The present disclosure relates to a proteinaceous powder comprising fungal biomass or proteins derived from fungal biomass and a method for preparing the proteinaceous powder. BACKGROUND

Proteins are important macronutrients as well as crucial ingredients in food and cosmetics applications where they are used as for example emulsifiers, stabilisers, gelling agents, or fat replacers. Traditionally, proteins from whey, a by-product in dairy processing, has been one of the most common proteins for different applications but also other animal proteins have been used. However, there is an increasing demand from consumers today for vegan alternatives to animal-based products due to health, sustainability, or ethical reasons. Therefore, traditional proteins from milk or egg are therefore being replaced with other protein sources. Common plant-based alternatives to animal proteins are for example derived from soybean, pea, hemp, or pumpkin seeds. An issue with some of the plant-based alternatives are that they are lacking essential amino acids needed in the human diet, as human bodies cannot produce these amino acids themselves. Furthermore, plant-based proteins can also be a source of allergens, where for example tree nuts, soy, gluten, and lupin are on EFSA’s list of 14 allergens required to be declared on labels in the European Union.

There is thus still need for an improved vegan based proteinaceous powder comprising a high yield of proteins, high-quality protein and/or a reduced number of allergens. SUMMARY

One or more of the above objects may be achieved with a proteinaceous powder in as described herein and a method for producing the proteinaceous powder as described herein.

A proteinaceous powder according to the present disclosure comprises fungal biomass of filamentous fungi and/or proteins derived from fungal biomass of filamentous fungi, a protein content of the proteinaceous powder being within the range of from 70 wt.% to 95 wt. % based on the total weight of the proteinaceous powder. Filamentous fungi are used for producing the fungal biomass, i.e. the fungal biomass of the present document is a fungal biomass of filamentous fungi, even when this is not explicitly mentioned. The remainder of the proteinaceous powder comprises e.g. remains of the fungal cells (e.g. fibres, carbohydrates, protein) and ash, i.e. salts and other inorganic material.

Preferably, the protein content of the proteinaceous powder is within the range of from 75 wt.% to 95 wt. %. Optionally, the protein content of the proteinaceous powder is within the range of from 75 wt.% to 80 wt. %. Mycoproteins (i.e. proteins derived from fungi) generally have a high protein content of about 50% in dry weight and may therefore be particularly suitable for use as an alternative to animal-based proteins. Additionally, mycoproteins contain all nine essential amino acids and as much as 54% of the amino acid content in mycoproteins constitutes of essential ones.

Furthermore, since mycoproteins are not linked to severe food allergies it is advantageous to use in food applications. Mycoproteins also have environmental benefits over some plant protein sources, especially since it does not require agricultural land to produce and overall has a low land requirement and water usage per kilogram of product.

It has been found by the present inventors that mycoproteins are an excellent source of vegan proteins, however, the mycoproteins also comprises dietary fibres. Due to the high fibre content, it is very hard to exceed a protein content of 60% protein per dry weight of fungal biomass. In the proteinaceous powder according to the present disclosure the protein content, based on the powder dry weight, is within the range of from 75 % wt. to 95 % wt.

A content of essential amino acids in the proteinaceous powder of the present disclosure may be 30 wt. % or more of the fungal protein content of the proteinaceous powder. This provides a highly nutritional and beneficial proteinaceous powder. The proteinaceous powder is also considered a complete protein source, meaning it contains all the essential amino acids in sufficient amounts to be consumed as the only protein source in a healthy diet. Furthermore, the proteinaceous powder according to the disclosure provides such a complete amino acids profile that no essential amino acid supplementation is required, which simplifies the proteinaceous powder production process and reduces production costs. According to the present document, all the content of essential amino acids in the proteinaceous powder may be derived from the filamentous fungi themselves, i.e. no addition of essential amino acids is necessary.

A content of branched-chain amino acids may be 15 wt. % or more of the fungal protein content of the proteinaceous powder. This is of particular benefit since branched amino acids have been shown highly beneficial and preferentially used in muscle building by the human body. As such, proteins to be used in sports nutrition should have high contents of branched amino acids.

The solubility of the proteinaceous powder in water may be within the range of from 10% to 60%, for instance as measured according to the protein solubility assay as described herein. The high solubility of the proteinaceous powder according to the present disclosure, and compared to the fungal biomass as such, makes the powder particularly suitable for use in food formulations in which a mixture of the proteinaceous powder with water prior to preparing the food formulation is required or for use of the proteinaceous powder in aqueous-containing cosmetical formulations. By isolating the fungal proteins instead of using the whole fungal biomass, the solubility may be increased.

The wettability of the proteinaceous powder is within the range of from 50 mg/min to 200 mg/min, for instance as measured according to the force imbitition measurement method as described herein. The high wettability of the proteinaceous powder according to the present disclosure, and compared to the fungal biomass as such, makes the powder particularly suitable for use in food formulation in which a mixture of the proteinaceous powder with water prior to preparing the food formulation is required or for use of the proteinaceous powder in aqueous-containing cosmetical formulations.

The fungal biomass used to prepare the proteinaceous powder of the present document may be obtained from food-safe filamentous fungi, such as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof. The food-safe filamentous fungi may for example be fungi of the species Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus, Rhizopus microsporus, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion or any combination thereof. Said genera and species of filamentous fungi provide a source of generally recognized as safe (GRAS) filamentous fungi that may readily be a source of high-quality proteins for the food industry.

The present disclosure also relates to an oil in water emulsion comprising the proteinaceous powder according to the present disclosure, the oil in water emulsion comprising oil droplets and an aqueous phase. The fungal proteins in the proteinaceous powder function as an emulsifier and will arrange in the interface between the oil and the water phase, thereby stabilizing the emulsion.

The oil droplets in the oil in water emulsion may each have an oil droplet size of less than 100 pm, optionally less than 10 pm. The oil droplet size may be measured as the (mean) diameter of said oil droplet size. A proteinaceous powder according to the present disclosure has been seen to function well as emulsifier and provides a stable oil in water emulsion with oil droplets of less than 100 pm.

The proteinaceous powder may have an emulsification capacity of from 0.1 to 1 m ² /mg of protein, preferably within the range of from 0.1 to 0.6 m ² /mg protein.

