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WO2025088342A1 - Procédé de production de protéines - Google Patents

Procédé de production de protéines Download PDF

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Publication number
WO2025088342A1
WO2025088342A1 PCT/GB2024/052743 GB2024052743W WO2025088342A1 WO 2025088342 A1 WO2025088342 A1 WO 2025088342A1 GB 2024052743 W GB2024052743 W GB 2024052743W WO 2025088342 A1 WO2025088342 A1 WO 2025088342A1
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WO
WIPO (PCT)
Prior art keywords
protein
carbon source
bsg
extracted
extraction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2024/052743
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English (en)
Inventor
Andrew Clayton
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Fermentation Technologies Ltd
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Fermentation Technologies Ltd
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Filing date
Publication date
Application filed by Fermentation Technologies Ltd filed Critical Fermentation Technologies Ltd
Publication of WO2025088342A1 publication Critical patent/WO2025088342A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/20—Proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
    • A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

Definitions

  • a human food product can be produced by a method comprising Solid-State Fermentation (SSF).
  • SSF Solid-State Fermentation
  • 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).
  • a method of producing protein suitable for use in a human food product 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.
  • SSF Solid-State Fermentation
  • a method of producing protein suitable for use in a human food product 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.
  • SSF Solid-State Fermentation
  • a method of producing protein suitable for use in a human food product 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.
  • SSF Solid-State Fermentation
  • the method further comprises providing the extracted protein in solution, suspension or as a powder.
  • the protein is solubilized in an aqueous medium and/or, optionally, precipitated.
  • extracted protein is provided in a powdered form for inclusion in a food product.
  • the carbon source is, or is derived from, waste materials from the food or agriculture industries.
  • 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.
  • 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.
  • the fungal species is Aspergilus sp. Rhizopus sp., Penicillium sp., and/or Neurospora sp..
  • an isolated or extracted mixture of proteins produced according to the present method is also provided. 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.
  • 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.
  • 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.
  • 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 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).
  • 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.
  • 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.
  • the fungus provides a high yield of edible protein. This is advantageous for delivering a low-cost method and resulting product.
  • the carbon source may be referred to as a feedstock or substrate.
  • 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.
  • SSF submerged fermentation
  • 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).
  • the carbon source is a waste material (for example BSG).
  • 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.
  • 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.
  • 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.
  • 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.
  • Solid-State Fermentation 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.
  • fungus e.g. one or more fungal species.
  • the carbon source is any source of carbon that meets the protein requirement to support growth of the fungal species.
  • 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.
  • 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.
  • SSF Solid-State Fermentation
  • the carbon source is provided as a solid. It may in some embodiments be moist, even to the point of being a slurry.
  • 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.
  • the BSG may be from brews for stout, IPA, and lager.
  • BSG from lager brews is preferred as it gives the best colour profile for the protein product.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mycotoxins are not generated by the fungus.
  • 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.
  • 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.
  • the fungus can be a genetically-modified organism (GMO) or genetically-engineered organisms (GEO) fungus.
  • GMO genetically-modified organism
  • GEO genetically-engineered organisms
  • reference herein to a fungus 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.
  • the fungus is Rhizopus sp., Penicillium sp., or Neurospora sp..
  • 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.
  • the fungus is, or includes one or more of::
  • the fungus is or includes Aspergillus sp., in particular Aspergillus oryzae or Aspergillus sojae.
  • the fermentation may be concluded at any time, for example by harvesting the fungus.
  • the protein extraction can be started immediately, or at least within 1 or 2 hours, in which case the fermentation is stopped by homogenisation.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • the enriched paste can be used as a suspension/emulsion or subsequently dried. Therefore, in some embodiments, the method includes any of these steps.
  • 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.
  • 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.
  • the protein is bleached.
  • 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.
  • the bleaching comprises addition of oxygen free radicals. In some embodiments, the free radicals are provided by hydrogen peroxide.
  • 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.
  • 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.
  • alternative food products such as food products comprising non-dairy protein, such as vegan food products especially ‘vegan cheese
  • the extracted protein is used in an alternative food product and such an alternative food product is also provided.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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®).
  • 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).
  • bleaching 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®).
  • testing for bleaching activity after any bleaching step e.g. checking for peroxide is provided.
  • the mixture may include, but not be limited to protein from both the fungal biomass and the carbon source.
  • the food product comprising the protein mixture.
  • the food product may be a vegan cheese, or protein shake, protein bar, plant-based patty, plant-based sausage, or plant-based mince.
  • 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.
  • HPH High-Pressure Homogeniser
  • 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.
  • the extraction of protein includes acid/base titration.
  • precipitation of protein occurs between pH3 and pH12. In some embodiments, this occurs at:
  • pH 11 • approx.. pH 11 (in some embodiments approx. 8.0 to 11 .5 or 10.5 to 1 1 .5) for secreted fungal protein.
  • 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%.
  • the substrate referred to in the Example is the carbon source described above.
  • SMF Submerged Fermentation
  • Plant protein extraction Using acid/base precipitation as well as centrifugation and filtration, to extract proteins from crops such as soy, pea, and wheat.
  • SSF Solid-State Fermentation
  • T/E model 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.
  • 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.
  • BSG Brewer’s spent grain, the waste material from beer brewing, acquired from a local brewer.
  • BSG was acquired, including from brews for stout, IPA, and lager.
  • 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).
  • Potato starch Waste by-product from a factory that extracts protein from potatoes.
  • Foam was tested as a physical substrate, with addition of extra nutrients for carbon and nitrogen sources.
  • Samples of each were procured and then compared by growing a range of organisms on each, and comparing for yield and product quality.
  • Sterilisation method Maintaining sterility is vital in SSF.
  • the substrate here is the feedstock, also referred to as the carbon source.
  • the feedstock also referred to as the carbon source.
  • 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
  • Microwave based drying of substrate 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).
  • Substrate are taken from dry and taken to a moisture level of 75%.
  • 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.
  • Plastic Rotary drum Plastic small drum with manual rotary handle to assist mixing.
  • Aluminium trays with air passage Bakery aluminium trays with holes in the wall covered with air permeable membrane to assist air passage.
  • Aluminium trays with stacked racks inside Mesh trays are stacked inside the aluminium trays.
  • Aluminium trays with air pipes • Aluminium trays with air pipes. Plastic pipes inserted inside the aluminium trys to assist air supply.
  • 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 with a humidity sensor used to conduct SSF.
  • Nitrogen Source Urea (5 g/L), ammonium sulphate (5 g/L, then 2.5 g/L), yeast extract (2.5 g/L).
  • 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.
  • 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.
  • This step also required the addition of liquid, to better facilitate physical breakdown, various quantities of water and PBS were tested for this.
  • 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.
  • 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.
  • 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.
  • 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.
  • Spray drying of the paste was also tried at pilot scale. Additional homogenisation of the past was required prior to spray-drying.
  • 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.
  • FIG. 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:
  • 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)
  • the first theoretical version of the process was tested through development of a Techno-Economic Model (T/E model), to test theoretical profitability.
  • T/E model Techno-Economic Model
  • the results of this first model showed a sufficient theoretical profit level. This profit level was deemed high enough to continue to laboratory testing.
  • 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:
  • un-milled BSG was selected as the substrate to move forward for second-round sample production.
  • 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.
  • 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.
  • 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:
  • 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.
  • Freeze drying may be used. However, we found that Spray-drying gives a preferred outcome in terms of solubility, appearance and taste.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de production de protéines appropriées pour une utilisation dans un produit alimentaire destiné à la consommation humaine. Le procédé consiste à réaliser une fermentation en milieu solide (SSF) d'une source de carbone à l'aide d'un champignon approprié pour la production d'aliments destinés à la consommation humaine, la source de carbone ayant éventuellement au moins 10 % de protéine en poids à sec (poids/poids) ; et extraire la protéine de la biomasse fongique et éventuellement de la source de carbone. L'invention concerne également un mélange de protéines isolées ou extraites et un produit alimentaire destiné à la consommation humaine contenant le mélange de protéines.
PCT/GB2024/052743 2023-10-25 2024-10-25 Procédé de production de protéines Pending WO2025088342A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011097266A1 (fr) * 2010-02-02 2011-08-11 Amano Enzyme Usa, Ltd. Utilisation de protéases pour intolérance au gluten
WO2012135646A2 (fr) * 2011-03-30 2012-10-04 Alvine Pharmaceuticals, Inc. Protéases pour la dégradation du gluten
EP4082355A1 (fr) 2021-04-30 2022-11-02 Svampsson Holding AB Procédé et système de fabrication d'une biomasse riche en protéines comprenant des champignons filamenteux comestibles
EP4190164A1 (fr) 2021-12-03 2023-06-07 Circular Food Solutions AG Protéines végétales texturées à faible teneur en humidité provenant de drêches de brasserie
WO2023198856A1 (fr) * 2022-04-14 2023-10-19 KONTOR N GmbH & Co. KG Procédé de production de protéines à partir de fractions de matière issues de l'industrie alimentaire
WO2024003323A1 (fr) 2022-06-29 2024-01-04 Mushlabs Gmbh Production d'un milieu de fermentation fongique à partir de drêches de brasserie

