Protein production method

Abstract

Provided is a method of producing protein suitable for use in a human food product. The method comprises Solid-State Fermentation (SSF) of a carbon source using a fungus suitable for human food production, the carbon source optionally having at least 10% protein by dry weight (w / w); and extraction of protein from the fungal biomass and optionally the carbon source. Also provided is an isolated or extracted mixture of proteins and a human food product comprising the protein mixture.

Description

[0001] Protein Production Method

[0002] Background

[0003] Animal farming is highly environmentally destructive, and a significant emitter of greenhouse gases. For humanity to reduce carbon emissions and avoid the worst impacts of climate change, alternatives must be found to animal agriculture as a source of human food.

[0004] The UK is legally committed to net-zero carbon emissions by 2050. Agriculture was the source of 49Mt CO2 UK GHG emissions in 2021 (>10% of total), including 49% of methane emissions. Without means to economically produce non-animal food proteins, countries like the UK will miss carbon goals for food and agriculture.

[0005] The most challenging nutrient to transition away from animal agriculture is protein. Alternatives to animal protein have been developed, such as plant proteins and novel technologies that used genetically-modified organisms to produce animal proteins (‘Submerged Fermentation’). However, these alternatives all have drawbacks.

[0006] Surprisingly, we have found that a human food product can be produced by a method comprising Solid-State Fermentation (SSF). This requires fermentation of a carbon source using a fungus. For example, the carbon source is a side stream (waste material) from the food or agricultural industry. The fungus is suitable for human food production. The carbon source may have at least 10% protein by dry weight (w / w).

[0007] Extraction of protein from the fungal biomass and, optionally, the carbon source, thereby provides a high-protein content product, suitable for human food, for application in human food products requiring or containing appropriate protein for human consumption.

[0008] Summary of the Invention

[0009] Provided is a method of producing protein suitable for use in a human food product, the method comprising: a. Solid-State Fermentation (SSF) of a carbon source using a fungus suitable for human food production, the carbon source optionally having at least 10% protein by dry weight (w / w); and b. extraction of protein from the fungal biomass and optionally the carbon source.

[0010] In some embodiments, there is provided a method of producing protein suitable for use in a human food product, the method comprising: a. Solid-State Fermentation (SSF) of a carbon source using a fungus suitable for human food production, the carbon source; and b. extraction of protein from the fungal biomass.

[0011] In some embodiments, there is provided a method of producing protein suitable for use in a human food product, the method comprising: c. Solid-State Fermentation (SSF) of a carbon source using a fungus suitable for human food production, the carbon source having at least 10% protein by dry weight (w / w); and d. extraction of protein from both the fungal biomass and the carbon source.

[0012] In some embodiments, the method further comprises providing the extracted protein in solution, suspension or as a powder. In some embodiments, the protein is solubilized in an aqueous medium and / or, optionally, precipitated. In some embodiments, extracted protein is provided in a powdered form for inclusion in a food product.

[0013] In some embodiments, the carbon source is, or is derived from, waste materials from the food or agriculture industries. In some embodiments, the carbon source is one or more of: Brewers’ Spent Grain (BSG), wheat bran, potato peel, bread waste or bread crusts, beet pulp, pea husks, fruit pomace, broken rice, soybean husk, sugar cane bagasse, koji waste, coconut coir, and / or oat sidings.

[0014] In some embodiments, the method comprises removal of allergens. In some embodiments, the method comprises removal of peroxide after bleaching by addition of a peroxidase and comprises removal of allergens. In some embodiments, the protein extraction further comprises bleaching the extracted protein. In some embodiments, the bleaching comprises addition of oxygen free radicals. In some embodiments, the free radicals are provided by hydrogen peroxide. In some embodiments, the method comprises removal of peroxide after bleaching by addition of a peroxidase. In some embodiments, gluten is removed enzymatically.

[0015] In some embodiments, the fungal species is Aspergilus sp. Rhizopus sp., Penicillium sp., and / or Neurospora sp..

[0016] Also provided is an isolated or extracted mixture of proteins produced according to the present method. Also provided is an isolated mixture of proteins comprising a mixture of fungal biomass and, optionally also proteins from a or the carbon source, for example wherein the carbon source is BSG.

[0017] Also provided is a food product comprising the protein mixture. In some embodiments, the food product is a nutritional protein powder for use in food and supplements such as vegan cheese, sports protein powder or nutritional supplement, confectionary and baked goods, condiments and seasonings, high protein snacks, ready meals, plant based meats, chocolate replacements, pet foods, soups, noodles, sauces and dressings.

[0018] Brief Description of the Figures

[0019] Aspects of the invention will now be described with reference to the following figures, wherein:

[0020] Figure 1 shows 5g of wheat bran in a 250ml Erlenmeyer flask;

[0021] Figure 2 shows example plastic trays used to conduct SSF;

[0022] Figure 3 is a diagram of one example of the process as exemplified herein. The second section of the figure shows the process of gluten removal from the product; Figure 4 shows the colouration of the protein, indicating that Untreated BSG (Brewers Spent Grain) would ideally be treated (e.g. bleached) to improve its colour, whilst Sample #4 was at the cusp of acceptability in terms of its colouration (for alternative / vegan use, as described above). Ideally, a beige, cream, off-white or white coloured protein is preferred for such alternative food product uses, e.g. vegan cheese. This needs to be matched against yield of protein (from the carbon source / substate) to make the process (and resulting food product) cost effective; and

[0023] Figure 5 shows a small pot with the extracted protein - protein fractions precipitated at different isoelectric points and were mixed together to provide the sample shown.

[0024] Detailed Description

[0025] The most challenging nutrient to transition away from animal agriculture is protein. Alternatives to animal protein have been developed, such as plant proteins and novel technologies that use genetically-modified organisms (GMO) to produce animal proteins (‘Submerged Fermentation’). However, these alternatives all have drawbacks: Therefore, there exists a considerable demand for a protein ingredient product that can mimic the benefits of animal protein in products such as vegan cheese or in protein shakes, such as flavour and texture. Furthermore, this needs to be producible at a cost base that would allow it to compete directly with animal and plant-based proteins. We have developed a process to produce such a product at a target yield and cost base that would make it economically viable for production.

[0026] Furthermore, this production process is advantageous as it is scalable for wide-spread industrial applicability. Low-cost protein, produced by the present method, also has the advantage of helping address climate change, through reduced ruminants, and also world food shortages.

[0027] In some embodiments, the fungus provides a high yield of edible protein. This is advantageous for delivering a low-cost method and resulting product.

