SE2350040A1

SE2350040A1 - A method of preparing a mycelium liquid extract and a dairy- free food product or food ingredient

Info

Publication number: SE2350040A1
Application number: SE2350040A
Authority: SE
Inventors: Anna Kristina Sofia Karlsson; Joan Lluch Casarramona; Paulo Gonçalves Teixeira; Sicong Zhu
Original assignee: Mycorena Ab
Priority date: 2023-01-17
Filing date: 2023-01-17
Publication date: 2024-07-18
Also published as: WO2024155225A1

Abstract

The present disclosure relates to a method of preparing a mycelium liquid extract comprising or consisting the steps of: a) providing a powder comprising filamentous fungi; suspending the powder comprising filamentous fungi of step a) in a liquid, thereby generating a suspension; adjusting the pH of the suspension of step b) to a value within the range of from 6.0 to 8.0, provided that the pH of the suspension of step b) is not already within said range; removing particles from the suspension of step b) or c), such as by filtration and/or centrifugation, thereby obtaining the mycelium liquid extract. The present disclosure also relates to a method of producing a dairy-free food product and/or dairy-free food ingredient comprising the mycelium liquid extract.

Description

A METHOD OF PREPARING A MYCELIUM LIQUID EXTRACT AND A DAIRY- FREE FOOD PRODUCT OR FOOD INGREDIENT Technical field id="p-1"

[0001] The present document relates generally to a method of preparing a mycelium liquid extract and a method of producing a daárgfﬂfree food product os' dairy-free food ingredient.

Background art id="p-2"

[0002] There is an increasing concern regarding the environmental impact of food production and consumption, particularly dairy products. Cattle growth for milk production is known for its high environmental impact and low resource efficiency. Replacement of dairy products by plant-based equivalents has been a rising trend among consumers for both health, environmental and ethical reasons, and several sources of plant-based milk replacement beverages are today available in the market. id="p-3"

[0003] The most common plant materials used for manufacturing of plant-based milk replacement products include soybeans, almonds, oats, rice and coconut. Even though all these sources are environmentally beneficial compared to milk, there are still some sustainability concerns when it comes to large scale supply of these. Fungi biomass has a lower climate impact compared to some of these plant-based sources in terms of carbon emissions, water use or land use. Health and nutritional benefits are also possible to achieve using a fungi-based material due to the presence of low sugar amounts, presence of healthy fibers composed of beta-glucans, and a complete protein with all the essential amino-acids. Some consumers are also concerned about allergenicity of selected plant-based sources, as well as possible presence of antinutrients. id="p-4"

[0004] Filamentous fungal mycelia, often referred to as mycoprotein, has been reported to be a high-quality protein. lt is also considered a non-allergen, contains a healthy amount of fibres and carbohydrates, and contains no known antinutrients. lts neutral taste is also an advantage to other plant-based sources, 108875SE since it reduces the need to add sugar and flavours in order to mask unpleasant taste notes. However, mycoprotein is a fibrous, resistant food product usually applied in meat replacements due to its natural form in a mycelial structure. id="p-5"

[0005] Furthermore, the production of dairy-free drinks using fungal biomass requires a series of steps to chemically and/or enzymatically break fungal cells in order to access the protein content and other nutrients inside fungal cells. Said steps may include a plurality of pH shifting steps and/or submitting the fungal cells to pH values of 10 or more, which may cause some undesirable flavor changes to the final product. Moreover, the pH shifting steps also requires a high quantity of chemical substances, which reduces the sustainability of the final dairy-free drink. Finally, the solubility of the fungal biomass subject to said chemically and/or enzymatically treatments may be compromised. id="p-6"

[0006] ln view of the above, the object of the present disclosure to provide an improved method for producing a mycelium liquid extract and/or a dairy-free food product and/or dairy-free food ingredient. Another object of the present disclosure is to provide a method which has a reduced environmental impact and is also simpler to reproduce than traditional methods for obtaining a mycelium liquid extract and/or a dairy-free food product and/or dairy-free food ingredient.

Summary [0007] The above problems are solved or at least mitigated by the present disclosure. id="p-8"

[0008] The present document is thus directed to a method of preparing a mycelium liquid extract comprising or consisting of the steps of: a) providing a powder comprising food-safe filamentous fungi; b) suspending the powder comprising or consisting of food-safe filamentous fungi in a liquid, thereby generating a suspension; 108875SE c) adjusting the pH of the suspension of step b) to a value within the range of from 6.0 to 8.0, such as about 7.5, provided that the pH of the suspension of step b) is not already within said range; d) removing particles from the suspension of step b) or c), such as by fi|tration and/or centrifugation, thereby generating a mycelium liquid extract. id="p-9"

[0009] Typical protein extraction protocols comprise pH shifting steps for increasing the protein yield, which requires the addition of several chemical substances for increasing and/or reducing the pH of the suspension comprising fungal biomass. However, the inventors have surprisingly found that a mycelium liquid extract having a good solubility is obtained by simply adjusting the pH of the suspension and removing insoluble particles from that suspension. The method as disclosed advantageously provides a simpler, cost effective and sustainable process for obtaining mycelium liquid extracts. Furthermore, by avoiding high pH values (e.g., pH values of 10 or more), undesirable flavor changes of the fungal biomass are avoided or significantly reduced. id="p-10"

