. This improvement occurs alongside an increase in solid content from about 22% in the composition at Day 0 to 44% in the Pleurotus ostreatus hyphae composition grown in okara at Day 25 (Fig.

[0142] 21). The starting Day 0 material, composed purely of soy pulp, resembles other soy-derived foods like tofu but remains the weakest and most hydrated, indicating that the soy pulp itself provides little mechanical resistance.

[0143] After fungal colonization, the Pleurotus ostreatus hyphae composition grown in okara at Day 25 becomes structurally closer to fungal materials such as oyster mushrooms andtempeh. Like tempeh, it is a mycelium-bound soy composite, but it differs in its hydration. Tempeh contains roughly 30% solids and gets its firmness from the compact arrangement of whole soybeans. In contrast, the Pleurotus ostreatus hyphae composition grown in okara at Day 25 achieves comparable or even higher stress values while retaining a much higher water content (approximately 56%), indicating its strength originates from the continuous mycelial network. Compared to oyster mushrooms (which are only -11% solids and remain soft), the Pleurotus ostreatus hyphae composition grown in okara at Day 25 forms a much denser and more cohesive matrix capable of sustaining higher stress.

[0144] When compared with the other tested foods, the Pleurotus ostreatus hyphae composition grown in okara at Day 25's mechanical profile is unique. It achieves a strength comparable to dense tofu and significantly greater than tempeh, all while retaining a substantial water content (56%). This is in stark contrast to the other high-water foods like zucchini, egg white, and mushrooms, which are exceptionally soft. The Pleurotus ostreatus hyphae composition grown in okara at Day 25's ability to be both firm and resilient (rather than brittle like tofu) suggests its structure is uniquely stabilized by the integrated mycelial network. This combination of moistness and firmness positions the boiled Pleurotus ostreatus hyphae composition grown in okara at Day 25 as a dense, structured solid, highlighting its potential as a novel, tunable food material.

[0145] Materials and Methods

[0146] Materials

[0147] Soybeans (German Origin, Rapunzel) were purchased from Galaxus, Switzerland. The fungal species Pleurotus ostreatus was obtained from Mycelia, Belgium.

[0148] Okara Production and Inoculation

[0149] The okara was obtained by sieving (150 mesh) homemade soymilk made using a Mila soymilk maker (Springlane Kitchen). The initial ratio of soybeans to water (filtered through a Brita filter) was 100 grams to 1 liter. The okara (solid content approximately 12.9 ± 0.7 %) was transferred to a 750 mL centrifuge bottle, and water was added until the total weight reached 750 grams. This mixture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded.

[0150] The incubation was carried out in 250 mL flasks containing 100 grams of sterilized (121°C, 60 min) 5% okara and three 7x7 mm squares cut from 7-day-old Pleurotus ostreatus agar plates (homogenized using a tissue grinder).The flasks were incubated in an orbital shaker at 150 rpm and 22°C for intervals of 0, 5, 10, 15, 20, and 25 days. After incubation, the contents were divided into two 50 mL falcon tubes and centrifuged at 4000 rpm for 5 minutes. The sediment and supernatant were separated.

[0151] Dynamic growth characterizations

[0152] The solid content of the sediment and supernatant was calculated by the weight change after drying under 50°C.

[0153] The pH and conductivity of the supernatant were also measured.

[0154] The sediments after different incubation days were diluted by adding 12g of PBS to 0.5g sediment (the final concentration is between 0.25% to 0.4%). The diluted sediments were poured into a 100mm petri dish and imaged using a camera (Sony Alpha 7).

[0155] The images were imported into ImageJ (1.53a) and converted to 8-bit format. Pellet size analysis was carried out on those images focusing on the pellets that have a ferret diameter higher than 0.04mm. The ferret diameter (the longest distance between any two points along a pellet’s boundary), circularity, and aspect ratio were selected to compare among different groups. Kernel density estimation (KDE) was applied to generate density plots, where the y-axis represents a normalized probability density rather than raw counts. The total area under each curve sums to 1, allowing for direct comparison of distribution shapes across different time points.

[0156] The sediments after different incubation days were fixed using 4% paraformaldehyde (PF A) for paraffin processing and using 1% glutaraldehyde for critical point drying. After fixation, they were washed thoroughly using phosphate-buffered saline (PBS).