The oil in water emulsion may have a foaming stability of at least 300 min before collapse of foam, for instance as measured according to the foam height measurement method as disclosed herein. A higher foaming stability advantageously provide an oil in water emulsion for use in food products where foaming is desired, such as egg or cream replacing, including foamed milk drinks, cappuccino, or merengue. The present document also discloses a method of preparing a proteinaceous powder comprising fungal biomass of filamentous fungi and/or proteins derived from fungal biomass of filamentous fungi, such as the proteinaceous powder described herein, the method comprising the steps of: a) providing an aqueous suspension of a fungal biomass of filamentous fungi, said aqueous suspension of a fungal biomass of filamentous fungi having a pH within the range of from 6 to 14, optionally within the range of from pH 9 to 11 ; b) performing a breakage of the fungal cells in the aqueous suspension of fungal biomass; c) optionally adjusting the pH of the suspension to a pH within the range of from 11 to 14, if the pH is not within said range, thereby solubilizing the proteins in the suspension; d) collecting a supernatant of the suspension obtained in step b) or c); e) precipitating the proteins in the supernatant of step d) by adjusting the pH of the supernatant to a pH within the range of from 3 to 5, thereby generating a suspension; f) optionally removing water from the suspension of step e), such as by filtration or by centrifugation, to prepare a dewatered suspension; and g) preparing a proteinaceous powder from the suspension of step e) or f) by further reducing the water content, such as by freeze drying, spray drying or chilled vacuum drying. Step b) may be performed by an enzymatic treatment such as by adding chitinase and/or b-glucanase, an ultrasonic treatment and/or mechanical treatment such as by grinding, cutting, high-pressure homogenisation, high-shear homogenisation. If two or more of these methods are used to break the cells, they can be performed in any order. The breakage of fungal cells enriches the suspension with fungal intracellular proteins, which allows obtaining a proteinaceous powder having a high protein concentration.

The pH adjustment in step c) may be to about pH 12. A higher pH advantageously solubilizes most of fungal proteins, thereby enriching the solution with said fungal proteins. The supernatant in step d) may be obtained by e.g. allowing the suspension of step b) or c) settle and/or by performing a centrifugation or filtration to pellet non-soluble constituents, such as remnants of cell walls etc. In this way the solubilised fungal proteins are separated from the rest of the fungal biomass, such as cell walls etc. Thus, in step d) the soluble proteins are separated from non-soluble matter and only the soluble fraction, comprising fungal proteins, is continued with in step e). This separation of soluble and non-soluble matter may e.g. take place via sedimentation, filtration, centrifugation and the like.

The pH adjustment to an acidic pH in step e) advantageously provides a fungal protein precipitation since the average isoelectric point of fungal proteins being within the pH of 3 to 5. Said precipitated fungal protein may be collected and isolated as a proteinaceous powder by dewatering and/or drying steps. In this way the proteins may be separated from other soluble components present in the supernatant of step d).

In step g), the water content is reduced to prepare a proteinaceous powder. Such reduction of water content may e.g. be performed by freeze-drying, chilled vacuum drying and/or spray-drying as using these techniques as is known to the skilled person. Preferably, step g) is preceded by a step f) of removing water, such as by filtration or centrifugation, from the precipitated proteins obtained in step e).

Regarding step g), the proteinaceous powder may e.g. be produced by the chilled vacuum drying, wherein the water content is reduced by a low-temperature vacuum dehydration, typically within 4 mbar to 50 mbar and 0 to 17 °C. Furthermore, in case of freeze drying, said technique may be performed on frozen material at -5°C to -35°C and e.g. vacuum values within 0.001 mbar to 6 mbar. Spray-drying is performed as generally known in the art by injecting the suspension containing the protein precipitate in the spray dryer. The pH of the suspension containing the protein precipitate may be adjusted up to about 7 before the drying step in some instances. In this way a neutral pH may be retained once the protein precipitate is dispersed in a liquid.

The present disclosure also relates to a method of preparing a proteinaceous powder, such as a proteinaceous powder disclosed elsewhere herein, the method comprising the steps of: a) providing a fungal biomass of filamentous fungi in an aqueous solution, such as cultivating fungi by liquid fermentation in an aerated bioreactor or by a solid state fermentation, to obtain a filamentous fungi biomass; b) adjusting the pH of the suspension to a pH within the range of from 6 to 14, optionally within the range of from 9 to 11 , c) optionally, adjusting the pH of the suspension to a pH within the range of from 4 to 7, preferably within the range of from 5 to 7, and subjecting the suspension to enzymatic breakage of the fungal biomass cell wall structure, such as by adding chitinase and/or b-glucanase to the suspension; d) optionally, subjecting the suspension to an ultrasonic treatment; e) subjecting the suspension to a mechanical breaking of the fungal cell walls, such as by grinding, cutting, high-pressure homogenisation and/or high-shear homogenisation; f) adjusting the pH of the suspension to a pH within the range of from 11 to 14, such as about 12, provided the pH is not within said range after step e); g) collecting the suspension from step f) and adjusting the pH to a pH within the range of from 3 to 5 and optionally removing water from the suspension, such as by filtration or by centrifugation; and h) preparing a proteinaceous powder from the suspension resulting from step g) by reduction of water content, such as by freeze drying, spray drying or chilled vacuum drying.