[0028] In some embodiments, the carbon source may be referred to as a feedstock or substrate. In some embodiments, the carbon source, in particular where the carbon source is a waste material, for example a waste material from food industry or agricultural industry process. These are so-called side streams. Using such waste products has the advantage of benefitting the circular economy. Examples include BSG. This is also advantageous for delivering a low-cost method and resulting product.

[0029] The use of SSF is also advantageous as it contributes to a significant cost reduction over submerged fermentation (SMF) systems.

[0030] In some embodiments, the fungus provides a product comprising a high yield of edible protein, suitable for human consumption, and the carbon source is a waste material (for example BSG). These, in combination with SSF, provide an advantageous low- cost method and resulting product.

[0031] EP4082355A1 relates to SSF, but does not describe any protein extraction from the fermented mixture. Carbohydrate Polymers (vol 199, 2018, Amorin et al., “Single Step production of Arabino-Xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewers’ spent grain” pages 46-554) relates to production of AXOS (arabino-xylooligosaccharides), complex sugars, and how these may be produced from fermentation of BSG by SSF. There is no disclosure of any protein extraction from the fermented mixture. Environmental Science & Pollution Research (vol 29, 2022, C. Eliopoulos et al., “Conversion of brewers’ spent grain into proteinaceous animal feed using solid state fermentation” pages 29562-29569) relates to BSG fermented with filamentous fungi by SSF, but focuses on fiber content for animal feed uses. It does not provide protein suitable for human consumption and does not describe any protein extraction from the fermented mixture. W02024003323A1 relates to a complex fermentation process. EP4190164A1 relates to fermenting plant proteins.

[0032] Although SSF has been used to produce animal feed and to produce specific enzymes (“Fungal Biovalorization of a Brewing Industry Byproduct, Brewer’s Spent Grain: A Review” by Marcus, A.; Fox, G.: Foods 2021 , 10, 2159. https: / / doi.Org / 10.3390 / foods10092159), it has not been used before for production of protein for use in human food products.

[0033] In particular, SSF has not been used before in combination with a carbon source from waste side streams for food production, especially human food production. SSF has not been used before in combination with a carbon source from having at least 10% protein (prior to fermentation) for food production, especially human food production.

[0034] The combination of SSF and waste side stream carbon sources (or those or having at least 10% protein (prior to fermentation)), especially BSG, followed by extraction of protein from the carbon source (for example BSG) and fungal biomass-derived protein is new and advantageous. In other words, taking the embodiment where the carbon source is BSG, the following key steps of the method together have not been seen together previously: solid-state (fungal) fermentation of BSG, with subsequent extraction of protein (optionally both BSG-derived protein and biomass-derived protein), and subsequent use of that protein for human food products. A human food product is, in some embodiments, a product that is suitable for human consumption. It may also have, in some embodiments, appropriate mechanical and / or mouth-feel properties and / or colour to meet the human consumer’s requirements.

[0035] The invention therefore provides for the use of waste materials as described herein as a substrate (or carbon source). In some embodiments, this is in addition to the use of solid state fermentation. In some embodiments, this is in addition to the use of protein extraction. In some embodiments, this to thereby produce a product for use as an ingredient for food. In some embodiments, this is combined to provide for the use of waste materials as described herein as a substrate (or carbon source), is in addition to the use of solid state fermentation, in addition to the use of protein extraction to thereby produce a product for use as an ingredient for food.

[0036] Solid-State Fermentation (SSF) is a well-known process which comprises growing, through a process called fermentation, fungus (e.g. one or more fungal species). Although moisture is helpful for growth, this is conducted in an environment that is not submerged, e.g. not in a tank such as a bioreactor tank, but instead it is conducted (for example in some embodiments) in a tray (preferably with a lid or seal to prevent contamination ) or chamber. In some embodiments, the surface of the carbon source is kept above the water line, if there is a water line at all.

[0037] The carbon source is any source of carbon that meets the protein requirement to support growth of the fungal species. In some embodiments, the carbon source is of at least 10% protein by dry weight (w / w). Where this protein by dry weight percentage is provided herein, it will be understood that this refers to the dry weight prior to fermentation.

[0038] In some embodiments, the carbon source is a, or comprises, seeds or grains, including a mixture of two or more seeds, a mixture of two or more grains, or a mixture of both. As the fermentation is Solid-State Fermentation (SSF), then in some embodiments the carbon source is provided as a solid. It may in some embodiments be moist, even to the point of being a slurry. In some embodiments, the carbon source is Brewers’ Spent Grain (BSG). Ikram et al., 2017 (Journal of Food Science - 2017 - Ikram - Composition and Nutrient Value Proposition of Brewers Spent Grain) indicates that BSG is typically in the region of approx. 15-26% protein w / w (dry weight). BSG is typically a waste material from beer brewing, and may be acquired in bulk from a brewer. In some embodiments, the BSG may be from brews for stout, IPA, and lager. In some embodiments, BSG from lager brews is preferred as it gives the best colour profile for the protein product.

[0039] In some embodiments, the BSG is provided in its un-milled form. This is advantageous as it was found to provide a greater yield of protein. Therefore, BSG in its un-milled state is preferred in some embodiments. Un-milled means not processed by grinding (or milling).

[0040] The carbon source must support fungal growth and may have at least approximately 10% protein. This is prior to fermentation in the present method. Some fermentation on the substrate may have occurred prior to the present method.

[0041] In some embodiments, the protein, derived from the carbon source, is helpful in contributing to the overall protein provided (following the present fungal fermentation and subsequent extraction) in the product, in addition to the protein provided (again following the present fungal fermentation and subsequent extraction) by the biomass of the fungus.

[0042] In some embodiments, the carbon source has at least approximately 10% protein, at least approximately 11 % protein, at least approximately 12% protein, at least approximately 13% protein, at least approximately 14% protein, has at least approximately 15% protein, at least approximately 16% protein, at least approximately 17% protein, at least approximately 18% protein, at least approximately 19% protein, or has at least approximately 20% protein.

[0043] In some embodiments, the carbon source has at least approximately 21 % protein, at least approximately 22% protein, at least approximately 23% protein, at least approximately 24% protein, has at least approximately 25% protein, at least approximately 26% protein, at least approximately 27% protein, at least approximately 27% protein, at least approximately 28% protein, has at least approximately 29% protein, at least approximately 30% protein, at least approximately 35% protein, at least approximately 40% protein, or at least approximately 50% protein.