[0157] The PFA fixed samples were dehydrated through graded alcohol, cleared with xylene, and infiltrated with paraffin at 60°C, then cooled down. The sectioning was carried out using microtone (Leica RM2265) at 2pm. Periodic acid schiffs (PAS) staining was carried out to detect polysaccharides, mucopolysaccharides, and glycolipids. Sections were de-waxed, hydrated, oxidized with 1% periodic acid, washed, and stained with Schiff’s reagent. After further washing, sections were stained with Harris Hematoxylin, differentiated in 1% acid-alcohol, and washed again. Finally, sections were dehydrated, cleared, and mounted. The slices were observed using an optical microscope (Nikon Eclipse 100NV).

[0158] Scanning electron microscopy (SEM) was conducted using Carl Zeiss Merlin microscope at IkV at the Interdisciplinary Center for Electron Microscopy (CIME) of Ecole Polytechnique de Lausanne (EPFL). The samples include were sputter-coated with 5 nm of gold-palladium.Rheology after different incubation days

[0159] Rheology was performed on a DHR-3 TA Instrument with an 8 mm diameter parallel plate steel geometry. All measurements were performed at 25°C, with a 1000 pm gap. Amplitude sweeps were performed at 1.0 rad- s'1for a 0.1% to 200% strain range. From this, the Linear Viscoelasticity (LVE) was determined by screening G7G” as a function of the strain. Frequency sweeps were then performed at the LVE limit strain value, here set at 1%, for a range of 0.001-200 Hz. The elastic recovery behavior of the samples was carried out by a step-strain sweep where low strain (1%) and high strain (200%) were cycled every 100s with an angular frequency of 1.0 rad s'1.

[0160] 3D printing of jammed mixture

[0161] Sediment from different groups was loaded inside a 3 mL syringe and centrifuged for 1 min at 4000 rpm to remove trapped air. 3D printing was performed with a commercial 3D printer (Bio X, Cellnk). The ink was extruded through 20G, 18G, and 16G nozzles (apertures: 0.63, 0.84, and 1.2 mm, respectively). The pressure tested were between 15 to 45 kPa with a printing speed of 10 mm s '.

[0162] The spreading factor was used to evaluate the shape fidelity of the printed filaments. Printed filaments were imaged, and their width was measured using ImageJ (1.53a). The spreading factor was calculated as the ratio of the measured filament width to the nozzle diameter.

[0163] To assess printability across different conditions, a printability map was generated based on nozzle size, applied pressure, and incubation time (see Fig. 12). Optimal printability with well-defined structures is indicated by circles, intermediate conditions where partial filament deformation or irregular extrusion was observed is indicated by triangles and non-printable conditions where the ink failed to form stable filaments is indicated by squares.

[0164] Moisture-responsive behaviour

[0165] Fresh printed Day 0 and Day 25 samples, as well as fresh oyster mushrooms, were used for moisture-responsive behaviour tests. Cylindrical samples (10 mm diameter x 8 mm height) were 3D printed using a 20G nozzle. For oyster mushrooms, same sized cylindrical sections were cut from the region between the stem and cap edge.Samples were first air-dried in a fume hood for two days until reached equilibrium. The initial and final dimensions and weights of the dried samples were recorded to calculate volume shrinkage (%).

[0166] To assess water absorption capacity, dried samples were immersed in two different conditions: room temperature soaking (cold water, ~20°C), and boiling water(100°C). For room temperature soaking, samples were submerged for 1, 3, 6, and 24 hours. For boiling water soaking, samples were immersed for 5, 10, 15, 29, 25 and 30 minutes. At each time point, samples were removed, surface water was gently wiped off and the weight was immediately recorded. The water uptake (%) was calculated based on the change in sample weight. The volume swelling (%) was determined by measuring dimensional changes. Swelling results were analysed for both soaked and boiled samples.

[0167] Compression Analysis

[0168] Compression tests were conducted using a DHR-3 rheometer (TA Instruments) equipped with a 20 mm parallel plate steel geometry. A constant strain rate of 3 pm / s was applied until a total strain of 40% was reached.

[0169] Fresh and rehydrated samples from the swelling tests were used for compression analysis. To compare their mechanical properties with conventional food materials, tofu, tempeh, egg white, and zucchini were also tested under the same conditions. Tofu was tested in its original form, while egg white, tempeh and zucchini were first boiled in water for 10 minutes. All samples were cut into cylindrical shapes (10 mm diameter x 8 mm height) for consistency.