In this method, steps c), step d) and step e) may be carried out in any mutual order, i.e. such that for example step e) of subjecting the suspension to a mechanical breaking of the fungal cell walls may be carried prior to or after step d) or c) and step e) may be performed prior to step d). Step f) of adjusting the pH of the suspension to a pH within the range of from 11 to 14 provides enhanced protein solubilization. As is explained above, this allows separation of the fungal proteins from the rest of the fungal biomass, such as cell walls etc. Thus, in step f) the soluble proteins are separated from non-soluble matter and only the soluble fraction, comprising fungal proteins, is continued with to step g). This separation of soluble and non-soluble matter may e.g. take place via sedimentation, filtration, centrifugation and the like. In step g) the adjustment of the pH provides protein precipitation and enhances concentration of the proteins. In this way the proteins may be separated from other soluble components present in the suspension of step f). Each of steps c) and d) are optional. The method may thus comprise none of these steps, only one of the steps or both steps. The pH of the suspension in step c) may thus be reduced when subjecting the cells of the fungal biomass to the enzymes for breaking the fungal cell walls and further enhance the extraction of protein. The pH of the suspension is advantageously adjusted to a value within the range of from 4 to 7, preferably within the range of from 5 to 7, to ensure a high enzyme activity and a high protein yield. Regarding step h), the details of these drying methods are as already disclosed above. The proteinaceous powder may thus e.g. be produced by the chilled vacuum drying, wherein the water content is reduced by a low-temperature vacuum dehydration, typically within 4 mbar to 50 mbar and 0 to 17 °C. Furthermore, in case of freeze drying, said technique may be performed on frozen material at -5°C to -35°C and vacuum values within 0.001 mbar to 6 mbar. Spray-drying is performed as generally known in the art by injecting the suspension containing the protein precipitate in the spray dryer. The pH of the suspension containing the protein precipitate may be adjusted up to about 7 before the drying step in some instances.

The fungal biomass provided in step a) in the methods of the present document may be fresh, frozen or dried, such as controlled low vacuum dried or freeze-dried, fungal biomass. Furthermore, the fungal biomass used in step a) may be in powdered form. The powdered fungal biomass may be obtained by first drying the fungal biomass and then breaking the dried mycelium and the cells into small particles in a pulverization step.

The fungal biomass used in step a) (whether in fresh, frozen or dried form) may e.g. be prepared by cultivating fungi by liquid fermentation in an aerated bioreactor or by a solid state fermentation. If solid state fermentation is used, the fungal cells are scraped off the surface of the growth medium and added to an aqueous solution to provide an aqueous suspension of fungal biomass. Furthermore, adjusting the pH of the aqueous suspension comprising the fungal biomass to within 6 to 14, preferably 9 to 11, may loosen the fungal cell pellet-like structure into a preferable mycelial state.

It has been found by the present inventors that mycoproteins are an excellent source of vegan proteins. But the mycoproteins also comprises dietary fibres. Due to the high fibre content, it is very hard to exceed a protein content of 60% protein per dry weight of fungal biomass. In the method according to the present disclosure a surprisingly high amount of proteins may be separated and concentrated from the dietary fibres of the mycoprotein and by a subsequent drying step a proteinaceous powder with an extended shelf-life which may be stored in higher storage temperatures. The proteinaceous powder is particularly advantageous for use in different food or cosmetics applications.

The present document is also directed to a proteinaceous powder comprising fungal biomass of filamentous fungi and/or proteins derived from fungal biomass of filamentous fungi obtained or obtainable by a method for producing a proteinaceous powder as disclosed elsewhere herein.

The present disclosure also relates to a cosmetic composition comprising a proteinaceous powder and/or an oil in water emulsion as described elsewhere herein. The fungal proteins advantageously provide an animal free source of high-quality proteins in high concentrations for use in cosmetic formulations.

The present document also discloses a food product comprising the proteinaceous powder and/or comprising the oil in water emulsion disclosed herein. The food product may e.g. be a drinkable product, a meat-replacement product, a seafood replacement product, noodles, and/or a powder product. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a bar chart illustrating a comparison of the water holding capacity of fungal biomass, dried with different drying methods;

Fig. 2 is a bar chart illustrating the protein content in the liquid phase after different cell degradation treatments, the different letters indicating significant difference (P < 0.05);

Fig. 3 is a diagram illustrating the extraction of protein at different pH; Fig. 4 is a bar chart illustrating the protein extraction yield at different pH;

Fig. 5 is a bar chart illustrating the forced imbibition rate of the freeze-dried biomass (FDB) powder, freeze-dried protein isolate (FDPI) powder, and spray-dried protein isolate (SDPI) powder, wherein different letters indicate significant differences (p<0.05);

Fig. 6 is a diagram illustrating particle size distribution of oil droplets stabilised by freeze dried protein isolate from fungal biomass;

Fig. 7 are two bar charts illustrating the oil droplet size of protein isolate stabilised emulsions, comparing freeze dried samples (left) and spray-dried samples (right); Fig. 8 are two diagrams illustrating the particle size distributions of emulsions with 128 mg powder/mL oil. The left image is mixed in high shear mixer and right image is mixed with high-pressure homogeniser. FDB; freeze-dried biomass, HPH; high-pressure homogeniser, FDPI; freeze-dried protein isolate, SDPI; spray-dried protein isolate;

Fig. 9 is a bar chart illustrating the emulsifying capacity of different emulsions.

FDB; freeze-dried biomass, FDPI; freeze-dried protein isolate, SDPI; spray- dried protein isolate; Fig. 10 is a bar chart illustrating protein solubility from different emulsion formulations. FDB; freeze-dried biomass, HPH; high-pressure homogeniser, FDPI; freeze-dried protein isolate, SDPI; spray-dried protein isolate;

Fig. 11 is a diagram showing the foaming properties of fungal biomass powder and protein isolates. FDB; freeze-dried biomass, FDPI; freeze-dried protein isolate, SDPI; spray-dried protein isolate;

Fig. 12 is bar chart illustrating dry matter content in supernatant and retentates from different extraction methods;

Fig. 13 is a bar chart illustrating protein content in supernatant from different extraction methods;

Fig. 14 is a bar chart illustrating the content of essential amino acids in different plant- and animal-based protein sources; and Fig. 15 is a bar chart illustrating the content of branched-chain amino acids in different plant- and animal-based protein sources. DETAILED DESCRIPTION

In the context of the present document, the terms “fungi biomass”, “fungal biomass” and the like may be used interchangeably. The fungi of the fungal biomass are filamentous fungi. The invention describes protein extraction from filamentous fungi biomass (containing mycoproteins) to obtain a proteinaceous powder. The protein extracts as described herein are composed of from 70 wt.% to 95 wt.% protein, or preferably 75 wt.% to 95 wt.% protein, based on the total weight of the proteinaceous powder, and can be used in formulations within food or cosmetics industries where an addition of protein is desired. For creating this isolate, mycelium biomass from fungal species with from 45 wt.% to 60 wt.% protein content in the dry mass may be used to ensure a high protein content in the extract.

