Protein production method

Abstract

A method suitable for producing a protein suitable for human consumption comprises solid state fermentation (SSF) of a carbon source such as Brewer’s Spent Grain (BSG) having at least 10% protein by d

Description

Background 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. The UK is legally committed to net-zero carbon emissions by 2050. Agriculture was the source of 49MtCO2e 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. 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. 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. The fungus is suitable for human food production and the carbon source must have at least 10% protein by dry weight (w / w). Extraction of protein from both the fungal biomass and the carbon source thereby provides a high-protein content product, suitable for human food, and in particular for use in so-called “vegan cheese” and other human food products containing appropriate protein for human consumption. Summary of the Invention 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 having at least 10% protein by dry weight (w / w); and b. extraction of protein from both the fungal biomass and the carbon source. In some embodiments, the carbon source is Brewers’ Spent Grain (BSG). 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, 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, gluten is removed enzymatically. In some embodiments, the fungal species is Aspergilus sp. Also provided is an isolated mixture of proteins produced according to the present method. Also provided is a food product comprising the protein mixture. In some embodiments, the food product is a vegan cheese. Brief Description of the Figures Aspects of the invention will now be described with reference to the following figures, wherein: Figure 1 shows 5g of wheat bran in a 250ml Erlenmeyer flask; Figure 2 shows example plastic trays used to conduct SSF; Figure 3 is a diagram of one example of the process as exemplified herein. The second step shows mixing and / or homogenisation; 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 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. Detailed Description 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 (GMO) to produce animal proteins (‘Submerged Fermentation’). However, these alternatives all have drawbacks: Advantages Disadvantages Plant protein • Affordable • Readily available • Lower carbon footprint that animal proteins • Flavours. Bring off-tastes. • Colours often not suitable to target products (e.g. vegan cheese • Allergens (e.g. soy, wheat / glutens) ‘Submerged fermentation’ • Can produce almost all proteins required • Cost. High capex and production costs make them unaffordable vs animal proteins alternative proteins • Significantly lower carbon footprint that animal proteins • GMO. Not possible to sell in many markets. 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, 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. Furthermore, this production process is advantageous as it is scalable for widespread 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. In some embodiments, the fungus provides a high yield of edible protein. This is advantageous for delivering a low-cost method and resulting product. 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 BSG, is a waste material. This is also advantageous for delivering a low-cost method and resulting product. The use of SSF is also advantageous as it contributes to a significant cost reduction over submerged fermentation (SMF) systems. In some embodiments, the fungus provides a high yield of edible protein 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. 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. htips: / / dos.org / 10.3390 / foods10092159), it has not been used before for production of protein for use in human food products. In particular, it has not been used before in combination with a carbon source 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. 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 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 properties and / or colour to meet the human consumer’s requirements. 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. The carbon source is any source of carbon that meets the protein requirement of at least 10% protein by dry weight (w / w). 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 gave the best colour profile for the protein product. 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). The carbon source must have at least approximately 10% protein. This is prior to fermentation in the present method. Some fermentation may have occurred prior to the present method. This 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. 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. 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. 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. 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). 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. 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 yogurt making. 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. In some embodiments, the fungus is, or includes: 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. In some embodiments, the fungus is, or includes: • Penicillium camembertii • Penicillium nalgiovense • Penicillium roqueforti • Fusarium domesticum • Geotrichum candidum • Aspergillus oryzae In some embodiments, the fungus is or includes Aspergilus sp., in particular Aspergillus oryzae. 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 formation is stopped by homogenisation. In some embodiments, the 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. Once the fermentation has been substantially concluded, the total protein is extracted from both the fungal biomass and the carbon source. Protein extraction, in some embodiments referred to as protein isolation may be according to standard methods. In some embodiments, these may include: • extraction of protein from the fermented mix using acid / base precipitation and, • optionally, centrifugation. 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. 