[0044] In some embodiments, the carbon source has approximately 10% - 15% protein, has approximately 10% - 20% protein, has approximately 10% - 25% protein, has approximately 10% - 30% protein, has approximately 10% - 35% protein, has approximately 15% - 20% protein, has approximately 15% - 25% protein, has approximately 15% - 30% protein, has approximately 15% - 35% protein, has approximately 18% - 20% protein, has approximately 18% - 25% protein, has approximately 18% - 30% protein, has approximately 18% - 35% protein, has approximately 20% - 25% protein, has approximately 20% - 30% protein, has approximately 20% - 35% protein, has approximately 25% - 30% protein, or has approximately 20% - 35% protein.

[0045] Environmental Science & Pollution Research (vol 29, 2022, C. Eliopoulos et al., “Conversion of brewers’ spent grain into proteinaceous animal feed using solid state fermentation” pages 29562-29569), cited above, indicates on page 29567 that the the initial protein content of unfermented BSG can vary between 10% and 30% by weight. Thus, in some embodiments, this range is preferred. In some embodiments, this range is preferred for BSG.

[0046] The above % are by dry weight, and may be measured in the standard way. For example, the carbon source may be dried and its mass measured. Extracted protein may also be dried and weighed. The two are then compared to provide the required protein by dry weight (w / w).

[0047] The carbon source is, in some embodiments, a plant-based carbon source. In some embodiments, it comprises one or more starches, in addition to the required protein.

[0048] The fungus is a fungus suitable for human food production. It should ideally therefore not result in the production of a pathogenic (to humans) substance, for example one that is active after the fermentation has concluded. In some embodiments, mycotoxins are not generated by the fungus. In some embodiments, mycotoxins are not generated by the fungus in combination with the carbon source. Many fungi are known to be used in the food industry, for example in cheese and yoghurt making.

[0049] In some embodiments, the fungus can be a single fungal species or a mixture of species and / or strains. In some embodiments, the fungus is a filamentous fungus.

[0050] In some embodiments, the fungus can be a genetically-modified organism (GMO) or genetically-engineered organisms (GEO) fungus. In particular, reference herein to a fungus (including a species, genera, order, phylum etc) also includes genetically- modified organisms (GMO) or genetically-engineered organisms (GEO) variants thereof. Such GM or GE variants may, in some embodiments, show, for example, at least 95%, preferably at least 98%, preferably at least 99%, preferably at least 99.5%, preferably at least 99.9%, preferably at least 99.95% or preferably at least 99.99% sequence homology in their genome to the reference organism.

[0051] In some embodiments, the fungus is Rhizopus sp., Penicillium sp., or Neurospora sp..

[0052] In some embodiments, the fungus is, or includes one or more of: Trichothecium roseum, Aspergillus fumigatus, Fusarium venenatum, Rhizopus delemar, Neurospora intermedia, Trichoderma reesei, Rhizopus oryzae, Penicillium camembertii, Aspergillus awamori, Mucor racemosus, Mucor lanceolatus, Penci Hi urn commune, Trichoderma harzianum, Monascus purpureus, Neurospora intermedia, Penicillium nalgiovense, Penicillium roqueforti, Fusarium domesticum, Geotrichum candidum, Rhizopus oligosporus and Aspergillus oryzae and / or Aspergillus sojae.

[0053] In some embodiments, the fungus is, or includes one or more of::

[0054] • Penicillium camembertii

[0055] • Penicillium nalgiovense

[0056] • Penicillium roqueforti

[0057] • Fusarium domesticum

[0058] • Geotrichum candidum • Aspergillus oryzae

[0059] • Aspergillus sojae

[0060] • Rhizopus oligosporus

[0061] In some embodiments, the fungus is or includes Aspergillus sp., in particular Aspergillus oryzae or Aspergillus sojae.

[0062] The fermentation may be concluded at any time, for example by harvesting the fungus. In some embodiments, the protein extraction can be started immediately, or at least within 1 or 2 hours, in which case the fermentation is stopped by homogenisation.

[0063] In some embodiments, the fermented mixture is instead cooled (for example placed in cold storage) for immobilisation, until extraction can start, with optional homogenisation as the start or prior to extraction.

[0064] Once the fermentation has been substantially concluded, the total protein is extracted from both the fungal biomass and the carbon source.

[0065] Protein extraction, in some embodiments referred to as protein isolation may be according to standard methods. In some embodiments, these may include:

[0066] • extraction of protein from the fermented mix using acid / base precipitation and,

[0067] • optionally, centrifugation.

[0068] In some embodiments, the total protein extracted is combined (if separate). This total protein, comprising the protein from both the fungal biomass and the carbon source, can then be used in the food product.

[0069] In some embodiments, the extracted protein is provided as an oven dried powder protein. In some embodiments, the extracted protein is provided as a spray dried powder protein. Therefore, in some embodiments, the method includes oven drying the extracted protein or spray-drying the extracted protein to thereby provide an oven dried or spray dried protein product. In some embodiments, the extracted protein is provided as a protein solution, for example a protein concentrate. In some embodiments, this may be by ultrafiltration of clarified lysed material with pH adjustment prior to ultrafiltration. Therefore, in some embodiments, the method includes any of these steps.

[0070] In some embodiments, the extracted protein is provided as a protein paste. In some embodiments, this is achieved using a pH4 - 6 for precipitation. In some embodiments, this provides the protein product as a suspension or emulsion. Therefore, in some embodiments, the method includes any of these steps.

[0071] In some embodiments, the method may include: selective removal of off-taste or low pH fraction and concentrated supernatant mixing with the higher pH paste to enrich it.

[0072] In some embodiments, the enriched paste can be used as a suspension / emulsion or subsequently dried. Therefore, in some embodiments, the method includes any of these steps.

[0073] In some embodiments, the extracted protein is provided as a spray dried powder by concentrating the clarified lysate and adding a carrier e.g. maltodextrin prior to spray drying. Therefore, in some embodiments, the method includes any of these steps.

[0074] In some embodiments, the extracted protein is provided as a combination of any of the above, thereby, in some embodiments, yielding an enriched protein paste. Therefore, in some embodiments, the method includes any of these combined steps.