[0170] Nutritional Analysis

[0171] The nutritional composition of Day 0 and Day 28 samples was analyzed by Eurofins Scientific AG (Schbnenwerd, Switzerland). The Day 28 sample was used instead of Day 25 due to an unintentional three-day longer incubation period. However, this sample was still considered valuable for representing a later stage of fermentation.

[0172] Freeze-dried samples were submitted for analysis, and the results were recalculated to reflect the nutritional values of the fresh state based on their respective solid content.

[0173] For comparison, the nutritional composition of other food materials was obtained from the U.S. Department of Agriculture FoodData Central database (https: / / fdc.nal.usda.gov / ).Viability analysis

[0174] To assess viability, 25 mL of the supernatant was transferred to a 100 mL flask and incubated at 22°C, 150 rpm for 7 days. After incubation, the solid fraction was collected by vacuum filtration using a 2.7 pm Whatman 542 filter, and the dry weight of the recovered solid was measured and compared.

[0175] Additionally, a portion of the sediment obtained after centrifugation was placed at the center of a malt extract agar (MEA) plate. The plate was incubated at 26°C, and the growth of the pellet was monitored to determine whether the pellets remained viable.

Claims

1. ClaimsI. A composition comprising pellets comprising vegetal fibres embedded in a network of hyphae of Pleurotus fungus,50% or less, preferably 45% or less, preferably 40% or less, preferably 35% or less, preferably 30% or less, preferably 25% or less of the pellets are characterized by a circularity of 0.8 or more; and30% or less, preferably 25% or less, preferably 20% or less, preferably 15% or less of the pellets are characterized by a ferret diameter of 0.75 mm or morewherein the percentages are defined by number, based on the total number of pellets in the composition and wherein the percentages are determined by image analysis.

2. The composition according to claim 1, wherein the vegetal fibres include cellulosic fibres, preferably cellulose and / or hemicellulose, preferably crystalline cellulose or hemicellulose, more preferably microcrystalline or nanocrystalline cellulose or hemicellulose, preferably cellulose nanocrystals, cellulose nanofibers, microcrystalline cellulose or combinations thereof.

3. The composition according to claim 1 or 2, wherein the flow point stress of the composition is of 5000 Pa or less, preferably 4500 Pa or less, more preferably 4000 Pa or less.

4. The composition according to any one of the preceding claims, characterized by a flow point strain of 20% or less, preferably 18% or less, preferably 15% or less, preferably 10% or less.

5. The composition according to any one of the preceding claims, characterized by a storage modulus G' of 30000 Pa or less, preferably 25000 Pa or less, most preferably 20000 Pa or less.

6. The composition according to any one of the preceding claims, characterized by a yield stress of 25000 Pa or less, preferably 20000 Pa or less.

7. The composition according to any one of the preceding claims, wherein the composition further comprises sugars.

8. The composition according to claim 7, wherein the fibre to lower molecular weight carbohydrates ratio by weight is of at least 10.

9. The composition according to any one of claims 1 to 8, wherein the Pleurotus fungus is Pleurotus ostreatus.

10. A 3D printing ink comprising a composition according to any one of claims 1 to 9.II. A method for the manufacture of a three-dimensional object comprisinga. providing a composition according to any one of claims 1 to 9; andb. forming the three-dimensional object byi. printing the composition using a 3D printer orii. extruding the composition.

12. A three-dimensional object comprising pellets comprising vegetal fibres embedded in a network of fungal hyphae.

13. A three-dimensional object according to claim 12, obtained or obtainable by the method of claim 11.

14. A culture medium for growing mycelium or a filamentous fungus comprising a source of vegetal fibres, preferably a source of cellulosic vegetal fibres, preferably a source of cellulose or hemicellulose, preferably a source of crystalline cellulose or hemicellulose, preferably a source of microcrystalline cellulose or hemicellulose, preferably a source of cellulose nanocrystals, cellulose nanofibers, microcrystalline cellulose or combinations thereof, more preferably okara.

15. A process for the preparation of a composition according to any one of claims 1 to 9, which comprises growing fungal mycelium or a filamentous fungus in a culture medium according to claim 16 under conditions suitable for the grown the mycelium or filamentous fungus.

Images