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. 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 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 1ppm gluten. In some embodiments, the allergen, in particular gluten, is removed to achieve less than 5, 10 or 20 ppm gluten. 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. In some embodiments, both the bleaching and the allergen removal occur after the protein has been precipitated. This is advantageous because treating too early in the process loses protein yield. 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 (as exemplified in Figure 3 by addition of catalase and / or 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). In any of the embodiments described herein, if bleaching is used, this may be in a single step or in multiple steps, using either the same enzyme in each case (e.g. a Catalase or 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. 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. 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. 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, optionally following homogenisation, straining or separation of solids, e.g. through a filter, sieve or cheese cloth, is also provided as part of the extraction of protein. 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: • approx.. pH3.5 (in some embodiments approx. pH3 to 4) for the BSG-derived protein fraction; • 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 • approx.. pH 11 (in some embodiments approx.. 8.0 or 10.5 to 11.5) for secreted fungal protein. 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 11 pprox.. 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%. Example The substrate referred to in the Example is the carbon source described above. Methodology The research was divided into 2 phases: • Desk research to develop a theoretical process for testing; followed by • Laboratory testing to run and optimise the process. Desk Research 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. • Plant protein extraction. Using acid / base precipitation as well as centrifugation and filtration, to extract proteins from crops such as soy, pea, and wheat. • Solid-State Fermentation (SSF). Growing filamentous fungi on solid substrates, often on waste materials such as the spent grains from breweries (BSG). 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. 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. 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. To test this hypothesis, a theoretical process was developed, consisting of 2 key stages: • Solid-state fermentation of BSG, using a filamentous fungus, • Followed by extraction of protein from the fermented mix using acid / base precipitation and centrifugation. 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. Laboratory Testing and Optimisation The process was broken into 2 parts, with separate research teams focusing on each: • Solid-State Fermentation • Protein extraction Solid-State Fermentation To test the SSF process, several key factors were tested and optimised for: • Choice of organism • Choice of substrate • Substrate preparation • Fermentation equipment • Fermentation conditions Choice of Organism A list of potential organisms was researched, covering filamentous fungi from numerous genera, including: Trichothecium roseum, Aspergillus fumigatus, Fusarium venenatum, Rhizopus delemar, Neurospora intermedia, Trichoderma reesei, Rhizopus oryzae, Penicillium camembertii, Penicillium nalgiovense, Penlcillium roqueforti, Fusarium domesticum, Geotrichum candidum, and Aspergillus oryzae These were then reviewed using the following requirements matrix, to select a shortlist of organisms for testing: Must haves t Strong preference tfMiee to have ihigh score hoes it jproduce jtwim? lean we get a IS ts ft geneticaiiy characterised son wi ipsuited tn genetic manipufatkx: Yes is It known w grow wii mi BSG? Yes bs it .used in ifood :¥es What flavour or aroma does it have? Cheesy What is Its native protem content? H<gh Can it grew on ‘Does ti ; alow mmstweJpcsxhire <40% -Kors t Negative srere ------i must have must have Maybe____ No tMoybe ;NO None Sad smell Medium low thigh 1 Based on these criteria, 6 organisms were shortlisted: • Penicillium camembertii • Penicillium nalgiovense • Penicillium roqueforti • Fusarium domesticum • Geotrichum candidum • Aspergillus oryzae 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. Choice of substrate A shortlist of substrates was researched for testing: • 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) • Fresh potatoes. Low-grade potatoes, acquired as sample from a potato wholesaler. The potatoes were cut into small cubes. • Potato starch. Waste by-product from a factory that extracts protein from potatoes. • Apple pomace. A waste by-product from cider pressing. Was provided both in fresh and dried forms. • Foam. Foam was tested as a physical substrate, with addition of extra nutrients for carbon and nitrogen sources. • Wheat bran. Tested in milled and un-milled forms. Figure 1 shows 5g of wheat bran in a 250ml Erlenmeyer flask. Samples of each were procured and then compared by growing a range of organisms on each, and comparing for yield and product quality. Substrate preparation Various methods and factors were compared for the preparation of substrate: • Sterilisation method • Moisture level • Nutrients • Inoculum quantity Sterilisation Maintaining sterility is vital in SSF. Several methodologies were developed and tested: • 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. • Sterilisation of substrate. The substrate here is the feedstock, also referred to as the carbon source. Several methods of sterilisation substrate were compared: o Autoclaving and drying o Drying only o Autoclaving only o Neither autoclaving nor drying • Maintaining sterility during inoculation. All inoculations were completed in a lateral flow hood with air extraction and sterilisation equipment on hand. • 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. Moisture level Substrate was taken from dry and taken to a moisture level of 75%. 