[0075] In some embodiments, further processing steps may also be included. In some embodiments, the protein is bleached. For example, the bleaching process comprises whitening the colour of the protein during the extraction to provide a whiter colour in the final extracted protein than otherwise would be the case without the bleaching. In some embodiments, this bleaching occurs after extraction of the protein from both the fungal biomass and the carbon source. In some embodiments, the bleaching comprises addition of oxygen free radicals. In some embodiments, the free radicals are provided by hydrogen peroxide. In some embodiments, the method further comprises removal of any residual peroxide activity after bleaching. In some embodiments, this is achieved by addition of a peroxidase. Bleaching affects the colour of the protein and is advantageous as is makes the protein more attractive to the human consumer. In some embodiments, the bleaching agent, in particular the hydrogen peroxide, is left for at least 6 hours. This improves colouration. This may be at least 7, 8, 9, 10, 11 or 12 hours. In some embodiments, this in combination with at least 60 degrees, at least 70, at least 80, or at least 90 C heating. At least 10 hours at 70 degrees C is preferred in some embodiments. It should be stressed that the colour is not related to the nutritional value of the extracted protein. Darkly coloured protein is useful in many scenarios, for example where the food product is already, or is intended to be, dark in colour. Where colour can be important is in uses in alternative food products (such as food products comprising non-dairy protein, such as vegan food products especially ‘vegan cheese) where currently consumers have certain expectations that the colour of the present (alternative) food product matches that of the substitute food product. Thus, in some embodiments, the extracted protein is used in an alternative food product and such an alternative food product is also provided. Ideally, a beige, cream, off-white or white coloured protein is preferred for such alternative food product uses, e.g. vegan cheese, and these are provided in some embodiments. This needs to be matched against yield of protein (from the carbon source / substate) to make the process (and resulting food product) cost effective. See the discussion in the Example regarding Sample 4.

[0076] In some embodiments, the method further comprises removal of allergens. Ideally, in some embodiments, this occurs after extraction of the protein from both the fungal biomass and the carbon source. In some embodiments, the allergen removal may occur before or after any bleaching step. Allergens that may be removed include, in some embodiments, gluten. Gluten may be removed, in some embodiments, through the addition of a suitable enzyme. In some embodiments, the enzyme, is a catalase. In some embodiments, the enzyme is a proline-specific endo-protease. In some embodiments, the enzyme is Brewers Clarex® . In some embodiments, allergens such as gluten are removed through single or multiple application of the allergen removal enzyme. In some embodiments, the allergen, in particular gluten, is removed to achieve less than 1 ppm gluten. In some embodiments, the allergen, in particular gluten, is removed to achieve less than 5, 10 or 20 ppm gluten.

[0077] Removal of allergens is advantageous as it allows the food product to be more widely acceptable to the human population, and this may also aid in distribution.

[0078] In some embodiments, both the bleaching and the allergen removal occurs after the protein has been precipitated. This is advantageous because treating too early in the process loses protein yield.

[0079] In some embodiments, the bleaching (as exemplified in Figure 3 by addition of peroxidase) may occur after the precipitation, then followed by the allergen removal may occur after the precipitation (as exemplified in Figure 3 by addition of catalase and / or Brewers Clarex®). Alternatively, in some embodiments, the allergen removal (as exemplified in Figure 3 by addition of catalase and / or Clarex) may occur after the precipitation, then followed by the bleaching (as exemplified in Figure 3 by addition of peroxidase).

[0080] In any of the embodiments described herein, if bleaching is used, this may be in a single step or in multiple steps, using the same enzyme in each case (e.g. Brewers Clarex®) or in a combination of two of more steps where one is applied, then another (for example Catalse then Clarex®). In some embodiments, testing for bleaching activity after any bleaching step (e.g. checking for peroxide) is provided.

[0081] Also provided is an isolated mixture of proteins produced according to the present method. The mixture may include, but not be limited to protein from both the fungal biomass and the carbon source.

[0082] Also provided is a food product comprising the protein mixture. In some embodiments, the food product may be a vegan cheese, or protein shake, protein bar, plant-based patty, plant-based sausage, or plant-based mince.

[0083] In some embodiments, the method further comprises breaking down the fermented mixture. In some embodiments, this occurs before the protein is precipitated (or titration is begun). This may be part of the extraction of protein or a separate step. In some embodiments, this breaking down may be achieved by homogenisation. In some embodiments, a High-Pressure Homogeniser (HPH) can be used. In some embodiments, a colloid mill can be used. In some embodiments, optionally following homogenisation, straining or separation of solids, e.g. through a filter, sieve or cheese cloth, centrifugation is also provided as part of the extraction of protein.

[0084] In some embodiments, the extraction of protein includes acid / base titration. In some embodiments, precipitation of protein occurs between pH3 and pH12. In some embodiments, this occurs at:

[0085] • approx.. pH3.5 (in some embodiments approx.. pH3 to 4) for the BSG-derived protein fraction;

[0086] • approx.. pH 4.5 to 5.5 (in some embodiments approx.. pH 4 to 6) for fungal protein (assumed mix of biomass and secreted) and / or

[0087] • approx.. pH 11 (in some embodiments approx. 8.0 to 11 .5 or 10.5 to 1 1 .5) for secreted fungal protein.

[0088] In some embodiments, the extraction of protein comprises removal of the liquid fraction. In some embodiments, this results in a protein product with a moisture level of approx.. 75%. In some embodiments, this may be at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.

[0089] Example

[0090] The substrate referred to in the Example is the carbon source described above.

[0091] Methodology

[0092] The research was divided into 2 phases:

[0093] • Desk research to develop a theoretical process for testing; followed by

[0094] • Laboratory testing to run and optimise the process.

[0095] Desk Research

[0096] The research started by comparing the primary production systems currently used, focusing on 3 technologies: • ‘Submerged Fermentation’ (SMF). Using genetically-modified microorganisms, such as yeast, grown in liquid-tank bioreactors, to produce target proteins.

[0097] • Plant protein extraction. Using acid / base precipitation as well as centrifugation and filtration, to extract proteins from crops such as soy, pea, and wheat.

[0098] • Solid-State Fermentation (SSF). Growing filamentous fungi on solid substrates, often on waste materials such as the spent grains from breweries (BSG).

[0099] During several months of research, the team learned the technologies involved in each, as well as their cost economics, and assessed their potential as production systems.

[0100] The first conclusion was that SMF systems would not be a viable technology set. Multiple examples emerged of such systems producing protein with cost unit economics in the region of $40-50 / kg, well above the target range. It was therefore discounted as an option. It was also noted that a significant component of this cost was due to high capex costs for the construction of such plants, which at the target volume was assessed to be in the region of $150m for one plant.

[0101] A hypothesis was then developed that combining the other 2 processes - SSF with protein extraction - which there was no evidence found had been tested elsewhere, could deliver a suitable product at the target unit cost level.

[0102] To test this hypothesis, a theoretical process was developed, consisting of 2 key stages:

[0103] • Solid-state fermentation of BSG, using a filamentous fungus,

[0104] • Followed by extraction of protein from the fermented mix using acid / base precipitation and centrifugation

[0105] The final stage of the desk research phase was to develop a techno-economic model (‘T / E model’) to test whether such a process would be economically viable at a scale of 500 tons per year. All relevant costs, including construction, equipment, and operating costs were considered, along with market pricing provided by large industrial purchasers of such ingredients. The T / E model demonstrated a hypothetical profit rate deemed sufficient to progress to laboratory testing.