[0010] Furthermore, the inventors have surprisingly found that suspending a powder comprising filamentous fungi in a liquid (e.g., water, buffer or any other formulated drink) in a pH of from about 6.0 to about 8.0, such as about pH 7 to about 8, such as about 7.5, and removing particles from the generated suspension creates a mycelium liquid extract having improved properties, including a smooth mouthfeel. id="p-11"

[0011] Adjusting the pH to from about 6.0 to about 8.0 provides a better protein extraction yield due to an increased amount of soluble fungal protein within said pH range. id="p-12"

[0012] The pH range of from 6.0 to 8.0 may be already present in the obtained suspension or may be reached by subsequent pH adjustment of the suspension. The skilled person knows which acids and/or alkalis are suitable for said pH adjustment, which include but are not limited to an inorganic base such as sodium 108875SE 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. id="p-13"

[0013] Particles, such as insoluble particles, are removed from the suspension by any technique known to the ski||ed person, including filtration and/or centrifugation. Such insoluble particles may comprise fungal cell wall debris, insoluble or denatured proteins, among other insoluble particles. The removal of the insoluble particles advantageously provides a myce|ium liquid extract which presents a high soluble fungal protein concentration and a smooth mouthfeel. The soluble fungal protein concentration may be from about 0.25 wt% to about 7.8 wt% based on the total weight of the myce|ium liquid extract. id="p-14"

[0014] The powder comprising filamentous fungi of step a) may comprise or consist of partly lysed fungal cells or fully lysed fungal cells. Said differences may generate a myce|ium liquid extract with different properties, such as different viscosity, soluble protein concentration and/or mouthfeel when used as such or for preparing a dairy-free food product. For a smoother myce|ium liquid extract, it is preferred that the fungal cells are at least partly lysed, more preferably fully lysed. id="p-15"

[0015] The myce|ium liquid extract may comprise soluble fungal proteins in an amount of from about 0.25 wt% to about 7.8 wt% based on the total weight of the myce|ium liquid extract. The fungal protein content is preferably a filamentous fungal protein content being soluble at a pH of from about 6.0 to about 8.0. id="p-16"

[0016] Previous to step a), a filamentous fungal biomass may be provided and subjected to a pre-treatment, such as dewatering and/or cell disruption. The powder comprising filamentous fungi may thus be prepared by subjecting a biomass of filamentous fungi to dewatering and/or cell disruption and may contain whole or broken fungal cells, in the latter case releasing intracellular components to the suspension, such as proteins, lipids, nucleic acids and carbohydrates. 108875SE id="p-17"

[0017] The pre-treatment of the filamentous fungal biomass may comprise a dewatering method selected from freeze drying, fluidized bed drying and/or low- temperature vacuum dehydration. Typically, any dewatering method known to the skilled person that does not increase the temperature of the filamentous fungal biomass to above about 17 °C may be used. Said dewatering methods advantageously provide a fungal biomass having functional fungal proteins, i.e., with a reduced amount of denatured proteins. Moreover, said dewatering methods may advantageously provide a higher fungal protein yield when compared with methods where higher temperatures are used. id="p-18"

[0018] Low-temperature vacuum dehydration may be performed at low temperatures, such as from about 0°C to about 17°C and within 4 mbar to 50 mbar. Said low-temperature vacuum dehydration advantageously reduces the water content of the filamentous fungal biomass. Furthermore, the water affinity and the ability to rehydrate of the powder are not compromised and dissolution is possible. id="p-19"

[0019] Furthermore, in case of freeze drying, said technique may be performed at -5°C to -35°C and vacuum values within 0.001 mbar to 6 mbar. id="p-20"

[0020] The pre-treatment of the filamentous fungal biomass may comprise a cell disruption method selected from milling, bead grinding and/or cryomilling. Said cell disruption methods may advantageously reduce the size of cell clusters and/or substantially break fungal cells. Said mechanical cell disruption method may be configured to generate average particle sizes within specific ranges. id="p-21"

[0021] The pre-treatment step may comprise dewatering of the filamentous fungal biomass followed by filamentous fungal biomass cell disruption. The de- watering step is preferably performed before the step of breaking the fungal cells, if such a step is present. lt is preferred that a dewatering method selected from freeze drying, fluidized bed drying and/or low-temperature vacuum dehydration is performed before a cell disruption method, such as milling, bead grinding and/or cryomilling. 108875SE id="p-22"

[0022] The powder comprising food-safe filamentous fungi may have an average particle size of about 500 um or less, preferably about 250 um or less, more preferably about 125 um or less, even more preferably about 74 um or less, most preferably about 37 um or less. Furthermore, the powder comprising filamentous fungi may be separated by size, such that the powder comprising filamentous fungi has an average particle size of about 500 um or less, preferably about 250 um or less, more preferably about 125 um or less, even more preferably 74 um or less, most preferably 37 um or less. Said separation by size may comprise any suitable method known to the skilled person, including filtering or by using a sieve. id="p-23"

[0023] The inventors have surprisingly found that powders comprising filamentous fungi having smaller average particle size comprise a higher concentration of soluble proteins, as shown in the experimental section. Thus, the fungal soluble protein concentration in the mycelium liquid extract may be adjusted by adjusting the average particle size of the powder comprising filamentous fungi. A specific average particle size or particle size range may be separated by sieving or any other technique known by the skilled person. Another advantage is that the protein concentration obtained is exceptionally high, for instance more than 30 g/L, which is 10x the protein amount in milk. Said high protein liquid mycelium extract may be a concentrated product to be diluted at the final destination, thereby simplifying and reducing costs of transportation. id="p-24"