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. Fermentation equipment 2 types offermentation equipment were used: • 250ml shake flasks for small scale comparison experiments (e.g. comparing growth rates at different temperatures) • Modified plastic trays for larger-scale comparisons, and sample production runs (see details below) Plastic trays 40x20cm plastic trays were purchased and modified for SSF: • 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. Figure 2 shows example plastic trays used to conduct SSF. Fermentation conditions Methods were tested and optimised for the fermentation itself, including: • Nitrogen Source: urea and ammonium sulphate • Carbon Source. 20g glucose • Temperature. Tested at 30 degrees 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. • Humidity. Humidity was monitored, but could not be adjusted, beyond setting initial moisture of the substrate. • Period. Fermentation period: 7 days. • Bed depth. Depth of substrate was 3cm • Substrate Sterilisation: drying and autoclaving (15 minutes) • Tray Volume: 5L Protein extraction To test the protein extraction process, certain key steps needed to be tested: • Physical breakdown of the fermented mix • Separation of unwanted solids • Protein extraction I precipitation and separation of protein fractions • Drying Breakdown of fermented mix 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. Several methodologies were tested for this step: • Sieve. The first breakdowns were performed by physically pushing the mix through a sieve • Hand-held ‘stick’ homogeniser • Manual ‘malt-muncher’ crushing unit • Either of the steps above, followed by squeezing with a cheese cloth • Either step above, combined with centrifugation to separate the solid fractions This step also required the addition of liquid, to better facilitate physical breakdown, various quantities of water and PBS were tested for this. 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. Protein extraction and precipitation The homogenised mix then had sodium hydroxide added to take the mix to pH 12, and it was heated to 70 degrees C. 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. 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. Drying The mixed or separate fractions were then dried, either by leaving at room temperature, or in a 70 degree C oven or drying cabinet. Extra processing steps - Colouration and Allergens 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: • Address Colouration; and optionally • Remove allergens. Surprisingly, the sequence of these steps within the whole process was found to be important in the overall yield. These steps were tested both prior to and subsequent to protein precipitation / extraction steps, and yields compared. Colouration 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: • Heating at 70 degrees C overnight with hydrogen peroxide • Removal of excess hydrogen peroxide using a peroxidase enzyme treatment • Testing for presence of hydrogen peroxide, using hydrogen peroxide test strips Removal of allergens 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: • Addition of Clarex (an enzyme treatment used in production of gluten-free beer) • Testing for presence of glutens, using gluten test strips. This is an example of the specific steps from a typical protein extraction: 1. Day 1 : Milling BSG with grinder 9 am Wednesday 2. 9:30 am Addition of 0.1 N NaOH, placement into the 70 C for 2h. To recover the secreted protein, for 1 tray do one wash with PBS, followed by centrifugation, then add 0.1M NaOH into the remaining BSG and leave it in the oven for 3H. 3. 11:30 take the beakers out of the oven and sift the BSG through the sift until 12:00 4. Place the BSG into centrifuge tubes, centrifuge it down at 4000 RPM done by 12:30 5. 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 for overnight. 6. Day 2: From 9:30 to 13:00 repeating the steps 1 -5 for second two trays 7. 13:00 centrifugation of tubes from day 1, collecting the pellet 8. 14:00 Addition of 12% , 1 % v / v peroxide to first portion of protein and leaving overnight till Friday at 70C 9. Day 3: 9am- take out the peroxide flask out of the oven, put into the cold room for 10 min to let it cool. io. Check the pH, it should be around 4 and add the catalase 3mI dissolved in small quantity of pH 4 PBS. Leave the protein at room temp for 3H n. Add 1 % v / v of Clarex, let the test tube sit at RT until 4pm. 12. For the second tray: steps 8-9 13. For tray 1: do the peroxide test. 14. Centrifuge the tube to remove the moisture. 15. Day 4: Steps 9-14 for the last 2 trays. Perform the gluten Test Figure 3 is a diagram of one example of the general process as exemplified herein. The second step shows mixing and / or homogenisation. The steps are as follows: 1) Ferment (SSF) the BSG with Aspergillus oryzae.', 2) mixing and / or homogenisation; 3) addition of 0.1M NaOH and heat at 70 degrees C for 3 hours. The aim is to solubilise the majority or preferably all the proteins; 4) Centrifuge and remove solids (the protein still be in solution) 5) Squeeze the BSG to remove protein 6) Centrifuge again 7) Add HCL to titrate at pH 10, pH 6 and pH 4, centrifuging after each pH / titration point 8) Incubate for approx, at least 5 hours (e.g. overnight) in the cold room 9) Centrifuge to remove liquids (e.g. at 4,000 rpm for 10 mins) 10) Add Peroixde 12% 11) Incubate at 70 degrees C to whiten the protein, (e.g. for approx, at least 5 hours e.g. overnight) 12) Add catalse to 1 % v / v, incubate at RTP for approx. 