[0106] Laboratory Testing and Optimisation

[0107] The process was broken into 2 parts, with separate research teams focusing on each:

[0108] • Solid-State Fermentation

[0109] • Protein extraction

[0110] Solid-State Fermentation

[0111] To test the SSF process, several key factors were tested and optimised for:

[0112] • Choice of organism

[0113] • Choice of substrate

[0114] • Substrate preparation

[0115] • Fermentation equipment

[0116] • Fermentation conditions

[0117] Choice of Organism

[0118] A list of potential organisms was researched, covering filamentous fungal species from different genera, including: Trichothecium roseum, Aspergillus fumigatus, Fusarium venenatum, Rhizopus delemar, Neurospora intermedia, Trichoderma reesei, Rhizopus oryzae. Penicillium camembertii, Penicillium nalgiovense, Penicillium roqueforti, Fusarium domesticum, Geotrichum candidum, and Aspergillus oryzae

[0119] These were then reviewed using the following requirements matrix, to select a shortlist of organisms for testing:

[0120] Based on these criteria, 6 organisms were shortlisted:

[0121] • Penicillium camembertii

[0122] • Penicillium nalgiovense

[0123] • Penicillium roqueforti

[0124] • Fusarium domesticum

[0125] • Geotrichum candidum

[0126] • Aspergillus oryzae

[0127] Each of these organisms was then acquired, sub-cultured on agar plates, and used as inoculum on the target substrates, to test for growth, yield, and flavour of produced protein. Fusarium domesticum was not sub-cultured.

[0128] Choice of substrate

[0129] A shortlist of substrates was researched for testing:

[0130] • BSG. Brewer’s spent grain, the waste material from beer brewing, acquired from a local brewer. Several types of BSG were acquired, including from brews for stout, IPA, and lager. Furthermore, BSG was tested in its natural (un-milled) form, and compared to a milled form of BSG. Comparative runs were done, comparing yield between SSF on BSG that had been milled, and BSG not milled. It was found that not milled had a better yield. Therefore BSG in its unmilled state is preferred in some embodiments. Un-milled means not processed by grinding (or milling).

[0131] • Fresh potatoes. Low-grade potatoes, acquired as sample from a potato wholesaler. The potatoes were cut into small cubes.

[0132] • Potato starch. Waste by-product from a factory that extracts protein from potatoes.

[0133] • Apple pomace. A waste by-product from cider pressing. Was provided both in fresh and dried forms. We collected this substrate but decided not to use it for further testing because the cost was high.

[0134] • Foam. Foam was tested as a physical substrate, with addition of extra nutrients for carbon and nitrogen sources.

[0135] • Wheat bran. Tested in milled and unmilled forms.

[0136] Figure 1 shows 5g of wheat bran in a 250ml Erlenmeyer flask.

[0137] Samples of each were procured and then compared by growing a range of organisms on each, and comparing for yield and product quality.

[0138] Substrate preparation

[0139] Various methods and factors were compared for the preparation of substrate:

[0140] • Sterilisation method. Maintaining sterility is vital in SSF. Several methodologies were developed and tested: o Equipment sterilisation. All equipment used was autoclaved prior to use, and any that with an ‘active surface’ as part of the SSF was further sterilised prior to handling, using ethanol wipes, UV light, and / or a live flame.

[0141] • Sterilisation of substrate. The substrate here is the feedstock, also referred to as the carbon source. For substrate sterilisation, two different autoclave settings were used, 15 minutes, 121 °C or 10 minutes at 115 °C. Several methods of sterilisation substrate were compared: o Drying and Autoclaving and drying o Drying only (70 °C ) could also be done o Autoclaving only o Neither autoclaving nor drying

[0142] • Maintaining sterility during inoculation. All inoculations were completed in a lateral flow hood with air extraction and sterilisation equipment on hand.

[0143] • Maintaining sterility during fermentation. All fermentation vessels were fully sealed before being taken out of the lateral flow hood, and kept sealed throughout the full fermentation period.

[0144] • Bag based inoculation. The substrate in the autoclave bag was sterilised and inoculum mixed after sterilisation.

[0145] • Non sterilisation method. The substrate was not autoclaved.

[0146] • Moisture level. Substrate was taken from dry and different volumes of water were added to reach different moisture levels. Volume of water mixed at level 1 ml / g, 2 ml / g, 2.6 ml / g, and 3 ml / g.

[0147] • Inoculum quantity. Spores were plated on agar plates which were stored in the fridge. Spores were then counted under the microscope and added by pipette to the substrate.

[0148] • Microwave based drying of substrate. Microwave was used to dry the substrate. Microwave drying was performed at different temperatures like 45°C, 60°C, 65 °Cand 75 °C. Different water activity of the substrate was tested (0.11 , 0.12, 0.26).

[0149] • Nutrients. Different concentrations (2g / L, 0.5 g / L) of mineral salts (Magnesium sulphate and Potassium dihydrogen orthophosphate) were tested.

[0150] Moisture level

[0151] Substrate are taken from dry and taken to a moisture level of 75%.

[0152] Inoculum quantity

[0153] Spores are plated on agar plates which were stored in the fridge. Spores are then counted under the microscope and added by pipette to the substrate.

[0154] Fermentation equipment

[0155] Different types of fermentation equipment (flask or tray) were used with modifications on the system. The size of the trays were used 5.5L, 11 L and 16L • 250ml shake flasks for small scale comparison experiments (e.g. comparing growth rates at different temperatures)

[0156] • Modified plastic trays for larger-scale comparisons, and sample production runs (see details below)

[0157] • Plastic Rotary drum. Plastic small drum with manual rotary handle to assist mixing.

[0158] • Disposable aluminium foil trays. Serving kitchen trays made of aluminium foil with a cover.

[0159] • Aluminium trays. Bakery aluminium trays with cover.

[0160] • Aluminium trays with air passage. Bakery aluminium trays with holes in the wall covered with air permeable membrane to assist air passage.

[0161] • Aluminium trays with stacked racks inside. Mesh trays are stacked inside the aluminium trays.

[0162] • Aluminium trays with air pipes. Plastic pipes inserted inside the aluminium trys to assist air supply.

[0163] • Aluminium trays with sensors. Tray covers are mounted with a moisture and temperature sensor to record moisture and temperature during fermentation.

[0164] Incubators

[0165] Different modifications in the incubator were performed to test the effect of environmental variables on the fermentation.