Furthermore, the content of essential amino acids of said proteinaceous powder may be for instance 30 wt.% or more of a total protein content of the proteinaceous powder. This provides a highly nutritional and beneficial proteinaceous powder, thereby avoiding supplementation of essential amino acids to the proteinaceous powder. The proteinaceous powder may comprise a content of branched-chain amino acids is 15 wt. % or more of a total protein content of the proteinaceous powder, which is particularly advantageous for increasing muscle building and for providing an improved proteinaceous powder for the sports nutrition field in general.

Any method commonly used for growing fungi may be used to produce the fungal biomass used in the present document. The fungal biomass may e.g. be obtained by cultivating fungi by a liquid or solid state fermentation. For example, the fungi may be grown under aerobic submerged fermentation conditions in a closed fermentation vessel with a liquid substrate medium with stirring. The fungi may also be grown under solid-state fermentation. The fungal cells may e.g. be inoculated either from plates or from a spore suspension into a pre-culture, which can be a flask or liquid bioreactor up to 30L working volume. The culture media may advantageously contain a carbon source, nitrogen source, phosphates and sulphates, and a trace metal solution to enable growth of the fungi and obtaining of the biomass. The preculture volume is then e.g., used to inoculate a production bioreactor, or a subsequent seed bioreactor with a volume 10-50 times larger than the preculture volume, with a culture media using the same pre-requirements as the preculture media. The bioreactor conditions may be kept at a pH between 4.0 and 6.0, with an aeration of at least 0.1 vvm and stirred using propeller blades. The fungi are preferably grown in a mycelial state, as opposed to pellet-like structures, even if it is possible to also use a pellet-like structure of the fungi. The growth can be done in a batch mode, in which fungi are harvested from the production tank after a 24h process or until less than 5% of the remaining carbon source is present, or as a continuous process, in which biomass is removed at a constant rate that matches growth and nutrient feed rate, or a semi-batch mode where biomass is partially harvested from one or several reactor and then such reactor(s) are filled with new media to continue fungal growth.

The fungal biomass may be used directly, i.e. as fresh fungal biomass, or frozen or dried before use as disclosed elsewhere herein.

Production of the fungal mycelium biomass entails the use of a species of filamentous fungi that can form mycelial structures in liquid fermentation conditions such as the ones belonging to the Zygomycota and Ascomycota phylum (excluding yeasts). Some exemplary fungi species that may be used to generate fungal mycelium biomass are as fungi of the genera Rhizopus, Neurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus, Cordyceps, Ustilago, Tuber, Fusarium, Pennicillium, Xylaria, Trametes, or any combination thereof. Examples of food safe filamentous fungal species include, but are not limited to, Aspergillus oryzae, Rhizopus oryzae, Rhizopus oligosporus, Rhizopus microsporus, Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti, Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion or any combination thereof.

For example, a fungal species selected from Neurospora intermedia, Neurospora crassa, Aspergillus oryzae, Rhizopus microsporus, Rhizopus oryzae and/or Rhizopus oligosporus may be used. For example, fungi of the genus Rhizopus may be used, such as of the species Rhizopus microsporus, Rhizopus oryzae and Rhizopus oligosporus.

The present document discloses a method of preparing a proteinaceous powder comprising proteins derived from fungal biomass. In said method, firstly, an aqueous suspension of fungal biomass is provided, said aqueous suspension having a pH within the range of from 6 to 14, optionally within the range of from 9 to 11. Said pH range can be considered a pre-treatment of the fungal biomass, which promotes a loosening the fungal cell pellet-like structure into a preferable mycelial state.

The fungal biomass can be then broken/disrupted using many techniques known by the skilled person, such as enzymatic treatment, mechanical breaking, ultrasonic treatment or combinations thereof. In case an enzymatic treatment step is performed, the pH of the suspension should be preferably adjusted to a range wherein said enzymes have maximum activity, for instance within the range of from 4 to 7 or 5 to 7. In case of mechanical breaking, suitable techniques known by the skilled person include grinding, cutting, high-pressure homogenisation, high-shear homogenisation, and combinations thereof. The skilled person knows how to adjust the parameters used in these techniques in order to effect a breakage of the fungal cells.

One example of a method to increase protein content is to dry fungal biomass to create a biomass powder. This powder can be created by for example freeze drying, which is a mild treatment that does not denature the proteins and thereby keeps their functionality. The powder can thereafter be resuspended in liquid or used as a dry ingredient. However, in such method a protein content of not more than 60% is obtained.

To further enhance the protein extraction in a method according to the present invention, intracellular proteins can be extracted after the fungal cells have been destroyed. This is achieved in the method of the present document by for instance mechanically breaking the cells in for example a high-pressure homogeniser. To further enhance the extraction of protein, the method may include the steps of changing the pH to loosen the cell structure, add enzymes that breaks the cells and/or applying ultrasonication. The pH is in a step according to the method adjusted a value within the range of from 6 to 14. The pH is preferably raised to between 9 and 11. Said pH range may loosen the fungal cell pellet like structure into a preferable mycelial state. However, the skilled person is aware that such high pH may also promote fungal cell disruption at some degree. The suspension may be homogenized after pH adjustment. To break fungal cells, enzymes such as b- glucanase and chitinase may be used, either on their own or in a mixture. In combination with enzymes, pH is then lowered to a value within the range of from 4 and 7, preferably within the range of from 5 to 7, to ensure a high enzyme activity. Ultra-sonication may be used at 48 W for 45 min, however the power and time parameters may vary according to the characteristics of the suspension in a specific batch. Subsequently, the suspension may be subjected to a mechanical treatment, such as high-pressure homogeniser with two passages at 900 bar, however the number of passages and pressure parameters may vary according to the characteristics of the suspension in a specific batch. Furthermore, the suspension obtained from the mechanical breaking may be subject to a pH within the range of from 11 to14 in order to promote protein solubilization.