1 hour 13) Perform a peroxide test 14)Add 1 % v / v Clarex to digest glutens, incubate at RTP for approx, at least 5 hours (e.g. overnight) 15) Perform the Gluten test 16) Package the product Results The research had the following key objectives: • Produce a product that would meet the approval of an industrial vegan cheese 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. • Use a process whereby the costs of production at that production scale would be profitable. Meeting these objectives required adapting each stage of the process to achieve the desired result, and often involved trade-offs between the desired results. The project went through 3 phases of development 1. Theoretical design 2. First round testing 3. Adaption and re-testing Theoretical design 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. First Round Testing For the first laboratory testing, key stages of the process required initial parameter setting and testing. The results of those tests were: Solid-State Fermentation Organism choice The shortlist of 6 organisms were put through the fermentation and extraction processes, which were compared for product quality and yield. Aspergillus Oryzae was chosen as the organism to move forward with sample production. It is advantageous, because: • It had the highest yield • 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 Substrate choice The results of the comparison of the different substrates was as follows: Substrate Pro’s Con’s D C? / "* DuU • Good organism growth and yield • Substrate contains protein, which further enhances yield • Dark colour of output protein • Presence of allergens (glutens) Potatoes • No allergens present • Low yields • Colour treatment still required Potato starch • As an industrial product, is very consistent • Very low organism growth Apple pomace • Accessible carbon content • Fresh apple pomace is seasonal, only available Aug-Nov. Dried apple pomace too expensive. Foam • Provided a purer, higher-quality product • Very low yield (hypothesised because the carbon source was too easy access for the organism, little protein production required) Wheat bran • Readily available substrate • Lower yield than BSG (hypothesised due to low intrinsic protein content of substrate) • Higher cost than BSG (due to being an agricultural product, rather then a waste stream) 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. 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: • Marginally higher yield • More general availability • Lightest colour 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. Fermentation equipment 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. Protein extraction Breakdown of fermented mix The most effective method for homogenisation and breaking down of the fermented mix at this scale of production was found to be: • Initial homogenisation using the malt-mulcher, followed by • Full homogenisation with a hand-stick blender, followed by • Separation of solids with a cheese cloth The correct choice of methods and equipment at this stage had an effect on final yield. This is because it determined 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. For example, by missing the hand blender step, yields would drop by over 50%. At industrial scale, it is anticipated that a homogeniser would be advantageous, for example a High-Pressure Homogeniser (HPH) as mentioned above. Due to their cost, we were not able to purchase one for this work. Protein extraction The optimum method was found to be to start at pH 12, and drop down 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: • pH 11. Secreted fungal protein. Generally the smallest fraction, this would also be the ‘highest quality’ with the lightest colour and highest protein functionality. • 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. • 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. Drying Initial sample runs produced a dried protein powder, however this was found to darken the colour and alter the chemical characteristics of the product. Therefore, it was decided not to proceed with a drying step, and to present the product post-centrifugation with liquid fraction removed, and a moisture level of 75%. Extra processing steps Colouration The hydrogen peroxide bleaching step and subsequent removal of hydrogen peroxide through enzyme treatment was found to give a satisfactory and desirable colour result. Advantageously, it was found that the longer the hydrogen peroxide step was maintained, the better colouration was achieved, with a minimum of overnight at 70 degrees C. Removal of allergens 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 1ppm. First round sample testing 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, colour treated with hydrogen peroxide, and catalase-treated to remove glutens. This product was then selected for second round sample production. 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. Second-Round Testing 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. There were several difficulties that had to be overcome: Enzyme treatment steps 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. Protein fractions Throughout the experiments, it was found that protein fractions precipitated at different isoelectric points. The fractions were tested separately, and mixed together, for their properties as a final product. It was decided to mix these fractions for the purpose of sample review and presentation. Samples were stored and presented in small pots for review, as shown in Figure 5. Yields Early in the experiments, yield was defined as ‘dry yield’: weight of dried protein product I weight of dried BSG substrate per fermentation. As drying was found to have negative consequences, the yield definition was then altered to ‘wet yield’: weight of 75% moisture protein product / weight of 75% moisture BSG substrate per fermentation. The target yield, above which production was economically viable, was determined to be 4-5%. Actual yields achieved from the research: • Phase 1 (early BSG samples without enzymatic treatment i.e. bleaching and / or removal of allergens): 8% • Phase 2 (BSG samples with enzymatic treatment to bleach and remove allergens): 2-3% • Phase 3 (BSG samples with enzymatic treatment (to bleach and remove allergens) at the end, i.e. after precipitation of the combined protein from the fungal biomass, secreted fungal protein and the BSG-derived protein): 8% 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. Updated economic modelling 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. Discussion and Conclusions The development of a methodology that is able to produce a viable product at sufficient yields is significant is several ways: Scaling production of this product 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