[0024] The suspension of step b) may comprise 2 wt% to 30 wt%, such as 6 wt% to 20 wt%, of the powder comprising filamentous fungi, based on the total weight of the suspension. id="p-25"

[0025] The pH adjustment of step c) may be made by 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 108875SE acid. Said pH adjustment advantageously solubilize fungal proteins, thereby promoting a better protein extraction yield of the method. id="p-26"

[0026] The filamentous fungi may comprise or consist 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, lnonotus, Neurospora, Pennicillium, Pleurotus, Rhizopus, Trametes, Trichoderma, Tuber, Ustilago, Xylaria, or any combination thereof. Said food-safe filamentous fungi advantageously provide a liquid mycelium extract for human consumption and having a smooth mouthfeel. id="p-27"

[0027] The filamentous fungi may comprise or consist of a food-safe filamentous 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, X ylaria h ypoxion, or any combination thereof. Said food-safe filamentous fungi advantageously provide a liquid mycelium extract for human consumption and having a smooth mouthfeel. id="p-28"

[0028] lt is preferred that the method for preparing a mycelium liquid extract does not comprise a heating step before the suspension of the powder in a liquid in step b). ln other words, the powder comprising food-safe filamentous fungi of step a) has preferably not been subjected to a heat treatment. A step may be considered a heating step if the temperature of the filamentous fungal biomass reaches about 40 °C or more, such as 50 °C or 60 °C or more. id="p-29"

[0029] Heating a fungal biomass after harvesting is very common in the field of fungal food products due to requirements that fungal RNA are to be degraded to avoid its accumulation, which may cause for instance gout or kidney stones. The inventors have surprisingly found that skipping the heating step in the beginning of the method of providing a mycelium liquid extract provides a substantial increase in fungal soluble proteins. Therefore, by not heat treating the fungal biomass before the preparation of the mycelium liquid extract, a surprisingly higher protein 108875SE yield is obtained. Moreover, the heating step may denature fungal proteins and reduce/suppress protein functionality, which is avoided by the method for preparing the mycelium liquid extract not comprising a heating step. ln other words, the entire method for preparing the mycelium liquid extract takes place at low temperature, i.e. at temperatures which do not denature the proteins, and advantageously allows the proteins to be preserved in their native form. id="p-30"

[0030] Furthermore, the inventors have surprisingly found that the combination of a low temperature dewatering technique as previously described with the lack of a heating step when preparing the mycelium liquid extract generates a liquid mycelium extract comprising a high fungal protein yield. Said high fungal protein yield may be particularly higher for powders comprising filamentous fungi subjected to a separation by particle size, wherein smaller particle sizes comprise a higher fungal protein concentration. Said particle size of the powder comprising filamentous fungi may be 500 um or less, preferably about 250 um or less, more preferably about 125 um or less, even more preferably 74 um or less, most preferably 37 um or less. id="p-31"

[0031] The mycelium liquid extract may be substantially free of insoluble particles. Said insoluble particles may be substantially or completely removed due to the particle removal step d), which may e.g. include filtration, decantation, centrifugation steps and/or any other suitable technique known by the skilled person. The absence of insoluble particles contributes for the improved mouthfeel of the mycelium liquid extract. id="p-32"

[0032] The mycelium liquid extract has a fungal protein content of about 30 g/L or more, such as 35 g/L or more or 40 g/L or more. lt was surprisingly found that said method is able to generate a high concentration of filamentous fungal protein, which may be used for producing a ciaëry-free 'food product andior (fairy-free 'food ingredäent having high protein coatceaftration. id="p-33"

[0033] The mycelium liquid extract may not be subject to a further pH adjustment step than the pH adjustment of step c). By avoiding further pH adjustments, a mycelium liquid extract having a good solubility is advantageously 108875SE obtained. Moreover, the method is simpler to perform and uses fewer chemical substances, which also increases the sustainability of the method as a whole compared with prior art methods. Finally, some undesirable flavor changes to the final product are avoided by not performing further pH adjustments. id="p-34"

[0034] The present document is also directed to a mycelium liquid extract obtained or obtainable by the method as defined in any one of the preceding claims. The mycelium liquid extract has a fungal protein content of about 30 g/L or more, such as 35 g/L or more, or 40 g/L or more. The protein content may consist of mostly native proteins (not denatured), which advantageously provide a mycelium liquid extract having a high concentration of functional fungal proteins. The fungal protein may consist essentially of food-safe filamentous fungal protein soluble proteins. Said soluble proteins are preferably soluble at a pH of about 6 to about 8, such as about 7.5. Furthermore, said food-safe filamentous fungal proteins are preferably in a native state, i.e., not denatured. id="p-35"

[0035] The present document also discloses a mycelium liquid extract comprising a food-safe filamentous fungal protein content of 30 g/L or more, such as 35 g/L or more, such as 40 g/L or more, such as 50 g/L or more, such as 60 g/L or more, and wherein substantially all or all of said food-safe filamentous fungal proteins are in a native state. The inventors have surprisingly found that a filamentous fungal high protein concentration can be obtained, wherein most of said filamentous fungal protein is in a native state, i.e., not substantially denatured. Said filamentous fungal high protein concentration in a native state advantageously provides a mycelium liquid extract having an increased protein functionality when compared to standard methods, since standard methods typically involve high temperature steps and/or multiple pH shifts for extracting and purifying proteins, thereby denaturing fungal proteins. Additionally, the mycelium liquid extract comprising filamentous fungal high protein concentration has improved foaming and/or emulsifying properties, as shown in the experimental section. Furthermore, the mycelium liquid extract comprising filamentous fungal high protein concentration has an improved aftertaste due to for instance lack of multiple pH shifts, lack high temperature steps and/or lack of additives added to 108875SE said mycelium liquid extract. The skilled person knows how to measure whether the proteins are in a native or denatured state. id="p-36"