[0166] • Closed chamber incubator. A close chamber incubator with controlled humidity and temperature and without air supply

[0167] • Incubator with fan. A fan placed inside the incubator to assist homogeneous air mixing

[0168] • Incubator with moisture on tray. A tray with water was placed inside the incubator to ensure high moisture content in the incubator. Humidity was measured using a humidity sensor.

[0169] Plastic trays

[0170] 40x20cm plastic trays were purchased and modified for SSF:

[0171] • Air holes were drilled for gas exchange and covered with porous film to prevent contamination. • Temperature and humidity sensors were bolted on, for measuring these variables.

[0172] Figure 2 shows example plastic trays with a humidity sensor used to conduct SSF.

[0173] Fermentation conditions

[0174] Methods were tested and optimised for the fermentation itself, including:

[0175] • Nitrogen Source: Urea (5 g / L), ammonium sulphate (5 g / L, then 2.5 g / L), yeast extract (2.5 g / L).

[0176] • Nutrient salts: Magnesium sulphate and Potassium dihydrogen orthophosphate (2g / L, 0.5 g / L).

[0177] • Carbon Source: Glucose (20 g / L) or without glucose

[0178] • Temperature: Fermentations were tested at 30 °C, 35 °C. Temperature was set and maintained using incubator units, and monitored both by observing the incubator unit temperature gauge occasionally, and constantly through wireless temperature sensors.

[0179] • Humidity. Humidity was monitored, but could not be adjusted, beyond setting initial moisture of the substrate, >85% and 95%

[0180] • Period. Fermentation period: 3 days to 7 days.

[0181] • Bed depth. Depth of substrate was2.5 cm, 3cm, 4cm, 6cm, or 7cm

[0182] Protein extraction

[0183] To test the protein extraction process, certain key steps needed to be tested:

[0184] • Physical breakdown of the fermented mix

[0185] • Chemical lysis of the fermented mix

[0186] • Separation of unwanted solids

[0187] • Protein extraction I precipitation and separation of protein fractions

[0188] • Drying

[0189] Breakdown of fermented mix

[0190] The first step after fermentation was to transfer the fermented mix to glass vessels then physically breakdown the fermented mix in order to be able to start extracting protein. A vital part of this step is to break down the cells of the microorganism to release the proteins inside, thereby maximising protein yield.

[0191] Several methodologies were tested for this step:

[0192] • Sieve: The first breakdowns were performed by physically pushing the mix through a sieve

[0193] • Hand-held ‘stick’ homogeniser

[0194] • Manual ‘malt-muncher’ crushing unit

[0195] • Either of the steps above, followed by squeezing with a cheese cloth

[0196] • Colloid mill, wet milling

[0197] • High shear mixer

[0198] • Either step above, combined with centrifugation to separate the solid fractions.

[0199] This step also required the addition of liquid, to better facilitate physical breakdown, various quantities of water and PBS were tested for this.

[0200] It was considered that at scale the best equipment item for this step would be a High- Pressure Homogeniser (HPH), however for reasons of budget constraints this was not tested.

[0201] Chemical lysis of the fermented mix

[0202] The homogenised mix then had sodium hydroxide added to take the mix to pH 10.502, and it was heated to 65 °C70 degrees C for 3 hours.

[0203] A number of other conditions were also tested with pH ranging from 8.5 to 12, temperature ranging from 50 °C, to 70 °C, and time from 3 hours to 12 hours. Sodium hydroxide lysis was tested at 0.05 M to 0.22M concentration (final), Bicarbonate of soda at 1 M was also tested. Dilution (solid: liquid) tested from 1 :2 to 1 :6.

[0204] Separation of the unwanted solids A cheesecloth was used to separate the BSG husks and other debris from the liquid. This was either done by manually squeezing the bag filled with the homgenised / lysed mix or using a press to push the liquid out. The partially clarified slurry was then centrifuged at 4000 rpm and the supernatant recovered. In some instances the cheesecloth was not used due to fouling.

[0205] Protein extraction and precipitation

[0206] Protein extraction then followed by gradual reduction of the pH through addition of hydrochloric acid. At any pH where protein precipitated, that fraction would then be spun off in the centrifuge, before continuing with the pH drop to identify other pH fractions for extraction. This step would be done at a chilled temperature, either through leaving in the fridge overnight, or packing in ice.

[0207] The outcome of this step would be a series of protein fractions extracted at different pH’s. These could either be combined as a ‘mixed’ final product, or tested as separate products.

[0208] As an alternative to acid precipitation salt precipitation was used. In this instance the cleared lysate was brought to a 50% saturation with ammonium sulphate. The precipitate was recovered by a combination of centrifugation and gravity fed filtration. The recovered precipitate showed very high protein content (~98%). However, to take this forward alternative salts would need to be trialled, with some sort of diafiltration step.

[0209] Drying

[0210] The mixed or separate fractions were then dried, either by leaving at room temperature, or in an oven at 50 °C, 55 °C or 70 °C or drying cabinet.

[0211] Freeze Drying of the paste was also investigated, due to time constraints and available throughput this has not been pursued further.

[0212] Spray drying of the paste was also tried at pilot scale. Additional homogenisation of the past was required prior to spray-drying.

[0213] In some instances the final product was left as a paste with a moisture content of approx 75%. Optional Extra processing steps: removal of Colouration and Allergens

[0214] To make the product suitable for use as an ingredient in vegan cheese, it was discovered that extra, unanticipated process steps would be required to:

[0215] • Address Colouration; and optionally

[0216] • Remove allergens.

[0217] Colouration

[0218] For use as an ingredient in vegan cheese, the product needed to have a light yellow colour. Untreated fractions would come out much darker than this, so a bleaching protocol was established, consisting of these steps:

[0219] • Heating at 70 degrees C overnight with hydrogen peroxide

[0220] • Removal of excess hydrogen peroxide using a peroxidase enzyme treatment

[0221] • Testing for presence of hydrogen peroxide, using hydrogen peroxide test strips

[0222] Removal of allergens

[0223] One clear requirement for the final product was that it include no common food allergens. As some of the substrates tested, particularly BSG, contained glutens, a gluten removal process was developed, consisting of:

[0224] • Addition of Brewers Clarex® (an enzyme treatment used in production of gluten-free beer)

[0225] • Testing for presence of glutens, using gluten test strips.

[0226] This is an example of the specific steps from a typical protein extraction:

[0227] Day 1 : Milling fermented BSG with grinder

[0228] • Addition of 0.05 M NaOH, heat treatment ate 65 °C for 3h. To recover the secreted protein, for 1 tray do one wash with PBS, followed by centrifugation, then add 0.05M NaOH into the remaining BSG and leave it in the oven for 3H.