Lastly, proteins that have been extracted/solubilized by cell destruction into a solution are isolated by changing pH to the isoelectric point of fungal proteins, i.e. , within the range of from 2 to 6, such as within the range of from 3 to 5, optionally between 3.5 and 4.5, which will advantageously make the proteins precipitate. The precipitate can thereafter be collected using centrifugation or filtration and thereafter be dried into a proteinaceous powder, using for example freeze drying, chilled vacuum drying or spray drying. The pH of the precipitate may alternatively be adjusted up to 6-8, such as to 7, before further removing the water from the precipitate by for instance freeze-drying, chilled vacuum drying or spray-drying the precipitate.

The protein precipitate may be freeze-dried e.g. to a temperature within the range of from -5°C to -35°C, then optionally subjected to a vacuum pressure such as within the range of from 0.001 mbar to 6 mbar until the water content is 10% by weight or lower, optionally 8% by weight or lower, thereby obtaining a freeze-dried proteinaceous powder. Alternatively, chilled vacuum dehydration may be used where the protein precipitate is chilled at a temperature between 0°C -17 °C and the water content is reduced using vacuum between 4 mbar and 50 mbar. Alternatively, the precipitate may be spray-dried by injecting the liquid containing the protein precipitate into any regular spray drying device.

Regardless of which technique(s) is/are used to promote cell breakage, the pH of the suspension may then be adjusted to the range within from 11 to 14, in case the pH is not within said range. Ensuring a pH within the range 11 to 14 advantageously promote protein solubilization and contributes to the high protein powder obtained by the end of this method.

Moreover, the solubilized proteins may be subject to precipitation in order to increase protein concentration and remove contaminants. Many techniques known by the skilled person may be used, such as isoelectric precipitation, salting out, flocculation and precipitation with miscible solvents. In case of the isoelectric precipitation technique, the pH of the suspension is adjusted to a pH within the range from 2 to 6, such as within the range of from 3 to 5, optionally between 3.5 and 4.5, which is the average isoelectric point of fungal proteins.

Precipitated proteins can be concentrated by removing or reducing water content from the suspension, for instance using filtration or centrifugation techniques. Said water content removal or reduction advantageously provide precipitated proteins with less water content, which reduces the time and costs related to the next steps of drying said precipitated proteins into a proteinaceous powder. The resulting suspension is therefore dewatered and may be subjected to further drying processes for generating a proteinaceous powder. Some techniques that may be used to produce the proteinaceous powder include freeze drying, chilled vacuum drying or spray drying. The final water content of the proteinaceous powder is typically from about 1 to about 10 wt.%, but in some embodiments the water content may be higher or lower.

Extracted proteins can be used in different formulations, for example as an emulsifier in oil in water emulsions. To prepare emulsions, the proteins can be dispersed in pure water or for example in a food graded aqueous buffer solution, such as a phosphate buffer. The oil phase can constitute of different vegetable oils, for example canola oil, and can be added up to third of the emulsion volume, depending on desired consistency. The proteinaceous powder according to the present disclosure is able to act as an emulsifier for the oil in water emulsions, promoting formation of small oil droplets, less than 100 pm in diameter. Small oil droplets are beneficial for emulsion stability, resulting in less phase separation in the end product. Here, it is shown that the proteinaceous powder can create emulsions with an emulsification capacity between 0.1 and 0.6 m ² /mg powder, indicating the ability to create small, stable oil droplets with low amount of powder. Droplet size can be measured using laser light scattering. Emulsion stability can be achieved using the proteinaceous powder without further additives. Emulsions can be used on applications such as in drink recipes, meat analogues or formulations of skin care.

The proteinaceous powder can also be used due to its foaming properties, in which the proteinaceous powder here is shown to create stable foams without further additives. High foaming properties is desired in applications of egg or cream replacing, for example foamed milk drinks, cappuccino, or merengue. Powders with proteins isolated from the biomass according to the present disclosure had better foaming properties compared to powders made of pure biomass, suggesting that the biomass powder is better suitable for applications where foaming is not desired, such as infant formula or sports nutrition. The fungal biomass was here shown to have higher contents of essential and branched-chain amino acids compared to other plant proteins and egg. This further proves the value to use the proteinaceous powder according to the present disclosure in sports nutrition.

The proteinaceous powder may be used to generate food products, such as a drink, a meat-replacement product, a seafood replacement product, noodles, a powder product, and/or an instant/rehydratable food product. Furthermore, the food product as a meat- replacement product may replace animal products such as a chicken, pork, beef and/or lamb. Further, the proteinaceous powder may be used to generate a cosmetic composition, such as by resuspending the proteinaceous powder in an aqueous solution or by preparing an oil in water emulsion as disclosed herein.

EXPERIMENTAL SECTION

In all the below examples, Rhizopus oligosporus was the fungal species used.

Example 1. Fungi biomass production

Production of fungi biomass

A fungal spore suspension 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 ^L 7 spores/mL) per 100 mL of growth medium, comprised of 20 g/L glucose, 5 g/L ammonium sulphate, 7 g/L of potassium phosphate and 1ml/L of a trace mineral solution, was added to each flask, followed by incubation at 30-35°C for 18-24h under shaking (100-150 rpm).

Sterilisation 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 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 dry matter of fungal biomass harvested from bioreactor cultivations was determined by first pressing all water down using a centrifuge and then drying samples at 105°C overnight and measuring the weight of the sample before and after drying, to determine the mass of water evaporated. Protein content was determined with Dumas combustion method (FlashEA 1112 Element Analyzer, Thermo Finningan, US) where nitrogen content was determined and converted to protein content with factor 6.25. The protein content of the biomass was used to calculate extraction yield in Example 3.

Example 2. Creating a fungi biomass powder as protein concentrate

Dehydration methods

Fungal biomass obtained from example 1 was cut in cubes of 1 cm and dehydrated using conventional hot air drying through wither a convection oven or a conduction oven. The fungal mycelium biomass was dried at 50°C or 70°C for 6h or overnight (until there was no significant change in sample weight). Samples dried through hot air drying resulted in a dark-coloured, compact and extremely hard mass.