[0036] The food-safe filamentous fungal protein may consist essentially of soluble proteins. Said soluble proteins are preferably soluble at a pH of about 6 to about 8, such as about 7.5. id="p-37"

[0037] The mycelium liquid extract comprising a filamentous fungal high protein concentration may be obtained due to the method of preparing a mycelium liquid extract as described elsewhere herein. Said method comprises steps that do not substantially denature filamentous fungal proteins, including freeze-drying, dewatering, cell disruption and/or grinding. The skilled person knows which steps can be combined such that filamentous fungal protein in a native state is obtained. id="p-38"

[0038] The present document also relates to a method of producing a dairy~free food product andlor daisy-free food ingredäent cornprising or consisting of the steps of: a) providing a mycelium liquid extract, such as the mycelium liquid extract as defined herein; b) mixing at least one vegetable oil and optionally at least one additive with the mycelium liquid extract provided in step a), thereby generating an emulsion; c) simultaneously or sequentially heating and homogenizing the emulsion provided in step b), thereby obtaining a dairy-free food product and/or dairy- free food ingredient; and d) optionally acidifying said dairy-free food product. id="p-39"

[0039] Said dairy-free food product may be a stable emulsion, i.e., an emulsion that does not have visual phase separation of oil from aqueous phase after homogenization step. Said stable emulsion may last for about 7 days or more at 4 °C. 108875SE 1 1 id="p-40"

[0040] ln step c) in the method of producing a dašæry-froe food product or dairy- free food ingredient, the emulsion is heated from about 60 °C to about 90°C in order to for instance degrade RNA in the fungal biomass and/or inactivate fungal cells. Said step c) of simultaneously or sequentially heating and homogenizing the emulsion advantageously provide a more stable emulsion, as defined in the previous paragraph. id="p-41"

[0041] ln step c) in the method of producing a dairy-free food product or dairy- free food ingredient the heating step may be made prior to the homogenization step. Heating the emulsion before the homogenization step advantageously provide the adequate disruption of any flocculation of cell wall fragments and denatured proteins. The homogenization may be a high shear homogenization. id="p-42"

[0042] After step c) in the method of producing a dairy-free food product or dairy-rfreo food ingredient, the dairy-free food product may be cooled to a temperature range of from about 40 °C to 80 °C. Said cooling may be provided by simply allowing the temperature of the dairy-free food product and/or food ingredient to reach said temperature range. id="p-43"

[0043] The at least one vegetable oil may be canola oil, olive oil, sunflower oil, coconut fat, palm fat, peanut oil, soybean oil, shea oil, and/or any combination thereof. Each vegetable oil may have a different saturation degree and fatty acid length, which may confer different properties for the dairy-free food product and/or food ingredient, including taste, smell and texture. id="p-44"

[0044] After step b), the at least one vegetable oil may have a concentration of from 1 wt% to 35 wt%, based on the total weight of the emulsion. id="p-45"

[0045] The at least one additive may comprise a stabilizing agent such as native starch, modified starch, pectin, alginate, guar gum, gellan gum, xanthan gum, konjac gum, curdlan gum, carrageenan, cellulose derivative, lecithin, polysorbate, and/or any combination thereof. Said stabilizing agent may change the viscosity of the dairy-free food product and/or food ingredient such that it is similar to an equivalent dairy food product 108875SE 12 id="p-46"

[0046] ln step b) in the method of producing a dairy~free food procïuot or dairy» free food ingredient, an additive is added in an amount of from about 0.01 % w/w to about 5 % w/w, based on the total weight of the emulsion. Furthermore in step b) a flavorant may be added in an amount of from about 0.01% w/w to about 3% w/w, based on the total weight of the emulsion, said flavorant being for instance vanilla extract and/or cocoa powder. id="p-47"

[0047] The at least one additive may comprise a tastant, such as sugar and/or salt. Said flavorant and/or tastant may improve the overall taste and/or mouthfeel of the dairy-free food product, thereby increasing its acceptance by consumers. id="p-48"

[0048] Step d) of the method of producing a dairgßfroo food procïuot anolor cïairy» 'free food ingredient may comprise a fermentation step using a microorganism such as Lactobacillus sp. and/or Streptococcus sp., Biﬁdobacterium sp., Lacticaseibacillus sp., and/or an addition of a pH regulating agent such as lactic acid, citric acid, acetic acid, hydrochloric acid, ascorbic acid or any combination thereof. The microorganism may be selected from S. thermophilus, L. bulgaricus, L. acidophilus, B. lactis and/or L. paracasei. The final pH obtained in step d) may be from about 4 to about 5, such as about 4.4 to about 4.8. The aoidšfioatšon än step d) advantageously provide an emulsiora having an improved flavor andfor taste, which is similar to food products such as cooking cream, yoghurt or cream cheese. Said improved flavor and/or taste is preferably obtained by fermentation of the dairy-free food product and/or food ingredient with the above-mentioned microorganisms. id="p-49"

[0049] Tho dašry~free food produot aodior dairyßfree food ingredient :way be a drink, a concentrated solution for diluting before use, a replacement product for yoghurt, cooking cream, or cream cheese. id="p-50"