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011097266A1 (fr) * 2010-02-02 2011-08-11 Amano Enzyme Usa, Ltd. Utilisation de protéases pour intolérance au gluten
WO2012135646A2 (fr) * 2011-03-30 2012-10-04 Alvine Pharmaceuticals, Inc. Protéases pour la dégradation du gluten
EP4082355A1 (fr) 2021-04-30 2022-11-02 Svampsson Holding AB Procédé et système de fabrication d'une biomasse riche en protéines comprenant des champignons filamenteux comestibles
EP4190164A1 (fr) 2021-12-03 2023-06-07 Circular Food Solutions AG Protéines végétales texturées à faible teneur en humidité provenant de drêches de brasserie
WO2023198856A1 (fr) * 2022-04-14 2023-10-19 KONTOR N GmbH & Co. KG Procédé de production de protéines à partir de fractions de matière issues de l'industrie alimentaire
WO2024003323A1 (fr) 2022-06-29 2024-01-04 Mushlabs Gmbh Production d'un milieu de fermentation fongique à partir de drêches de brasserie

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AHMAD MUHAMMAD IJAZ ET AL: "A review on mycoprotein: History, nutritional composition, production methods, and health benefits", TRENDS IN FOOD SCIENCE & TECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, GB, vol. 121, 19 January 2022 (2022-01-19), pages 14 - 29, XP086971024, ISSN: 0924-2244, [retrieved on 20220119], DOI: 10.1016/J.TIFS.2022.01.027 *
C. ELIOPOULOS ET AL.: "Conversion of brewers' spent grain into proteinaceous animal feed using solid state fermentation", ENVIRONMENTAL SCIENCE & POLLUTION RESEARCH, vol. 29, 2022, pages 29562 - 29569, XP037794917, DOI: 10.1007/s11356-021-15495-w
IKRAM ET AL.: "Ikram - Composition and Nutrient Value Proposition of Brewers Spent Grain", JOURNAL OF FOOD SCIENCE, 2017
MARCUS, A.FOX, G.: "Fungal Biovalorization of a Brewing Industry Byproduct, Brewer's Spent Grain: A Review", FOODS, vol. 10, 2021, pages 2159, Retrieved from the Internet <URL:https://doi.orq/10.3390/foods10092159>

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