The fungal biomass from example 1 was also freeze dried. For this, the biomass was cut in cubes of 1 cm and frozen for 24h at -20°C. After frozen, samples were placed in an Alpha 1-4 LSCplus freeze dryer set to shelf temperature of -10°C, vacuum between 1 and 3 mbar using a rotary vacuum pump, and condenser temperature at -86°C. The product temperature was monitored and the drying was deemed complete when the product did not show a cooling from ongoing sublimation. The time to a dry product averaged at 64h. Products from freeze drying showed a bright white colour similar to the fresh product, with an intact structure similar to the original product.

For chilled vacuum dehydration, the fungal biomass from example 1 was subjected to a customized vacuum process at low temperature. The samples were cut in 1cm cubes and chilled in a fridge to a stable temperature of 10°C, 15°C or 20°C. The samples were then placed in a vacuum chamber with shelves regulated to be kept at 10°C, 15°C or 20°C respectively. The samples were spread among the shelves so that all cubes would be in contact with the regulated surface. The chamber was also connected to a condenser with a temperature between -50°C and -86°C. The chamber was subjected to a vacuum pressure of 4 mbar. Samples were collected every hour and water content was calculated for these samples as explained in Example 1.

Water holding capacity

Dehydrated fungal mycelium biomass obtained from chilled vacuum dehydration at 10°C, freeze drying and convective oven drying at 50°C was grinded through a mill in a “fine” particle setting. One gram of powder was hydrated with excess water for 5 minutes and filtered. The wet mass was weighted and the water holding capacity (WHO) was calculated as:

WHO = (weight of rehydrated biomass - weight dry biomass) / weight dry biomass

Example 3. Extraction of fungi protein into a liquid media

Pre-treatment of biomass

Different methods to break fungi cells were applied but the samples first went through the same pre-treatment. Ten grams of frozen biomass were defrosted in 250 ml_ distilled water for 30 min at room temperature. After thawing, pH was adjusted to 10 with NaOH (1 M) and mixed for 5 min at 25,000 rpm (Ultra Turrax T25, Staufen im Breisgau, Germany), before different methods to break cells were applied. A sample from the pre-treatment was collected and used as a reference when comparing protein extraction (Figure 2) using Dumas combustion method.

Enzymatic degradation

After homogenization, samples were incubated with three different enzyme mixtures in ratio 1:100 enzyme to wet biomass ratio. pH was adjusted based on the enzyme, and the following three enzyme mixtures used were: Chitinase from Streptomyces griseus (pH adjusted to 6), b-glucanase from Trichoderma longibrachiatum (pH adjusted to 5.5) or a combination of the two enzymes (pH adjusted to 6). After enzymatic addition, the samples were incubated at 37°C for 2h. After incubation, there was a centrifugation step (5250 x g, 20°C, 90 min), whereafter the supernatant was collected for protein quantification using Dumas combustion method.

Ultrasonication

Ultrasonic treatment was conducted for 45 min at 48 W using a horn-type ultrasonic probe (40 kHz, Branson Sonifier, U.S.), in a cooling bath that was adjusted to be lower than 30°C. After the treatment, the samples were centrifuged (5250 x g, 20°C, 90 min), whereafter the supernatant was collected for protein quantification using Dumas combustion method. High-pressure homogenisation

Samples were feed into a high-pressure homogeniser (Niro Soavi Lab Homogenizer PandaPLUS 2000, GEA, Germany) and passed through two passages at 900 bar. After the homogenisation, the samples were centrifuged (5250 x g, 20°C, 90 min), whereafter the supernatant was collected for protein quantification using Dumas combustion method. As the high-pressure homogeniser resulted in most extracted proteins (Figure 2), this was used as a step for further protein extraction explained below.

Adjustment of pH for cell disruption

After the high-pressure homogeniser, different extraction pH was tried. pH was adjusted to between 2-14 using NaOH or HCI, both 1 M. Samples were thereafter centrifuged (5250 x g, 20°C, 90 min) and the supernatant was collected.

Protein content was measured both according to the Dumas combustion method explained in Example 1 and using bicinchoninic acid assay (BCA) assay kit. For the BCA analysis, 500 pL sample were diluted in 4.5 mL distilled water. From this, 100 pL was added to 2 mL BCA working reagent and gently mixed. The samples were incubated for 30 min at 37°C and thereafter cooled to ambient temperature before the absorbance was measured at 562 nm. The absorbance was compared to a standard curve to calculate protein concentration. The protein concentrations from the Dumas and BCA methods had a Pearson correlation coefficient of 0.99. The result of the protein concentration in the supernatants is presented in Figure 3.

Figure 3 shows that above pH 6, the protein concentration will increase with increasing pH, and the maximum concentration was found in the highest pH measured, pH 12. This was due to limitations in the Dumas method which could not handle samples at pH 13 or 14. The minimum protein concentration was found around pH 4 and it is therefore assumed that the isoelectric point is close to pH 4.

The protein concentration from the BCA kit was used to calculate the protein extraction yield according to the following equation.

Protein in supernatant

Protein extraction yield (%) = - - - xlOO

Protein in biomass

Protein yield from different extraction pH’s can be seen in Figure 8.

The protein extraction curve is closely linked with the protein concentration curve (Figure 3) and shows again that the minimum yield is for pH 4, while the highest is for pH 12.

From Figure 8 it is also shown that after pH 12, the yield will start to decrease and 12 is therefore assumed as the pH where most proteins are extracted.

Example 4. Creating a powdered isolated from the liquid extraction

Protein precipitation and concentration

The supernatant from Example 3 was pH adjusted to 4 which was believed to be the isoelectric point, to precipitate the proteins to a protein isolate. The protein isolate was thereafter re-adjusted back to 7 before drying using freeze-drying or spray-drying. The samples were freeze dried for 6 days in a laboratory freeze dryer (Hetosicc, Freeze dryer CD 12, Birkerod, Denmark) and spray-drying was performed in a Buchi Mini Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, Switzerland) with an inlet temperature of 150°C, outlet temperature of 80°C, aspiration 90-100% and pump at 35%.