[0050] The present document also relates to a dairy-free food product and/or dairy-free food ingredient obtained or obtainable by any one of the methods as defined herein. 108875SE 13 id="p-51"

[0051] The dairy-free food product and/or dairy-free food ingredient may be a drink, a concentrated solution for diluting before use, a replacement product for yoghurt, cooking cream, or cream cheese. id="p-52"

[0052] The dairy-free food product and/or food ingredient may comprise properties obtained due to the method of producing said food product and/or food ingredient. For instance, said dairy-free food product and/or food ingredient does not comprise undesirable flavor changes that may occur when the fi|amentous fungus is submitted to multiple pH shifts, as in traditional methods. Furthermore, an increased concentration of soluble fungal proteins and which also have their functionality preserved may be present in said dairy-free food product. The increased protein functionality may be due to the lack of a heating step, the reduced number of pH shift steps or combinations thereof. id="p-53"

[0053] The dairy-free food product and/or dairy-free food ingredient may be a cooking cream that does not coagulate when boiled at about 100°C for about 5 min. id="p-54"

[0054] The dairy-free food product and/or daily-free food ingredient may comprise one or more of the microorganisms as defined elsewhere herein.

Brief description of drawinqs id="p-55"

[0055] Fig. 1 illustrates a method of preparing a mycelium liquid extract. id="p-56"

[0056] Fig. 2 illustrates a dairy-free drink prepared by using mycelium liquid extract (left side) and by using heat treated biomass powder (right side). id="p-57"

[0057] Fig. 3 illustrates a method of producing a daisy~free food product, such as a cream or a drink. [0058] Fig. 4 illustrates a method of producing a dairyf-free yoghurt. Definitions id="p-59"

[0059] Singular references do not exclude a plurality, i.e. terms such as "a", "an", "first", "second" etc. do not preclude a plurality. 108875SE 14 id="p-60"

[0060] "Dairy-free food product" or "dairy-free food ingredient" as used in the present document is an edible product for humans and/or other animals which does not comprise ingredient(s) derived from mammals, such as cows, sheeps or goats. id="p-61"

[0061] "Mycelium liquid extract" as used in the present document refers to a liquid extract of filamentous fungi that does not comprise any substantial amount of fibers and/or mycelium from the fungi. Substantial amount may be for instance more than 0.1 g/L of fibers and/or mycelium from the fungi. id="p-62"

[0062] ""Emulsion"" as used in the present document is regarded as an emulsion that does not have visual phase separation of oil from aqueous phase after homogenization step. The emulsion may be stable for about a week at 4 °C.

Detailed description Experimental section id="p-63"

[0063] Example 1. Production of fungal biomass id="p-64"

[0064] 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 (10^7 spores/mL) per 100 mL of growth media was added to each flask, followed by incubation at 30-35°C for 18-24h under shaking (100-150 rpm). id="p-65"

[0065]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 20min. Upon sterilization, a volume of 30 L of fungi culture obtained from a 16-24h 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 108875SE 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 24h and biomass was harvested after this period. 50L 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 24h. The obtained fungalbiomass was filtered and washed with tap water. [0066] Example 2: Production of mycelium liquid extract id="p-67"

[0067]Fungal biomass was freeze-dried and grinded into fine powder using an electric grain grinder (RRH-800A, Huanyu, China) to obtain freeze-dried powder comprising filamentous fungi. 20 g of freeze-dried powder comprising filamentous fungi was added to 280 g drinkable water, and the dispersion was adjusted to pH 7.5 to increase protein solubility. The dispersion was then centrifuged at 4500 rpm for 25 min (Thermoscientific Sorvall ST8) to remove big particles. The obtained supernatant was referred to as mycelium liquid extract. [0068]A general graph of production of mycelium liquid extract is listed in Figure 1. id="p-69"

[0069] Example 3: Soluble protein content in dispersion of mycelium protein isolate (MPI) and dispersion of freeze-dried non-heated (FDNH) powder comprising filamentous fungi id="p-70"

[0070]Mycelium protein isolate (MPI) was prepared using a conventional pH shifting method for extraction plant protein: 200 g biomass was produced and harvested as mentioned in Example 1. The obtained biomass was blended with 1 kg of water using kitchen blender (multitalent 8, BOSCH) for 30 s at maximum speed, and the slurry was adjusted and kept above pH 9 using 1M sodium hydroxide (NaOH). The slurry was left in room temperature for 1 hour to allow better protein solubilization. The slurry was aften/vards transferred into 50 ml falcon tubes and centrifugated at 4000 RPM for 30 min to separate the protein-rich supernatant from broken-cell pellets. The supernatant was collected and adjusted to pH 4 using 1 M hydrochloric acid (HCl) to sediment majority of the protein. The 108875SE 16 protein sediment was collected by centrifugation at 4000 RPM for 30 min and discarding liquid phase. The obtained protein sediment was resuspended into equal amount of water, and the suspension was adjusted to pH 7. The protein suspension was then freeze-dried using a freeze drier (Alpha 2-4 LSC, Martin Christ) and grinded with a spice grinder to obtain mycelium protein isolate powder. id="p-71"

[0071]Freeze-dried non-heated powder comprising filamentous fungi (FDNH) was prepared according to the following procedure: 1000 g biomass was produced and harvested as mentioned in Example 1. The obtained biomass was freeze-dried using freeze dryer (Alpha 2-4 LSC, Martin Christ) and aftervvards grinded into fine powders using an electric grain grinder (RRH-800A, Huanyu, China). [00?2]BCA analysis was performed to determine the soluble protein content of MPI suspension and FDNH suspension. Both MPI suspension and FDNH suspension were prepared by dispersing 0.1 g obtained powders in iii rrrl water and pH iiiras adjusted to pH if, followed by contrifugatiosi at 4000 rpm for 10 rnin. ššupernatant iii/ere colieoted and solubie protein content »vas deterrnined. Mycelium liquid extract made frorn iviPš and FÜNi-i irvere simiiar in soluble protein content tfviiich »vore both around 2.2 irigirni.. id="p-73"

[0073]Tab|e 1. soluble protein content in the mycelium liquid extract made from mycelium-protein-isolate and mycelium-freeze-dried powder.