[0229] • Take the beakers out of the oven and sift the BSG through the sift.

[0230] • Place the BSG into centrifuge tubes, centrifuge it down at 5300 RCF

[0231] • Adjust the pH down to 10 then 6 and finally to 3.5 with subsequent centrifugation and protein recovery. After adjusting the pH to 3.5, leave the beaker at the cold room overnight.

[0232] Day 2: Repeating the steps 1 -5 for successive trays • Centrifugation of tubes from day 1 , collecting the pellet for oven drying or Clarex treatment.

[0233] Day 3: Add 1 % v / v of Clarex, let the test tube sit at RT until 7h.

[0234] • Centrifuge the tube to remove the moisture.

[0235] Day 4: Steps 7-9 for the successive, trays. Perform the gluten Test.

[0236] Figure 3 is a diagram of one example of the general process for protein extraction as exemplified herein. The steps show raw material handling, mixing and / or homogenisation, alkaline lysis, protein precipitation and drying . The steps are as follows:

[0237] 1 ) Ferment (SSF) the BSG with Aspergillus oryzae

[0238] 2) Mixing and / or homogenisation with water;

[0239] 3) Addition of 0.05M NaOH and heat at 65 °C for 3 hours. The aim is to assist in cell lysis and solubilise the majority or preferably all the proteins

[0240] 4) Squeeze the BSG to separate the bulk solids from the protein

[0241] 5) Centrifuge and remove fine solids (the protein still be in solution)

[0242] 6) Add HCL to titrate at pH 6, pH 5 and pH 4, centrifuging after each pH I titration point

[0243] 7) Incubate for approx, at least 1 hour (or overnight) in the cold room

[0244] 8) Centrifuge to remove liquids (e.g. at 4,000 rpm for 10 mins) and collect the paste

[0245] 9) Dry in oven at 50 °C

[0246] For catalase treatment: a) Add catalase to 1 % v / v, incubate at RTP for approx. 1 hour b) Add 1 % v / v Clarex to digest glutens, incubate at RT for approx, at least 5 hours (e.g. overnight)

[0247] 10) Package the product

[0248] Results

[0249] The research had the following key objectives:

[0250] • Produce a product that would meet the approval of an industrial food ingredient manufacturer, in terms of taste, texture, nutrition and colour. • Achieve a yield that would allow for profitable production, based on a production quantity of 500 tons per year.

[0251] • Use a process whereby the costs of production at that production scale would be profitable.

[0252] Meeting these objectives required adapting each stage of the process to achieve the desired result, and often involved trade-offs between the desired results.

[0253] The project went through 3 phases of development

[0254] 1. Theoretical design

[0255] 2. First round testing

[0256] 3. Adaption and re-testing

[0257] 4. Third party

[0258] Theoretical design

[0259] The first theoretical version of the process was tested through development of a Techno-Economic Model (T / E model), to test theoretical profitability. The results of this first model showed a sufficient theoretical profit level. This profit level was deemed high enough to continue to laboratory testing.

[0260] First Round Testing

[0261] For the first laboratory testing, key stages of the process required initial parameter setting and testing. The results of those tests were:

[0262] Solid-State Fermentation

[0263] Organism choice

[0264] The shortlist of 6 organisms were put through the fermentation and extraction processes, which were compared for product quality and yield.

[0265] Aspergillus Oryzae was chosen as the organism to move forward with sample production. It is advantageous, because:

[0266] • It had the highest yield

[0267] • Product produced had a suitable texture and flavour • It is well known and characterised in food production systems, with no mycotoxins produced, regardless of conditions

[0268] Substrate choice The results of the comparison of the different substrates was as follows:

[0269] Based on these results, un-milled BSG was selected as the substrate to move forward for second-round sample production. Advantageously., a better yield was found in non-milled vs milled BSG. It was hypothesised that this was due to better gas exchange in the un-milled form outweighing the better carbon access of the milled form. Avoiding the milling step was noted to also confer a cost advantage.

[0270] Within this, a further comparison was made between yields of different BSG’s, including from BSG for stout, IPA, and lager. Advantageously, of these, three, the lager BSG was selected, because of:

[0271] • Marginally higher yield

[0272] • More general availability

[0273] • Lightest colour

[0274] Though the BSG substrates of other brews (such as IPA and stout) were also deemed acceptable, if colour of the food product is less of a requirement.

[0275] Fermentation equipment

[0276] Trays showed excellent performance as the key equipment unit for fermentation. It was decided to continue with this equipment item at pilot scale, and that larger trays would be tested. The process may be scaled up using an automated fermentation system.

[0277] Protein extraction

[0278] Breakdown of fermented mix

[0279] The most effective method for homogenisation and breaking down of the fermented mix at this scale of production was found to be use of a Colloid mill (wet milling) and recirculating the mix for several minutes, until a foam starts to appear.

[0280] •

[0281] The correct choice of methods and equipment at this stage had an effect on final yield. This is because it determines whether the protein fraction from the BSG substrate was subsequently extracted or not. Experimentation showed that approximately 50-60% of protein extracted was from this source. At industrial scale, it is anticipated that a homogeniser would be advantageous, for example a High-Pressure Homogeniser (HPH), high shear mixer and / or colloid mill as mentioned above. Due to their cost, we were not able to purchase or trial a high pressure homogeniser (HPH). Protein extraction

[0282] The optimum method was found to be to start at pH 10.50, 65 °C for 3 hours. After mechanical homogenisation and chemical lysis the pH was dropped through the full pH range, and centrifuging out any fraction that precipitated. Over time, regular precipitations were found at the following pH points, and assumptions made about them:

[0283] • pH 11 . Secreted fungal protein. Generally the smallest fraction, this would also be the ‘highest quality’ with the lightest colour and highest protein functionality.

[0284] • pH 4.5-5.5. Fungal protein (assumed mix of biomass and secreted). Generally the largest or second largest fraction, this was identified as the ‘core product’ of interest.

[0285] • pH 3.5. BSG fraction. Generally the largest, or second-largest fraction, this was identified as contributing significantly to yield, but also as the ‘lowest quality’ and requiring the highest degree of processing for colour and allergens.

[0286] Drying

[0287] Freeze drying may be used. However, we found that Spray-drying gives a preferred outcome in terms of solubility, appearance and taste.

[0288] Optional Extra processing steps

[0289] Colouration

[0290] The hydrogen peroxide bleaching step and subsequent removal of hydrogen peroxide through enzyme treatment was found to give a satisfactory and desirable colour result.