The protein content of the final isolate powders was determined using Dumas method explained above, and was 77.2 % of the dry matter for both isolates, compared to 54.1% of the dry matter in freeze-dried fungal biomass. The moisture content in all powders were less than 2%. Forced imbibition of powders

Forced imbibition was measured according to the method adapted from Andersson et al. 2020 (Andersson, I.M, Bergenstahl, B., Alexander, M., Paulsson, M., and Glantz, M. 2020. Effects of feed composition, protein denaturation and storage of milk serum protein/lactose powders on rehydration properties. International Dairy Journal, 110). 150 ml_ of distilled water was poured into a 400 ml_ beaker (75 mm in diameter). Powder (0.1% w/v) was evenly distributed on the water surface using a sieve after the measurement started and constant stirring was applied using magnetic stirrer at 250 rates per minutes. The mixture was pumped through a flow-through cuvette with a flow rate of 60 mL/min which was placed in a spectrophotometer (Varian Cary 50 Bio UV-Visible). Distilled water was used as the blank and the absorbance was measured at 600 nm at ambient temperature (25°C). Each measurement was run for 30 minutes, where absorbance was measured manually every 5 second and each powder was measured in duplicates. The forced imbibition rate of the powders was calculated based on:

Where v is forced imbibition rate, t _Abs,2 is the time when the absorbance stabilized, t _Abs,i is the time when the absorbance started to deviate from 0, and m is the mass of the powder. Results from forced imbibition rate experiments can be seen in Figure 5.

The forced imbibition rate was found to be significantly faster for the freeze-dried biomass powders compared with the isolate powders (p<0.05; Figure 5).

Creating oil-in-water emulsions using isolated extracted protein powder Emulsions with protein isolate powders were made and evaluated according to method described in Ostbring et al., 2020 (Ostbring, K., Nilsson, K., Ahlstrom, C., Fridolfsson, A.& Rayner, M. (2020) Emulsifying and Anti-Oxidative Properties of Proteins Extracted from Industrially Cold-Pressed Rapeseed PressCake. Foods, 9(5):678. Available from: https://doi.org/10.3390/foods9050678). Emulsions were prepared by mixing 2 ml_ phosphate buffer (0.005 M, 0.2 M NaCI, pH 7) with 1 ml_ canola oil and adding biomass powder in the following concentrations 4, 8, 16, 32, 64 and 128 mg powder/m L oil. The emulsions were thereafter mixed in a high-shear homogeniser (Ystral, D-79282, Ballrechten-Dottingen, Germany) for 1 min at 24,000 rpm. After mixing, the emulsions were incubated at 4°C for 1 h to stabilize.

After incubation, particle size distribution was measured by static laser light (Mastersizer 2000, Malvern Instruments, Worcestershire, UK). Emulsions were added to an obscuration rate of between 10-20% and refractive indexes were set to 1.33 and 1.46 for the serum and oil phase, respectively. The particle size distributions of emulsions made with freeze dried protein isolate can be seen in Figure 6 and further emulsion data is presented in Figure 7.

The particle size distribution of emulsions made with freeze dried protein isolate shows a decrease in droplet size with an increase of powder concentration. However, with increasing concentration there is also an increase in larger particles (> 100 pm) which is likely to be powder aggregates. In-order to try and minimise these aggregates and create even smaller emulsion droplets, a high-pressure homogeniser was used. Emulsions with 1 ml_ canola oil and 99 ml_ distilled water and 64 or 128 mg freeze dried fungal biomass/mL oil samples were mixed. First, the samples were vortexed for 5 min before they were put in a high-pressure homogeniser (Niro Soavi Lab Homogenizer PandaPLUS 2000, GEA, Germany) circle run for 3 min at 200 bar. The droplet sizes of emulsions made with freeze-dried and spry-dried powders are presented in Figure 7.

The particle size of emulsions made with protein isolates shows a decrease in droplet size with an increase of powder concentration. The freeze-dried powders, has a better emulsifying capacity as the oil droplets are smaller. By using high-pressure homogenisation, it is possible to reduce the droplet size further. Size distributions of emulsions with 128 mg powder/mL oil are compared in Figure 8.

Figure 8 shows that the spray-dried isolate powder has less aggregates compared to the other two powders. However, the distributions of the emulsions from the high-shear mixer are quite similar and might be limited in size by the mixer. From the size distributions of the emulsions mixed with the high-pressure homogeniser it is clearer that the isolates can create smaller emulsions than the freeze-dried biomass as they have more particles with a size smaller than 1 pm. From the measured droplet sizes, the emulsion capacity (EC) can be calculated using the following equation: where, C _p is the calculated absorbed protein concentration (see below), d3,2 is the surface weighted mean, and f is the dispersed phase volume fraction. Emulsifying capacity for the different powders and concentrations can be seen in Figure 9.

The emulsifying capacity was better for the protein isolates compared to the freeze-dried fungal biomass. For most concentrations, the freeze-dried protein isolate powder showed the highest emulsifying capacity.

Protein solubility

To evaluate the solubility of the isolate powders and the freeze-dried fungal biomass in Example 3, the absorbed protein concentration in an emulsion was determined. First, powders were dissolved in phosphate buffer, as explained above, and centrifuged at 5000 x g for 30 min. The protein concentration in the supernatant was thereafter determined using BCA assay kit explained above. From this, the percentage of insoluble proteins were determined by the following formula: protein content in supernatant

Soluble protein (%) = - - - protein content in powder

Soluble protein at different concentration of emulsifier is presented in Figure 10.

After the first centrifugation, the powders were used to make an emulsion which was also centrifuged at 5000 x g for 30 min whereafter the oil phase was discarded. The serum phase was thereafter centrifuged again at 5000 x g for 30 min and the protein concentration in the supernatant was determined using BCA assay kit. The absorbed protein concentration (C _p ) was determined as the difference in proteins dissolved in the phosphate buffer and in the emulsion serum phase.

Figure 10 shows a decrease in soluble proteins with an increased concentration of the different powders. The freeze-dried fungal biomass has the smallest content of soluble proteins in the phosphate buffer, which could indicate that too much processing can affect the properties of the proteins.