Povr-tiei' type Scriubie protein cinretent in the niyfceiitsin iicgiricš extract (nig/'rnEQi The MPI method is a method known in the art that denatures proteins and/or generate protein aggregates due to the multiple pH shifts of said method. Said protein denaturation and/or aggregates do not return to their original native state, even after returning the mycelium liquid extract to a pH where most proteins would be soluble. On the other hand, the FDNH method does not significantly denature 108875SE 17 proteins since steps that promote protein denaturation, such as pH shifts and high temperature incubation, are avoided. Thus, FDNH method provided a mycelium liquid extract having soluble filamentous fungai proteins in a native state. [0074] Example 4: Nutritional content of the mycelium liquid extract id="p-75"

[0075]Funga| biomass was freeze-dried and grinded into fine powder using an electric grain grinder (RRH-800A, Huanyu, China) to obtain freeze-dried non- heated powder comprising filamentous fungi (FDNH). 200 g freeze dried powder comprising filamentous fungi was added to 800 g demi water, and the dispersion was adjusted to pH 7 to increase protein solubility. The dispersion was then centrifuged at 4500 rpm for 25 min (Thermoscientific Sorvall ST8) to remove big particles and a mycelium liquid extract was gained. The obtained mycelium liquid extract was sent to Eurofins Scientific testing laboratories for essential amino acids and micronutrient analysis. Table 2 shows the essential amino acids and micronutrient content in produced mycelium liquid extract. id="p-76"

[0076]Tab|e 2. Essential amino acids and micronutrient analysis in the mycelium liquid extract Parameter Result Unit Crude Protein Kjeldahl (Nx6.25) 4.23 g/100 g Tryptophan 0.0431 g/100 g Histidine 0.0893 g/100 g lsoleucine 0.182 g/100 g Leucine 0.240 g/100 g Lysine 0.282 g/100 g Phenylalanine 0.152 g/100 g Threonine 0.166 g/100 g Valine 0.221 g/100 g Methionine 0.0790 g/100 g 108875SE 18 Calcium (Ca) 330 mg/kg Zinc (Zn) 3.1 mg/kg Riboflavin (vitamin B2) 0.369 mg/100 g Pyridoxine (vitamin B6) 0.145 mg/100 g Niacin (vitamin B3) 2.78 mg/100 g Magnesium (Mg) 170 mg/kg Potassium (K) 2300 mg/kg id="p-77"

[0077] Example 5: Comparison of soluble protein content from FDNH powder comprising filamentous fungi with different particle sizes id="p-78"

[0078]Comparison of soluble protein content in the mycelium liquid extract made from freeze-dried non-heat-treated (FDNH) powder comprising filamentous fungi with different particle sizes. id="p-79"

[0079]Funga| biomass was harvested and freeze-dried using freeze dryer (Alpha 2-4 LSC, Martin Christ). The freeze-dried biomass was then grinded into fine powders using an electric grain grinder (RRH-800A, Huanyu, China). The obtained FDNH powder was separated by particle size using sieves with mesh sizes of 125 um, 74 um and 37 um. Four fractions of the grinded powder were obtained: G) between 74 um and 125 um, ® below 74 um, and (S) below 37 um. id="p-80"

[0080] Powder dispersions of 20 wt% sieved powders were prepared by blending 0.5 g powder into 2 g distilled water. The dispersions were well mixed and were adjusted to pH 7.2. The dispersions were centrifuged at 4500 rpm for 25 min (Thermoscientific Sorvall ST8) to remove big particles. The obtained supernatant was collected, and protein content was measured using bicinchoninic acid assay (BCA assay). 108875SE 19 id="p-81"

[0081]BCA assay results are listed in Table 3. Soluble protein content in the mycelium liquid extract depends greatly on the particle size of grinded powder. The smaller the particle size, the more soluble protein will be extracted in the mycelium liquid extract. Protein content in the mycelium liquid extract extracted from powders with particle size smaller than 37 um was 44 mg/mL, which is approximately 50% more than that obtained from powders of particle sizes between 74 um and 125 um. id="p-82"

[0082]Tab|e 3. soluble protein content in the mycelium liquid extract made from freeze dried fungi powder of different particle sizes.