[0291] Removal of allergens

[0292] The clarex application was found to achieve acceptable gluten removal, however in some cases the step would have to be repeated to achieve less than 1 ppm.

[0293] Removal of lipds / fat Testing of protein content by BCA or Kjeldahl assay gave a result of 40 - 50% purity. A number of post lysis methods were tested to improve purity. This includes cold treatment, and cold treatment to clarify the lysate and separating the higher fat content from the lower fat content layer. Results showed this method offered no improvement and wouldn’t be practical at scale. T reatment with hexane on the final product did show a potential to improve purity, though with significant loss to overall yield.

[0294] First round sample testing

[0295] Samples from fermentations on different substrates were reviewed with an industrial producer of vegan cheese, and the sample chosen for progression was ‘Sample #4’: Aspergillus oryzae, grown on BSG, and catalase-treated to remove glutens. This product was then selected for second round sample production.

[0296] Figure 4 shows the colouration of the protein, indicating that Untreated BSG would ideally be treated (e.g. bleached) to improve its colour, whilst Sample #4 was at the cusp of acceptability in terms of its colouration (for alternative / vegan use, as described above). Ideally, a beige, cream, off-white or white coloured protein is preferred for such alternative food product uses, e.g. vegan cheese. This needs to be matched against yield of protein (from the carbon source / substate) to make the process (and resulting food product) cost effective.

[0297] Second-Round Testing

[0298] In the second round of testing, the product chosen from the first round (‘Sample #4’) was produced at higher batch volumes (350g @ 75% moisture per batch), and the process was optimised for yield.

[0299] There were several difficulties that had to be overcome:

[0300] Enzyme treatment steps

[0301] It was initially found that the choice of BSG as a substrate meant that 2 enzyme treatment steps had to be included in the process: to remove hydrogen peroxide and glutens. It was found that the addition of these steps caused a significant drop in extracted protein yield, from 8% down to 2-3%. It was then determined to change the sequence of steps, and do enzyme treatment at the end of the process. This caused yields to return to 8% according to this example extraction process.

[0302] It was subsequently found that enzyme treatment to remove allergens should be applied at the point of recovering the precipitated paste, prior to drying. This gave the best recovery in terms of final yield. Allergens are not always removed.

[0303] Protein fractions

[0304] Throughout the experiments, it was found that protein fractions precipitated at different isoelectric points. The fractions were tested separately In some instances the recovered fractions were mixed and stored together. In most instances the fractions are stored separately as the high pH fraction 4 - 6 gives a superior taste score. Samples were stored and presented in small pots for review, as shown in Figure 5.

[0305] Yields

[0306] The best total recovered yield using a colloid mill gave a yield of 13.1 % on a dry weight to dry weight basis, final product to dry substrate. The current techno-economic model shows an economical model becomes viable at 12% by dry weight.

[0307] By yield, we mean extracted protein, i.e. protein recovered from the extraction that can ultimately be used in a food product. This will typically be in solid form. In some embodiments, it may then be later solubilised if useful.

[0308] Updated economic modelling

[0309] Based on the final process selected, the techno-economic model was amended to include the optimisations and process refinements identified. This led to the following conclusion: that the present method produces alternative protein at a significantly lower cost point than liquid (SMF) bioreactor fermentation methods.

[0310] Discussion and Conclusions

[0311] The development of a methodology that is able to produce a viable product at sufficient yields is significant in several ways. Scaling production of this product

[0312] Having established a methodology that can successfully and economically produce a useful product at laboratory scale can now lead directly to the establishment of a scaled production process.

Claims

Claims1 . A method of producing protein suitable for use in a human food product, the method comprising: a. Solid-State Fermentation (SSF) of a carbon source using a fungus suitable for human food production, the carbon source optionally having at least 10% protein by dry weight (w / w); and b. extraction of protein from the fungal biomass and optionally the carbon source.

2. The method of claim 1 , further comprising: c. providing the extracted protein in solution, suspension or as a powder.

3. The method of claim 1 or 2, wherein the extracted protein is solubilized in an aqueous medium and / or, optionally, precipitated.

4. The method of any one of the preceding claims, wherein extracted protein is provided in a powdered form for inclusion in a human food product.

5. The method of any one of the preceding claims, wherein the carbon source is, or is derived from, waste materials from the food or agriculture industries.

6. The method of claim 5, wherein the carbon source is one or more of: Brewers’ Spent Grain (BSG), wheat bran, potato peel, bread waste or bread crusts, beet pulp, pea husks, fruit pomace, broken rice, soybean husk, sugar cane bagasse, koji waste, coconut coir, and / or oat sidings.

7. The method of claim 6, wherein the carbon source is Brewers’ Spent Grain (BSG).

8. The method of any one of the preceding claims, wherein the protein extraction further comprises bleaching the extracted protein.

9. The method of any one of the preceding claims, further comprising removal of allergens, optionally wherein gluten is removed enzymatically.

10. The method of any one of the preceding claims, wherein the fungal species is Aspergilus sp. Rhizopus sp., Penicillium sp., and / or Neurospora sp..11 . The method of any one of the preceding claims, wherein the fungus is, or includes one or more of: Trichothecium roseum, Aspergillus fumigatus, Fusarium venenatum, Rhizopus delemar, Neurospora intermedia, Trichoderma reesei, Rhizopus oryzae, Penicillium camembertii, Aspergillus awamori, Mucor racemosus, Mucor lanceolatus, Pencillium commune, Trichoderma harzianum,Monascus purpureus, Neurospora intermedia, Penicillium nalgiovense, Penicillium roqueforti, Fusarium domesticum, Geotrichum candidum, Rhizopus oligosporus and Aspergillus oryzae and / or Aspergillus sojae.

12. An isolated or extracted mixture of proteins produced according to the method of any one of the preceding claims.

13. An isolated or extracted mixture of proteins comprising a mixture of fungal biomass and, optionally also proteins from a carbon source.

14. The isolated mixture of proteins according to claim 12 or 13, wherein the carbon source is one or more of: Brewers’ Spent Grain (BSG), wheat bran, potato peel, bread waste or bread crusts, beet pulp, pea husks, fruit pomace, broken rice, soybean husk, sugar cane bagasse, koji waste, coconut coir, and / or oat sidings.

15. A human food product comprising the protein mixture according to any one of claims 13-14.

16. The food product of claim 15, which is vegan cheese, sports protein powder or nutritional supplement, confectionary and baked goods, condiments and seasonings, high protein snacks, ready meals, plant based meats, chocolate replacements, pet foods, soups, noodles, sauces and dressings.

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