Foaming Properties

Foaming properties were determined using a modified version of the method described in Lonchamp et al. 2019 (Lonchamp, J., Clegg, P.S., and Euston, S.R. 2019. Foaming, emulsifying and rheological properties of extracts from a co-product of the Quorn fermentation process. European Food Research and Technology, 245, 1825-1839). 15 ml_ solution of 1% w/v spray-dried protein isolate, freeze-dried protein isolate, and freeze- dried fungal biomass were prepared in 50 ml_ glass beakers, respectively, and stirred for 1 h. The solutions were then frothed for 3 min using handheld whisk-type frother (Ikea, Sweden). The height of the resulting foam was measured immediately after whisking and every 10 min until the foam collapsed. The foaming ability was expressed as the initial height of the foam while the foam stability was measured as the time required for the foam to fully collapse. All samples were measured in duplicate.

From Figure 11, it is shown that using freeze-dried fungal biomass to create a foam is not as beneficial as using any of the protein isolate, where the freeze-dried isolate has the best foaming stability.

Example 5. Protein extraction using grinding of biomass

Extraction of proteins to liquid media

Fungal biomass obtained as in Example 1 was grinded twice in a meat grinder using a disk with pore size 3 mm and thereafter diluted in water ratio 1 :4. The pH of the biomass mixture was adjusted to 10 or 12 and the solution was thereafter filtered through a 40 pm filter where the filtrate was collected. The filtrate was either left at the extraction pH or the pH was adjusted back to 7. Additionally, a control sample at extraction pH 7 was also performed.

Dry matter content of both filtrate and retentate was carried out like the method explained above and was measured in duplicates. Protein content was determined using BCA kit as explained above, but with an incubation for 2 h at room temperature instead of 30 min at 37°C and was measure in triplicates. Dry matter content is presented in Figure 12 and protein content in Figure 13. Figure 12 shows that a higher extraction pH will result in slightly more solids in both filtrate and retentate.

In Figure 13 it is shown that a higher extraction pH will lead to a higher protein content in the supernatant which agrees with Figures 3 and 4. Adjusting the pH after extraction will lead to a slightly lower protein content due to dilution but will not result in major changes.

Example 6. Use of mycoprotein isolate as sports nutrition

Fungal biomass was sent away for determination of amino acid content using HPLC with fluorescence detection at a certified laboratory (ALS Scandinavia AB, Danderyd,

Sweden). Figure 14 shows the content of essential amino acids (EAA) in % of total protein content for different crops and animal-based proteins. Values for fungal biomass is calculated from analytical results while the rest are from Gorissen et al. 2018 (Gorissen,

S., Crombag, J., Senden, J., Waterval, W., Bierau, J., Verdijk, L. B., and van Loon, L. (2018). Protein content and amino acid composition of commercially available plant-based protein isolates. Amino acids, 50(12), 1685-1695). Using data from Gorissen et al. 2018, the branched-chained amino acid (BCAA) content was calculated and compared with calculated results for fungal biomass (Figure 15). Figures 14 and 15 show that fungal biomass contains more EAA and BCAA than most other plant sources as well as more than egg. When isolating and drying fungal biomass into a protein powder, it is assumed that the content of amino acids will be similar to the initial content and that using this product as a sports nutrition would therefore be very valuable.

CLAUSES

1. A proteinaceous powder comprising fungi biomass or proteins derived from fungi biomass, wherein a protein content of the proteinaceous powder is within the range of from 70 wt.% to 95 wt. %.

2. The proteinaceous powder according to clause 1 , wherein the protein content of the proteinaceous powder is within the range of from 75 wt.% to 95 wt. % 3. The proteinaceous powder according to clause 1 or 2, wherein a content of essential amino acids is 30 wt. % or more of a total protein content of the proteinaceous powder.

4. The proteinaceous powder according to any one of the preceding clauses, wherein a content of branched-chain amino acids is 15 wt. % or more of a total protein content of the proteinaceous powder.

5. The proteinaceous powder according to any one of the preceding clauses, wherein the solubility of the proteinaceous powder in water is within the range of from 10% to 60%, as measured according to the protein solubility assay as described herein.

6. The proteinaceous powder according to any one of the preceding clauses, wherein the wettability of the proteinaceous powder is within the range of from 50 mg/min to 200 mg/min, as measured according to the force inhibition measurement method as described herein.

7. An oil in water emulsion comprising the proteinaceous powder according to any one of the preceding clauses, wherein the oil in water emulsion comprises oil droplets and an aqueous phase, the proteins in the proteinaceous powder being arranged in an interface between the oil droplets and the aqueous phase.

8. The oil in water emulsion according to clause 7, wherein the oil droplets each has an oil droplet size of less than 100 pm, optionally less than 10 pm. 9. The oil in water emulsion according to clause 7 or 8, wherein the proteinaceous powder has an emulsification capacity of from 0.1 to 1 m ² /mg of protein, preferably within the range of from 0.1 to 0.6 m ² /mg protein. 10. The oil in water emulsion according to any one of clauses 7-9, wherein the oil in water emulsion has a foaming stability of at least 300 min before collapse of foam, as measured according to the foam height measurement method as disclosed herein.

11. A method of preparing a proteinaceous powder according to anyone of the preceding clauses, the method comprising the steps of a) cultivating fungi of in a liquid fermentation in an aerated bioreactor or by a solid state fermentation, to obtain a fungi biomass; b) providing the fungi biomass obtained in step a) in an aqueous solution to form a suspension; c) adjusting the pH of the suspension to a pH within the range of from 6 to 14, optionally within the range of from 9 to 11, and homogenizing the suspension; d) optionally, adjusting the pH of the suspension to a pH within the range of from 4 to 7, preferably within the range of from 5 to 7, and subjecting the suspension to enzymatic breakage of the fungi biomass cell wall structure, such as by adding chitinase and/or b-glucanase to the suspension; e) optionally, subjecting the suspension to an ultrasonic treatment; f) subjecting the suspension to a mechanical breaking of the fungi cell walls, such as by grinding, cutting, high-pressure homogenisation, high-shear homogenisation; g) adjusting the pH of the suspension to a pH within the range of from 11 to 14; h) collecting the suspension from step g) and adjusting the pH to a pH within the range of from 3 to 5 and removing water from the suspension, such as by filtration or by centrifugation; and i) preparing a proteinaceous powder from the suspension resulting from step h) by reduction of water, such as by freeze drying, spray drying or chilled vacuum drying.