Freeze-cšried p-awxfder Soluble protein c-arztent in the rnyceiiun: Eäquäd extract (mgínﬂ) E) ïïßi pm, 125 pan] Eüﬂtï 1G. 55 D IG pm, ?'4 pmi 41.411 6.2 E) ïü pm, 3? pm] 4436 1135 [0083] Example 6: Comparison of soluble protein content in the mycelium liquid extract id="p-84"

[0084]To compare the soluble protein content of the mycelium liquid extract made from powder comprising filamentous fungi produced with different drying and/or heat treatment, three different powders comprising filamentous fungi were produced from @freeze-dried non-heat-treated biomass (FDNH), @air-dried non- heat-treated biomass (ADNH), and ®freeze-dried heat-treated biomass (FDHT). id="p-85"

[0085]FDNH powder was produced by freeze-drying 1 kg of harvested biomass produced according to the procedure in Example 1, and dried using freeze drier (Alpha 2-4 LSC, Martin Christ). The obtained freeze-dried biomass was then grinded into fine powder using an electric grain grinder (RRH-800A, Huanyu, China) and went through sieves with a mesh size of 250 um. 108875SE id="p-86"

[0086]ADNH powder was produced by air drying 1 kg of harvested biomass produced according to the procedure in Example 1, and dried using an electronic food dehydrator (Gastronoma) at 40°C for 24h. The obtained freeze-dried biomass was then grinded into fine powder and went through sieves with a mesh size of 250 um. id="p-87"

[0087]FDHT powder was produced by harvesting biomass produced according to the procedure in Example 1 and heat up to 80°C for 15 min. The heat-treated biomass was then freeze-dried using freeze drier (Alpha 2-4 LSC, Martin Christ). The obtained freeze-dried biomass was then grinded into fine powder using an electric grain grinder (RRH-800A, Huanyu, China) and went through sieves with a mesh size of 250 um. id="p-88"

[0088] For each powder type, dispersions of 20 wt% sieved powders were prepared by blending 0.5 g powder into 2 g distilled water. The dispersions were well mixed and were adjusted to pH 7.2. The dispersions were centrifuged at 4500 rpm for 25 min (Thermoscientific Sorvall ST8) to remove big particles. The obtained supernatant was collected, and protein content was measured using bicinchoninic acid assay (BCA assay). id="p-89"

[0089] BCA assay results are listed in Table 4. Soluble protein content in the mycelium liquid extract depends largely on the drying and heating history of powder. Soluble protein content in the mycelium liquid extract extracted from ADNH (36 mg/mL) was smaller than soluble protein that from FDNH (42.1mg/mL). Soluble protein content in the mycelium liquid extract extracted from FDHT has the least soluble protein content (1 .6 mg/mL). id="p-90"

[0090]Tab|e 4. soluble protein content in the mycelium liquid extract made from freeze-dried non-heat-treated biomass (FDNH), air-dried non-heat-treated biomass (ADNH), and freeze-dried heat-treated biomass (FDHT). rnyceiitirta povvfier Soluble protein content in the rnyceiiurrt iicgtsicš extract iintgirnL) 108875SE 21 FpNi-a 42.1 i 078. ABNH 3:51; FDHT m5 i 0.01 [0091] Example 7: mycelium-based dairy-free drink using mycelium liquid extract id="p-92"

[0092]3 g canola oil was added to 97 g of mycelium liquid extract produced as described in Example 1, and the whole mixture was premixed at 90°C for 150 s in Thermomix® TM6 at speed level 2. The obtained liquid was cooled to 60°C before 6 g vanilla sugar powder was added in. The flavored liquid was then homogenized using a high shear blender (CAT X120 (33000 RPM)) for 30 seconds to reach a stable emulsion. The emulsion has appearance and texture resembling milk. A general graph of production of dairy-free drink is in listed in Figure 3. id="p-93"

[0093]Another sample was made of 3 g canola oil and 97 g of supernatant obtained from heat-treated biomass powder described in Example 3. Same dairy- free drink procedure was applied as stated above. The obtained emulsion phase separated, indicating a poorer and less stable emulsion compared to the one using mycelium liquid extract. A comparison photo of dairy-free drink using mycelium liquid extract and using heat treated biomass powder is listed in Figure 2. id="p-94"

[0094] Example 8: mycelium-based dairy-free cooking cream id="p-95"

[0095]0.04 g gellan gum, 0.08 g xanthan gum, 0.08 g sunflower lecithin and 13 g canola oil was added to 87 g of mycelium liquid extract produced as described in Example 1, and the whole mixture was premixed at 90°C for 150 s in Thermomix® TM6 at speed level 2. The obtained liquid was cooled to 60°C and homogenized using a high shear blender (CAT X120 (33000 RPM)) for 30 seconds to reach a stable emulsion. The stable emulsion has a mouthfeel resembling cooking cream. The cooking cream is stable upon pan boiling at 100°C for 5 min without coagulation. A general graph of production of dairy-free cream is in listed in Figure 3. id="p-96"

[0096] Example 9: mycelium-based dairy-free yoghurt id="p-97"

[0097]0.6 g pectin (CM901) was mixed with 3 g canola oil and together added to 90.4 g of mycelium liquid extract. 6 g sucrose was added to the obtained mixture. The whole mixture was premixed at 90°C for 120 s. The obtained liquid was cooled to 60°C and homogenized using a high shear blender (CAT X120) at 33000 RPM for 30 seconds to reach a stable emulsion. The emulsion was cooled down to 43 °C. Culture (VEGE061, Danisco) containing strains S.thermophilus, L.bulgaricus, L.acidophilus, B.lactis, L.paracasei was added to the mixture. The whole mixture was then fermented at 43 °C until pH reached 4.8. The fermented emulsion has a mouthfeel resembling yogurt. A general graph of production of dairy-free yoghurt is listed in Figure 4. id="p-98"

[0098]Viscosity of different commercial dairy-based yoghurt and plant-based yoghurt as well as the above prepared mycelium-based dairy-free yoghurt were measured using a viscosity meter (IKATM ROTAVISC lo-vi, Germany) at rotating speed of 100 rpm. The results are shown in Table 5. Mycelium-based yoghurt have similar viscosity as dairy-based yoghurt. id="p-99"

[0099]Tab|e 5. Viscosity of commercial yoghurts, plant-based dairy-free yoghurts and mycelium-based dairy-free yoghurt prepared using mycelium liquid extract. lt 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.