1. 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 having at least 10% protein by dry weight (w / w); andb. extraction of protein from both the fungal biomass and the carbon source.

2. The method of claim 1, wherein the carbon source is Brewers’ Spent Grain (BSG).

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

4. The method of claim 3, wherein the bleaching comprises addition of oxygen free radicals.

5. The method of claim 4, wherein the free radicals are provided by hydrogen peroxide.

6. The method of claim 5, further comprising removal of peroxide after bleaching by addition of a peroxidase.

7. The method of any one of the preceding claims, further comprising removal of allergens.

8. The method of claim 7, wherein gluten is removed enzymatically.

9. The method of any one of the preceding claims, wherein the fungal species is Aspergilus sp.

10. An isolated mixture of proteins produced according to the method of any one of the preceding claims.

11. An isolated mixture of proteins comprising a mixture of fungal biomass and proteins from a carbon source.

12. The isolated mixture of proteins according to claim 10 or 11, wherein the carbon source is BSG.

13. A food product comprising the protein mixture according to any one of claims 11-13.

14. The food product of claim 13, which is a vegan cheese.Application No: GB2316337.1Examiner: Mr David J EvansClaims searched: 1-14Date of search: 19 April 2024Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X,E 1-14 WO 2024 / 003323 Al (MUSHLABS GMBH) whole document of interest, especially see processes illustrated by figures 1 &2. X 1-14 EP 4082355 Al (SVAMPSSON HOLDING AB) especially refer to the process shown in figure 1 and paragraphs [0016]-[0028], X 1-14 EP 4190164 Al (CIRCULAR FOOD SOLUTIONS) in particular see figure 3 and paragraph [0009] onwards.Categories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family J7 Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From A23J 0001 / 00 01 / 01 / 2006 A23J 0003 / 14 01 / 01 / 2006 A23J 0003 / 30 01 / 01 / 2006 A23L 0033 / 17 01 / 01 / 2016 C07K 0001 / 00 01 / 01 / 2006 C12N 0001 / 14 01 / 01 / 2006Application No: GB2316337.1Examiner: Mr David J EvansClaims searched: 1-14Date of search: 19 April 2024Patents Act 1977Corrected Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance v A 1-14 EP 4082355 Al (SVAMPSSON HOLDING AB) in particular refer to process shown in Figure 1 and paragraphs [0016]-[0028], X 1-14 Carbohydrate Polymers, vol. 199, 2018, Amorin et al, "Single-step production of arabino-xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewer' spent grain", pages 546-554. whole brief paper of interest, especially pages 548-552. X 1-14 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. whole brief disclosure of interest, especially see page 25967 onwards.Categories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From A23J 0001 / 00 01 / 01 / 2006 A23J 0003 / 14 01 / 01 / 2006 A23J 0003 / 30 01 / 01 / 2006 A23L 0033 / 17 01 / 01 / 2016Subclass Subgroup Valid From C07K 0001 / 00 01 / 01 / 2006 C12N 0001 / 14 01 / 01 / 2006

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