Claims

1. A method of preparing a mycelium liquid extract comprising or consisting of the steps of: a) providing a powder comprising food-safe fiiamentous fungi; b) suspending the powder comprising food-safe fiiamentous fungi of step a) in a liquid, thereby generating a suspension; c) adjusting the pH of the suspension of step b) to a value within the range of from 6.0 to 8.0, such as about 7.5, provided that the pH of the suspension of step b) is not already within said range; d) removing insoluble particles from the suspension of step b) or c), such as by filtration and/or centrifugation, thereby obtaining a mycelium liquid extract.

2. The method according to claim 1 wherein the mycelium liquid extract comprises soluble fungal proteins in an amount of from about 0.25 wt% to about 7.8 wt% based on the total weight of the mycelium liquid extract.

3. The method according to claim 1 or 2, wherein said powder comprising food-safe fiiamentous fungi of step a) is prepared by providing a fiiamentous fungal biomass and subjecting said fiiamentous fungal biomass to a pre-treatment comprising a dewatering method selected from freeze drying, ﬂuidized bed drying, and/or low-temperature vacuum dehydration, and/or a cell disruption method selected from milling, bead grinding and/or cryomilling.

4. The method according to claim 3, wherein said pre-treatment step comprises dewatering of the fiiamentous fungal biomass followed by fiiamentous fungal biomass cell disruption.

5. The method according to any one of the preceding claims, wherein the powder comprising food-safe fiiamentous fungi of step a) has an average particle 108875SE size of about 500 um or less, such as about 250 um or less, about 125 um or less, about 74 um or less or about 37 um or less.

6. The method according to any one of the preceding claims, wherein the suspension of step b) comprises 2 wt% to 30 wt%, such as 6 wt% to 20 wt%, of the powder comprising filamentous fungi, based on the total weight of the suspension.

7. The method according to any one of the preceding claims, wherein the fungi of said filamentous 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, lnonotus, Neurospora, Pennicillium, Pleurotus, Rhizopus, Trametes, Trichoderma, Tuber, Ustilago, Xylaria, or any combination thereof.

8. The method according to any one of the preceding claims, wherein said method does not comprise a heating step.

9. The method according to any one of the preceding claims, wherein the mycelium liquid extract has a fungal protein content of about 30 g/L or more, such as 35 g/L or more or 40 g/L or more.

10. A mycelium liquid extract obtained or obtainable by the method as defined in any one of the preceding claims.

11. A mycelium liquid extract comprising a food-safe filamentous fungal protein content of 30 g/L or more, such as 35 g/L or more, such as 40 g/L or more, such as 50 g/L or more, such as 60 g/L or more, and wherein substantially all or all of said food-safe filamentous fungal proteins are in a native state.

12. A method of producing a dairy-free food product and/or dairy-free food ingredient comprising or consisting of the steps of: a) providing a mycelium liquid extract as defined in claim 10 or 11; 108875SEb) mixing at least one vegetable oil and optionally at least one additive with the mycelium liquid extract provided in step a), thereby generating an emulsion; and c) simultaneously or sequentially heating and homogenizing the emulsion of step b), thereby obtaining a dairy-free food product and/or dairy-free food ingredient.

13. The method of producing a dairy-free food product and/or dairy-free food ingredient according to claim 12, wherein in step c) the emulsion is heated at a temperature from about 60 °C to about 90°C.

14. The method of producing a dairy-free food product and/or dairy-free food ingredient according to claim 12 or 13, wherein in step c) the heating step is made prior to the homogenization step.

15. The method of producing a dairy-free food product and/or dairy-free food ingredient according to any one of c|aims 12-14, wherein the emulsion of step b) comprises about 1 wt% to about 35 wt% of the at least one vegetable oil, based on the total weight of the emulsion.

16. The method of producing a dairy-free food product and/or dairy-free food ingredient according to any one of c|aims 12-15, wherein in step b) an additive is added in an amount of from about 0.01 % w/w to about 5 % w/w, based on the total weight of the emulsion.

17. The method of producing a dairy-free food product and/or dairy-free food ingredient according to any one of c|aims 12-16, further comprising a step d) of acidifying the dairy-free food product and/or dairy-free food ingredient of step c).

18. The method of producing a dairy-free food product and/or dairy-free food ingredient according to claim 17, wherein step d) comprises: a fermentation using a microorganism such as Lactobacillus sp. Streptococcus sp., Biﬁdobacterium sp., and/or Lacticaseibacillus sp.; and/or 108875SEan addition of a pH regulating agent such as Iactic acid, citric acid, acetic acid, hydrochloric acid, ascorbic acid or any combination thereof.

19. The rnethod according to any one of claims 12-18, wherein said dairy- free food product and/or dairy-free food ingredient is a drink, a concentrated solution for di|uting before use, a replacement product for yoghurt, cooking cream, or cream cheese.

20. A dairy-free food product and/or a dairy-free food ingredient obtained or obtainable by the method as defined in any one of claims 12-19.